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Agilent Nanospray HPLC-Chip/MS Protein Identification Solutions LC/MS Application Guide

1. Solvent tray n ___Degasser Analytical system Conventional or HPLC Chip MS Orthogonal nanospray source and nanocolumn EE a 10 po ji anah ler MSD lon Tra __solvent tray P ae Ramo n Figure 15 Offline SCX in combination with nanoflow LC MS MS The fraction collection system consists of e Agilent 1100 Series capillary pump with micro vacuum degasser e Agilent 1100 Series thermostatted micro well plate autosampler e Agilent 1100 Series thermostatted column compartment with micro 2 position 6 port valve e Agilent 1100 Series diode array detector DAD with 500 nL flow cell e Agilent 1100 Series thermostatted micro collector spotter e Agilent ChemStation B 01 03 software 66 Protein ID Solutions LC MS App Guide Optional Peptide Fractionation with Offline SCX 6 Safety WARNING Always take proper precautions for handling and disposing of solvents and other chemicals Consult the material data safety sheets supplied by the vendors See the manuals that you received with the 1100 Series modules for additional safety precautions Procedure summary Schematic overview Figure 16 shows a schematic diagram of offline 2D LC MS You separate peptides in the first dimension by offline SCX with fraction collection the topic of this chapter You then separ
2. Search public databases at www matrixscience com or search proprietary databases using an in house server Search a second database if your first choice fails to provide significant data For faster searches select a species or group of species rather than All entries Be aware that some databases do not rigorously classify taxonomy so if you specify taxonomy you may obtain fewer legitimate hits You may also miss homologous proteins from related species If your search fails to give a result try the Species information unavailable or All entries selections Protein ID Solutions LC MS App Guide 91 8 92 Data Analysis with Mascot Protein Database Search Enzyme Missed cleavages Fixed modifications Variable modifications Peptide charge Data file Instrument Specify the enzyme used for protein digestion If you are unsure you can select None but the search will take much longer because all cleavages must be considered Unless your digestion is perfect choose 1 or at most 2 You can choose larger numbers if you have confidence this is necessary but larger numbers will increase search time and will increase the chance of false positives These modifications are applied universally and add no additional search time Avoid specifying a large number without cause These modifications are not applied universally and add significantly to the search time Select 1 2 and 3 unless you know you can more narrowl
3. 90 acetonitrile 0 1 formic acid in water Flow rate 300 nL min Table1 Nanoflow pump solvent gradient Time min B 0 3 10 3 30 45 33 65 33 01 3 40 min 5 min Table 2 Injector program Step Action 1 Draw default amount from sample 10 pL min speed default offset 2 NEEDLE wash in flush port 5 0 sec 3 INJECT 4 WAIT 10 00 min 5 VALVE bypass Protein ID Solutions LC MS App Guide Needle flush solvent Bottom sensing Pre and post run macros Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 20 methanol 0 1 formic acid in water On 0 9 mm offset for plastic conical micro vials Agilent p n 9301 0978 or 0 2 mm for 96 well Eppendorf well plates Agilent p n 5042 8502 For direct injection mode when the 40 uL injection loop is installed use pre run and post run macros to reduce the pressure flow fluctuation The setup is shown in Figure 12 The macros are available at www agilent com chem techsupport Look under Status Bulletins and Patches Copy the macros to hpchem core Run Time Checklist Instrument 1 X m Check Method S R macro picontrl mcx go IV Data Acquisition I Standard Data Analysis I Customized Data Analysis Macro a Save GLP Data M Post Bun Command Macro macro pictloff mcx go I Save Method with Data Cancel Hep Figure 12 Pre run and post run macros for direct injection with 40 uL loop Protein ID So
4. Ser The peptide bond on the N terminal side of Pro is particularly labile high local proton affinity and usually results in an MS MS spectrum dominated by the y ion ending in Pro 215er 100 0 200 400 600 800 100 1200 1400 1600 1800 Mass m z My Mo f ono yH2o a bonis ynia M y vero V bte yo bes V benzo yHsros y2HsPo4 Highlight None wi e f tbH20 f tbNHs ka Rank gt 256 93 LVGEDVHNImT 362 24 Go oS A l ce l e w a o ts I Ls ts A 32FM 1085 1087 2 pk MHs 1443 592 mie 722 9 2 2 Reset Labels Grown 347et5 100 Grow Shrink Full Alby Mass m z My Mb M bno M yno Ma M bni V yni M ye V yeo V bee M oya M bes Y beH20 V yapos V y2H3P04 Go Highlight None or f b tbH2o f rbnna ba Rank lt gt l ese as1votovunmTee224 wat n o s v AE EAT PR JT N ua SPSON1 1375 1375 0 pkl 3 MH 1420 972 mie 710 99 2 2 Labels 1 23046 h 100 4 Grow Shrink v A by i bie Full o 200 400 600 800 1000 1200 1400 Mass m z Niby My Mo bao yizo a onia yNHs V we yeto V bee a brs V benzo yHsPog y2H3Poq 258 93 LVGEDVHNImT 362 24 Go Highlight None z me roho ronka ra Rank lt gt Protein ID Solutions LC MS App Guide 79 7 Data Analysis with Spectrum Mill MS Proteomics Workbench Table 20 Guidelines for manual results validation Guideline Example E r m MH 712 9
5. The LC MS Application Guide presents the information you need to conduct the LC MS steps for the Nanospray and HPLC Chip MS Protein Identification Solutions In this guide you will learn e How to fractionate peptides by liquid chromatography e How to perform MS MS analyses of peptides e How to analyze the resulting data with the Spectrum Mill MS Proteomics Workbench or the Mascot protein database search software Agilent Technologies Inc 2005 Printed in USA Second edition August 2005 G2458 90002 fhe Agilent Technologies
6. To set a lower threshold go to the MS n tab on the Trap control screen and set Threshold Abs and or Threshold Rel to a lower number Click the Fragmentation button on the MS n page of the Trap control screen Make sure SmartFrag is checked and set to 30 200 See If you have poor sensitivity on page 61 of this chapter See Troubleshooting on page 95 of Chapter 8 Data Analysis with Mascot Protein Database Search See To use Mascot protein database search on page 90 of Chapter 8 Data Analysis with Mascot Protein Database Search See the documentation you received with the Spectrum Mill MS Proteomics Workbench See the documentation you received with the Spectrum Mill MS Proteomics Workbench Try searching another database 62 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with HPLC Chip MS 5 Tips See the tips in the Agilent Nanoflow LC System for Mass Spectrometry MS G2229 Quick Start Guide See also the operation and maintenance chapters in the Agilent 1100 Series G4240A HPLC Chip MS Cube User s Guide Protein ID Solutions LC MS App Guide 63 5 Peptide Separation and Detection with HPLC Chip MS 64 Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide ee ece 6 7 e Optional Peptide Fractionation with e Offline SCX System components 66 Safety 67 P
7. Safety 7 Additional materials required 7 To prepare solvents 7 To plumb the system 8 To prepare and install the nanospray needle 12 To prepare the LC and MS just before an analysis 15 The conventional nanoflow LC MS system used for the Nanospray Protein Identification Solution includes the Agilent nanoflow LC modules and nanoflow LC columns coupled with Agilent s orthogonal nanospray source to the LC MSD Trap XCT Plus or XCT Ultra The LC MSD Trap software is used for complete system control and automation The system includes everything you need to run successful 1D and 2D nanoflow LC MS MS analyses including the enrichment column analytical column connection capillaries and low dispersion fittings This chapter describes how to set up this system and how to prepare it for an analysis If you wish to use the HPLC Chip MS Protein Identification Solution skip to Chapter 4 phe Agilent Technologies 2 Setup of Conventional Nanoflow LC MS MS System System components Solvent tray Degasser Column E MM Compartment KA E a mp Figure 2 TS boler Orthogonal nanospray source and nanocolumn y a O eS 10 port micro valve and holder aa MSD lon Trap Nanoflow LC MS MS sys
8. a MS MS gt 7 Search Spectrum Autovalidation Summary of best matches a y validation with Protein Peptide De novo Summary sequencing End For more details see the Spectrum Mill Application Guide Figure 17 Summary of iterative processing with the Spectrum Mill workbench 74 Protein ID Solutions LC MS App Guide Data Analysis with Spectrum Mill MS Proteomics Workbench 7 Figure 17 shows the iterative MS MS search strategy with the Spectrum Mill workbench Assuming the goal is to identify as many proteins as possible the following summarizes the process 1 Copy or move data to the Spectrum Mill server 2 Preprocess the raw data files with the Data Extractor 3 Search a database in identity mode preferably a species subset database for the first search 4 Autovalidate the results with the highest scores Validation means that the proposed database match is accepted as the correct match for the MS MS spectrum 5 Optional Manually review the medium quality spectra and validate additional results 6 Use Tool Belt to create a saved results file of validated protein hits res file 7 Search the spectra that have not been validated a Search against validated protein hits for the combination of oxidized methionine and pyroglutamic acid b If you suspect phosphorylation search against validated protein hits for the combination of phosphorylated S phosphorylated T and phosphorylated Y c If you su
9. 0 1 formic acid in water to remove hydrophobic peptides 2 In the Sequence Parameters set the post sequence command macro to macro SHUTDOWN MAC go Note that the Standby command stops the LC pumps and ejects the Protein ID Chip but does not put the ion trap into standby mode The Shutdown macro stops the LC pumps and puts the Protein ID Chip and the ion trap into standby mode 3 Before you run your next sequence open the data files for the blanks and check for peptide peaks If there were substantial peaks in the last injection inject more blanks CAUTION If you do not plan to use the system for the next eight hours turn off the LC pumps and put the ion trap in standby mode This extends the life of the electron multiplier 58 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with HPLC Chip MS 5 Troubleshooting If you have flow path blockages Cause Solution 1 Particles from solvent or sample Always filter solvents through 0 4 um filters e Clean up samples per Sample Preparation Guide If you have unstable flow Cause Solution 1 Flow blockages See If you have flow path blockages above 2 System pressure too low Keep system pressure higher than 20 bar at the pump outlet 3 EMPV needs maintenance Run the EMPV cleaning procedure See the nanoflow pump manual 4 Air bubbles in liquid flow path a Purge pumps if you have not already done so b If bubbles persis
10. 0 30 x 35 mm 3 5 um particle size p n 5065 9912 e ZORBAX BioSCX Series II 0 80 mm x 50 mm 3 5 um particle size p n 5065 9942 A 5 acetonitrile 0 03 formic acid B 5 acetonitrile 500 mM ammonium formate 0 03 formic acid 5 uL min for the 0 3 mm column or 15 uL min for the 0 8 mm column See Table 18 for a protein mixture of moderate complexity e g a subcellular fraction or Table 19 for a highly complex protein mixture e g a whole cell lysate In both tables the gradient balances the need to separate proteins with the need to optimize analysis time Table 18 SCX gradient for fraction collection for moderately complex peptide mixture Time min B 0 0 30 20 36 100 44 100 44 01 0 Table 19 SCX gradient for fraction collection for highly complex peptide mixture Time min B 0 0 60 20 72 100 86 100 86 01 0 Protein ID Solutions LC MS App Guide Injection volume Optional Peptide Fractionation with Offline SCX Up to 8 uL or 40 uL depending on loop installed Typically you set your injection volume to inject no more than 100 to 150 ug total protein per run Fraction Time based first fraction 0 5 min remaining fractions each 3 min liquid collection contact control mode with distance of 0 mm fraction size 15 uL cooling 4 C Troubleshooting 6 Problem Cause Solution SCX column plugs frequently All peptides elute in the first few fractions Some peptides
11. 9 9 0 005 10 0 0 74 0 74 01 0 005 Injector program i Table5 Injector program Step Action 1 Draw default amount from sample 10 uL min speed default offset 2 NEEDLE wash in flush port 5 0 sec 3 INJECT Needle flush 20 methanol 0 1 formic acid in water solvent Bottom sensing On 0 9 mm offset for plastic conical micro vials Agilent p n 9301 0978 or 0 2 mm for 96 well Eppendorf well plates Agilent p n 5042 8502 Protein ID Solutions LC MS App Guide 27 28 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Protocol for 1D enrichment LC long method for more complex samples Columns Enrichment column ZORBAX 300SB C18 0 3 x 5 mm 5 um particle size e Reversed phase column ZORBAX 300SB C18 75 um x 150 mm 3 5 um or ZORBAX 300SB C18 75 um x 50 mm 3 5 um particle size The longer column gives better separations when there are more peaks Maximum 8 uL or 40 uL depending on loop installed njection volume Enrichment Position 1 at 0 min position 2 at 5 min then return to position 1 at 146 min column switch Nanoflow pump A 0 1 formic acid in water B 90 acetonitrile 0 1 formic acid in water Primary flow 200 to 500 uL min From the Instrument menu choose More pumps gt Auxiliary Table6 Nanoflow pump solvent and flow gradient Time min B Flow pL min 0 3 0 3 5 3 0 3 11 15 0 3 131 45 0 3 138 90 0 4 143 90 0 6 146 3 0 4 160 3 0 3 Stop time 1
12. Always filter solvents through 0 4 um filters e Clean up samples per Sample Preparation Guide Ifyou do not plan to use the system for several days remove the nanospray needle column holder assembly and store in protective plastic sleeve Ifthe needle is blocked flush with 100 B1 for 30 min This sometimes removes the blockage If you have unstable flow Cause 1 Flow blockages can be especially troublesome in direct injection mode with sampler in mainpass System pressure too low EMPV needs maintenance Air bubbles in liquid flow path Solution See If you have flow path blockages above Keep system pressure higher than 20 bar at the pump outlet Run the EMPV cleaning procedure See the nanoflow pump manual a Purge pumps if you have not already done so b If bubbles persist pump 500 nL min of 50 B for 20 min c lf bubbles still persist disconnect first capillary at pump end of system and purge at higher flow rate Reconnect first capillary and disconnect next one Purge again Continue disconnecting capillaries sequentially and purging until bubbles are gone Protein ID Solutions LC MS App Guide 35 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS If you have unstable spray Cause Solution 1 Capillary voltage not set correctly Adjust the capillary voltage Using the 8 um needle tips and liquid flow between 175 and 300 nL min the capillary voltag
13. Analysis with Mascot Protein Database Search D Auto_MIS_MagfExport m 30FM1_D DataAnalysis Script File Edit View Layout Help eale Object Form 7 Events x Auto MIS _MgfExport Processing Script C Agilent Technologies 2003 MSD Trap SW 5 2 This Script is used to generate compound mass Spectra from AutoMS n data deconvolute that data and then export it to a mgf data file name containing the same filename as the original d file and located in the same d folder Can be applied to Mb 78 AutoMS n D example data file Dim DataFilePath DataFileName SubExt TargetPath Dim fso Set fso CreateObject Scripting FileSystemObject DataFilePath Analysis Path DataF i leName Analysis Name SubExt Len DataFileName 2 ITargetPath DataFilePath Mid DataFileName 1 SubExt e mgf Check if export MGF file already exists and delete if true if fso0 FolderExists TargetPath then fso deletefolder TargetPath lend if Carry out the data processing of a protein digest ClearResults Chromatograms Clear Compounds Clear Chromatograms Add daTIC da ll daBoth Chromatograms Add daTIC daallMSn daBoth Chromatograms Add daBPC dadllMS daBoth FindautoNsn Compounds Deconvolute export to d datafilename mgf Analysis Compounds Export TargetPath damgf Save processed results Analysis save iform close Figure 19 Visual Basic script to automate data analysis Fo
14. Guide 61 5 Peptide Separation and Detection with HPLC Chip MS If you have low database search scores or poor sequence coverage Cause Solution 1 Sample preparation or data acquisition problem 2 Number of precursors for data dependent MS set too low 3 Threshold for triggering MS set too high 4 SmartFrag turned off resulting in inadequate fragmentation 5 Poor sensitivity 6 Default settings for LC MSD Trap data analysis software not appropriate for sample applies only if you use Mascot search 7 Mascot parameters not optimized applies only if you use Mascot search 8 Spectrum Mill Data Extractor parameters not set properly applies only if you use Spectrum Mill workbench 9 Spectrum Mill Search parameters not set properly applies only if you use Spectrum Mill workbench 10 Chosen database did not give good results Open the data file and see how many peaks you have in the MS BPC If you have very few peaks you have a problem with the sample preparation or with the data acquisition settings See causes 2 through 5 below as well as the troubleshooting in the Sample Preparation Guide If you have a reasonable number of peaks your problem resides in the data processing See causes 6 through 10 below Go to the MS n tab on the Trap control screen and set No of Precursor lons to 5 or 6 Examine your data and set a lower threshold if it appears inadequate MS experiments were generated
15. Online Plot Soran st Figure 6 Desired stability of flow Figure 7 Desired stability of pressure Protein ID Solutions LC MS App Guide Setup of Conventional Nanoflow LC MS MS System 2 To prepare the second pump If you are using a second pump for the enrichment column mode or 2D LC mode prepare it as follows 1 Purge the second capillary or standard flow pump Open the manual purge valve and purge the solvent channel s you will use at 2 5 mL min for four minutes each 2 Switch to your composition and flow rate for sample loading 3 Close the manual purge valve To prepare the well plate sampler If the well plate sampler has not been used for two days prime the needle wash for 120 seconds Go to Instrument gt More Injector gt Prime Flush Pump To prepare the LC columns The first time you use a new SCX enrichment or nanoflow LC column condition it as follows 1 Make several injections of a high level digest sample e g 500 fmol BSA digest 2 Inject several solvent blanks To calibrate the MS A separate needle holder is provided so you can keep the tuning mix infusion assembly intact and easily interchange it with the column needle holder assembly Calibrate the ion trap mass spectrometer as directed in the Orthogonal Nanospray Ion Source User s Guide e Use the ESI tuning mix G2431A e Use the nanospray source e Be sure to check the ion trap mass axis calibrations scan isolation a
16. a Comparison on Yeast Agilent publication number 5989 0212EN 32 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS MS MS method for LC MSD Trap XCT Plus or XCT Ultra lonization mode Drying gas flow Dry gas temperature Vcap Skim1 Capillary exit Trap drive Averages ICC Maximum accumulation time Smart target MS scan range Scan mode Number of precursor ions Averages Fragmentation amplitude SmartFrag Source and ion optics conditions Positive nanospray with Agilent orthogonal source G1982B 5 L min 300 C Typically 1600 2000 V Check for stable current of 80 100 nA at starting solvent composition and flow 30 V 100 V lon trap conditions MS scan 85 500 000 300 2200 lon trap conditions automatic MS MS Peptide 3 or 4 for XCT Plus 5 or 6 for XCT Ultra 1 1 3 V On 30 200 Protein ID Solutions LC MS App Guide 33 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Active exclusion Prefer 2 MS MS scan range Ultra scan ICC target On 2 spectra 1 min On 100 2000 On 500 000 To shut down the nanoflow LC MS MS system CAUTION To shut down the system do the following at the end of your sample sequence 1 Inject two solvent blanks to remove residual peptides from the injector Acquire data for these injections For simple samples inject the solvent you used to di
17. a lot of samples use a 2 L bottle Agilent p n 9301 6342 for the aqueous solvent The solvent channels used for the nanoflow pump are Al and B1 2 Prepare 0 1 formic acid in water for solvent Al 3 Prepare 90 acetonitrile 0 1 formic acid in water for solvent B1 CAUTION Replace the solvent for the B1 channel at least weekly The A1 solvent is stable longer Remember to check your waste bottle frequently The bottle fills more quickly because you split your flow Protein ID Solutions LC MS App Guide 7 2 Setup of Conventional Nanoflow LC MS MS System To plumb the system 1 Decide which LC configuration is most appropriate for your samples If you are unsure Read Choosing the LC method below 2 Read and follow all instructions in the Agilent Nanoflow LC System for Mass Spectrometry MS G2229 Quick Start Guide Skip this step if you did it when the system was installed 3 Arrange your LC modules as shown in Stacking diagrams on page 10 4 Read Plumbing overview on page 11 5 Plumb the system For the capillaries to use refer to the diagram and table in the Agilent Nanoflow Proteomics Solution Quick Reference Guide These provide an overview of all the capillaries needed for the installation Be sure to select only the plumbing components you need for your mode of operation direct injection enrichment column 2D LC For more details refer to diagrams in Direct injection mode 1D LC on pag
18. an enrichment column The Protein ID Chip is preconfigured for the enrichment column mode The choice of direct injection mode versus enrichment column mode depends upon sample characteristics injection volume chromatographic considerations and peptide recovery needs Apg Agilent Technologies 97 9 Reference Sample Analysis Strategy Direct injection mode 1D LC The following discussion applies only for the Nanospray Protein Identification Solution The direct injection mode is not applicable for the HPLC Chip MS Protein Identification Solution A diagram of the direct injection mode for the conventional nanoflow LC MS MS system is shown in Figure 24 The direct injection mode provides the best chromatography There is no enrichment column to compromise peak resolution The direct injection mode also gives better sequence coverage and best sensitivity You may lose peptides if you use the enrichment column The unrecovered peptides are generally those of lower chain length with less diagnostic value but they may be important for your particular analysis Nanoflow pump G z Analytical column nt as MS detection Waste 1100 Micro well plate sampler Figure 24 Direct injection mode Protein ID Solutions LC MS App Guide Reference Sample Analysis Strategy 9 Enrichment column mode 1D LC The enrichment column is sometimes called a trapping column or a pre column Flow diagrams f
19. dimensional LC with salt plugs Flow diagrams for the conventional nanoflow LC MS MS system are shown in Figure 28 through Figure 31 Flow diagrams for the HPLC Chip MS system are similar but some components are contained within the Protein ID Chip For details on these microfluidic components see the flow diagrams in the Agilent 1100 Series G4240A HPLC Chip MS Cube User s Guide Protein ID Solutions LC MS App Guide 101 9 Reference Sample Analysis Strategy In the first step of online 2D LC you load the digest onto a strong cation exchange SCX column See Figure 28 You use the Agilent 1100 well plate sampler and a second standard or capillary LC pump for sample loading The loading solvent is typically 96 9 water 0 1 formic acid and 3 acetonitrile The enrichment column is in line during sample loading to capture any peptides that are not retained on the SCX column Second pump Contained within Protein ID Chip of HPLC Chip MS Protein Identification Solution Inject sample Nanoflow pump 5 z m MS detection 1100 Micro well plate sampler 1100 Column compartment Figure 28 2D LC step 1 load sample on SCX column In the second step Figure 29 you elute the sample from the strong cation exchange column onto a C8 C18 enrichment column You do this by injecting a sequential salt gradient with the autosampler For LC MS the volatile salts such as ammonium acetate and ammonium formate are generally
20. do not elute from the SCX column Particles from biological Filter or use Agilent Spin Filters p n 5185 5990 to remove particulates samples If your column is plugged you can sometimes recover by backflushing the column or replacing the screen at the head of the column Check your column information sheet for the part number for the screen Salts from biological Remove salts with Agilent Cleanup C18 Spin Tubes or Cleanup C18 samples Pipette Tips Urea or guanidine HCI Remove salts with Agilent Cleanup C18 Spin Tubes or Cleanup C18 was used inthe tryptic Pipette Tips digestion Excess ammonium Dry sample Add 100 to 200 pL water Repeat drying and water addition bicarbonate from once or twice ending with a dry sample Dissolve sample in 0 1 formic digestion acid or other appropriate buffer Highly charged peptides Runa pH gradient in combination with a salt gradient The SCX method is optimized for tryptic peptides which typically carry no more than 3 or 4 charges For more highly charged peptides you may need to run a combination salt pH gradient For details see the note on page 104 Protein ID Solutions LC MS App Guide For additional troubleshooting tips see the manuals you received with the modules in your fraction collection system 71 72 Optional Peptide Fractionation with Offline SCX Tips Gradient for fraction collection Preventing degradation of fractions Plumbing of capillaries Opti
21. more details on 1D and 2D LC configurations see Chapter 9 Reference Sample Analysis Strategy starting on page 97 Protein ID Solutions LC MS App Guide Setup of HPLC Chip MS lon Trap System To configure the Chip Cube From the Instrument menu choose More Chip Cube gt Configuration Refer to Figure 14 and configure the Chip Cube as follows Injection Flush 8 uL for 1D LC or 16 uL for 2D LC with 75 um needle seat capillary Volume Loading Pump G1376A Analytical Pump G2226A Pump Configuration Instrument 1 X m Used Pumps Injection Flush Volume g pl Loading Pump p2 G1376A DE33200942 Analytical Pump pl G22264 DE40500281 Cancel Help Figure 14 Chip Cube configuration Explanation To allow maximum reuse of methods some configuration parameters are specific to the system and are not stored with the method Examples include the injector loop configuration and the plate configuration for the micro well plate sampler When you establish the above configuration settings for the Chip Cube the system automatically calculates based on injection flush volume and sample injection volume when to switch the valve between sample loading and sample analysis Also when you wish to eject the Protein ID Chip the system calculates how long to wait to release LC pump pressure before it actually ejects the HPLC Chip So it is critical that you properly configure the Chip Cube Protein I
22. preferred The salt step gradient typically consists of individual salt solutions ranging from 15 millimolar to 500 millimolar These are injected in aliquots related to the size of the SCX column For example 20 uL aliquots are used for a 0 30 x 35 mm column Each injected salt solution elutes a fraction of the peptides from the SCX column for analysis by nanoflow LC MS MS The result is an individual data file for each step of the salt gradient You can optimize the spacing of the salt concentrations for your sample Some samples fractionate more evenly with smaller concentration differences at the beginning of the stepwise gradient For example you might use 15 30 45 60 75 90 120 300 and 500 millimolar concentrations as opposed to concentrations spaced at 50 millimolar increments 102 Protein ID Solutions LC MS App Guide Reference Sample Analysis Strategy 9 Second pum Contained within Protein ID Chip of HPLC Chip MS Protein Identification Solution Inject salt gradient Nanoflow pump k a ral Waste MS detection 1100 Micro well plate sampler 1100 Column compartment Figure 29 2D LC step 2 step elute sample from SCX to enrichment column In the third step you wash and desalt the fraction on the enrichment column To do this you switch the six port valve in the micro well plate sampler and pump loading solvent or water with the second pump See Figure 30 Second pum ore Contained within Protei
23. use Spectrum Mill workbench 10 Chosen database did not give good results Open the data file and see how many peaks you have in the MS BPC If you have very few peaks you have a problem with the sample preparation or with the data acquisition settings See causes 2 through 5 below as well as the troubleshooting in the Sample Preparation Guide If you have a reasonable number of peaks your problem resides in the data processing See causes 6 through 10 below Go to the MS n tab on the Trap control screen and set No of Precursor lons to 3 or 4 for the XCT Plus or 5 to 6 for the XCT Ultra Examine your data and set a lower threshold if it appears inadequate MS experiments were generated To set a lower threshold go to the MS n tab on the Trap control screen and set Threshold Abs and or Threshold Rel to a lower number Click the Fragmentation button on the MS n page of the Trap control screen Make sure SmartFrag is checked and set to 30 200 See If you have poor sensitivity on page 37 of this chapter See Troubleshooting on page 95 of Chapter 8 Data Analysis with Mascot Protein Database Search See To use Mascot protein database search on page 90 of Chapter 8 Data Analysis with Mascot Protein Database Search See the documentation you received with the Spectrum Mill MS Proteomics Workbench See the documentation you received with the Spectrum Mill MS Proteomics Workbe
24. ways Both separations can be done online online 2D LC In this case the peptides are eluted from the SCX column by injection of salt plugs are further separated by RPLC and are then subjected to MS MS analysis All this is automated on a single LC MS MS system Alternatively the SCX can be done offline with a continuous salt gradient and fraction collection Then those fractions are separated online with RPLC and analyzed by MS MS The offline approach requires two LC systems one for SCX and one for RPLC It often yields more protein identifications but can take significantly more time depending on the number of fractions collected and analyzed An intermediate technique in terms of separating power and time commitment is online 2D separation with a semi continuous salt gradient Mass spectrometry Following LC separation the peptides are analyzed by mass spectrometry MS With the Agilent system the LC eluent enters the ion source of an ion trap mass spectrometer via an orthogonal nanospray source The molecular weights of the peptides are determined by MS precursor ion scans MS MS scans are then triggered on selected precursor ions according to data dependent acquisition rules that are established prior to the analysis The MS spectra provide molecular weights of the peptides while ion fragments in the MS MS spectra provide amino acid sequence information Protein ID Solutions LC MS App Guide 3 1 Overview of LC MS Steps with th
25. 0 35 80 0 30 37 80 0 30 37 01 30 0 45 45 min Protein ID Solutions LC MS App Guide Post time Capillary pump Needle flush solvent Bottom sensing Peptide Separation and Detection with HPLC Chip MS none 3 acetonitrile 0 1 formic acid in water Capillary pumps use no flow gradient just set flow to 4 uL min 20 methanol 0 1 formic acid in water On 0 9 mm offset for plastic conical micro vials Agilent p n 9301 0978 or 0 2 mm for 96 well Eppendorf well plates Agilent p n 5042 8502 Protein ID Solutions LC MS App Guide 5 53 Peptide Separation and Detection with HPLC Chip MS Protocol for online 2D LC method with Protein ID Chip SCX column HPLC Chip Injection volume 54 Recommended salt steps LC details ZORBAX BioSCX Series II 0 30 x 35 mm 3 5 um particle size Use Protein ID Chip p n G4240 62001 The chip integrates the following columns e Enrichment column ZORBAX 300SB C18 40 nL 5 um particle size e Reversed phase column ZORBAX 300SB C18 75 um x 43 mm 5 um particle size 20 uL for salt steps must have 40 uL loop installed 0 15 30 45 60 75 90 120 300 500 mM ammonium formate prepared in 3 acetonitrile 0 1 formic acid in water Choose the number of salt steps based on sample complexity Prepare salt solutions from 5 M ammonium formate stock Agilent p n G1946 85021 Prepare the solutions directly into the 2 mL glass autosampler vials supplied with the HPL
26. 0 Flow gradient for second non capillary pump Time min Flow mL min initial 0 005 0 01 0 1 0 50 0 1 1 0 0 05 1 01 0 01 10 0 01 10 01 0 005 15 0 005 15 1 0 74 short method or 159 long method 0 74 01 short method or 159 01 long method 0 005 Protein ID Solutions LC MS App Guide 31 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Injector program Sequence table Table 11 Injector program Step 1 Action Draw default amount from sample 10 pL min speed default offset NEEDLE wash in flush port 5 0 sec INJECT A typical sequence table for salt steps is given in Table 12 Table 12 Sequence table to inject salt steps Line Location Sample Method Inj Sample Type DataFile Inj Volume Name Name Location 1 P1 A 01 Sample 2DLCX M 1 Sample 0mM 5 2 Vial 1 15mM salt 2DLCX M 1 Sample 15mM 20 3 Vial 2 30mM salt 2DLCX M 1 Sample 30mM 20 4 Vial 3 45mM salt 2DLCX M 1 Sample 45mM 20 5 Vial 4 60mM salt 2DLCX M 1 Sample 60mM 20 6 Vial 5 75mM salt 2DLCX M 1 Sample 75mM 20 7 Vial 6 90mM salt 2DLCX M 1 Sample 90mM 20 8 Vial 7 120mM salt 2DLCX M 1 Sample 120mM 20 9 Vial 8 300mM salt 2DLCX M 1 Sample 300mM 20 10 Vial 9 500mM salt 2DLCX M 1 Sample 500mM 20 11 Vial 10 1000mM salt 2DLCX M 1 Sample 1000mM 20 Protocol for 2D LC method with semi continuous salt gradient For this method refer to the Agilent application note Improved 2D Nano LC MS for Proteomics Applications
27. 52 miz 366 93 2 2 f Labels Y4 between the first two N terminal aE It is typical to lack fragmentation D amino acids i e no by or y ions l 9 68245 100 Grow Shrink Full 700 Mass m z awy My Mb f bno yoo a onis ynia V ye vero JY b ys ors Y benzo yHsroa y2haPoa 256 93 LVGEDVHNImT 362 24 Go Highlight None lt b PF kenzo f tbnHs ka Rank gt Look for coverage over a significant portion of the backbone i l SPSON1 1049 1040 0 pkl 350e 100 Grow Good lunar Shrink i Full 600 Marz miz Alby My Me f ono yho a T buns ynia V y yeo V be ys bea V benzo yHsroa y2Hsroa 256 92 LVGEDVHNIMT 362 24 Go Highlight None f th f keho f kenia ka Rank lel usTeg 1 K SPSON2 1363 1365 0 pkt MH 779 772 mz 390 39 2 2 6 80 0 450 Mass m z My Mo T bno yho a bni ynia V y T yero Y be T ya bes Y benzo J ytsroa y2H3Po4 256 93 LVGEDVHNImT 362 24 Go Highlight None tb f tbH2o f tbnHs ba Rank lt l Reset s l K MHs 773 762 me 387 98 2 2 Labels The peptide bond on the C terminal 1 l E L L at a side of acidic residues D E may a 7 3 00e 6 show enhanced cleavage a sa Shrink 400 450 500 550 600 650 700 750 Mass m z awy My Mo ono yno a bonis ynia M e vero M oe yes bes V bezo yHarog y2Hsro4 258 93 LVGEDVHNImT 362 24
28. 60 min Post time 5 min Second pump 3 acetonitrile 0 1 formic acid in water Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 Isocratic quaternary or binary pumps use flow gradient given in Table 7 on page 29 Capillary pumps use no flow gradient just set flow to 15 uL min Table 7 Flow gradient for second non capillary pump Time min Flow mL min initial 0 005 0 01 0 1 0 50 0 1 1 0 0 05 1 01 0 01 8 0 0 01 8 01 0 005 9 9 0 005 10 0 0 159 0 159 01 0 005 Injector program prog Table8 Injector program Step Action 1 Draw default amount from sample 10 uL min speed default offset 2 NEEDLE wash in flush port 5 0 sec 3 INJECT Needle flush 20 methanol 0 1 formic acid in water solvent Bottom sensing On 0 9 mm offset for plastic conical micro vials Agilent p n 9301 0978 or 0 2 mm for 96 well Eppendorf well plates Agilent p n 5042 8502 Protein ID Solutions LC MS App Guide 29 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Protocol for online 2D LC method with salt steps Columns Injection volume 30 Recommended salt steps LC details e SCX column ZORBAX BioSCX Series II 0 30 x 35 mm 3 5 um particle size e Enrichment column ZORBAX 300SB C18 0 3 x 5 mm 5 um particle size e Reversed phase column ZORBAX 300SB C18 75 um x 150 mm 3 5 um or ZORBAX 300SB C18 75 um x 50 mm 3 5 um parti
29. Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide Agilent Technologies Notices Agilent Technologies Inc 2005 No part of this manual may be reproduced in any form or by any means including elec tronic storage and retrieval or translation into a foreign language without prior agree ment and written consent from Agilent Technologies Inc as governed by United States and international copyright laws Manual Part Number 62458 90002 Edition Second edition August 2005 Printed in USA Agilent Technologies Inc 5301 Stevens Creek Blvd Santa Clara CA USA 95052 Warranty The material contained in this docu ment is provided as is and is sub ject to being changed without notice in future editions Further to the max imum extent permitted by applicable law Agilent disclaims all warranties either express or implied with regard to this manual and any information contained herein including but not limited to the implied warranties of merchantability and fitness for a par ticular purpose Agilent shall not be liable for errors or for incidental or consequential damages in connec tion with the furnishing use or per formance of this document or of any information contained herein Should Agilent and the user have a separate written agreement with warranty terms covering the material in this document that conflict with these terms the warranty terms i
30. App Guide 5 56 Peptide Separation and Detection with HPLC Chip MS MS MS method for LC MSD Trap XCT Ultra lonization mode Drying gas flow Dry gas temperature Vcap Skim1 Capillary exit Trap drive Averages ICC Maximum accumulation time Smart target MS scan range Scan mode Number of precursor ions Averages Fragmentation amplitude Source and ion optics conditions Positive nanospray with Agilent orthogonal source included within HPLC Chip MS interface G4240A 4 L min 300 C Typically 1800 V Check for stable current of about 40 50 nA at starting solvent composition and flow 30 V 100 V lon trap conditions MS scan 85 500 000 300 2200 lon trap conditions automatic MS MS Peptide 5 or6 1 3 V Protein ID Solutions LC MS App Guide Peptide Separation and Detection with HPLC Chip MS 5 SmartFrag On 30 200 Active exclusion On 2 spectra 1 min Prefer 2 On MS MS scan 100 2000 range Ultra scan On ICC target 500 000 Protein ID Solutions LC MS App Guide 57 5 Peptide Separation and Detection with HPLC Chip MS To shut down the HPLC Chip MS system To shut down the system do the following at the end of your sample sequence 1 Inject two solvent blanks to remove residual peptides from the injector Acquire data for these injections For simple samples inject the solvent you used to dissolve your peptides For complex samples inject 60 acetonitrile
31. C Chip MS Protein Identification Solution Table 15 Preparation of salt solutions for 2D LC with salt steps Concentration mM Volume of 5 M stock pL Volume of water pL 15 3 997 30 6 994 45 9 991 60 12 988 75 15 985 90 18 982 120 24 976 300 60 940 500 100 900 1000 100 400 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with HPLC Chip MS Nanoflow pump Use the nanoflow pump settings from either the short or long enrichment methods for the Protein ID Chip See page 51 or page 52 Choose the appropriate method based on the complexity of the sample Capillary pump 3 acetonitrile 0 1 formic acid in water Capillary pumps use no flow gradient just set flow to 4 uL min Sequence table A typical sequence table for salt steps is given in Table 16 Table 16 Sequence table to inject salt steps Line Location Sample Method Inj Sample Type DataFile Inj Volume Name Name Location 1 P1 A 01 Sample 2DLCX M 1 Sample OmM 5 2 Vial 1 15mM salt 2DLCX M 1 Sample 15mM 20 3 Vial 2 30mM salt 2DLCX M 1 Sample 30mM 20 4 Vial 3 45mM salt 2DLCX M 1 Sample 45mM 20 5 Vial 4 60mM salt 2DLCX M 1 Sample 60mM 20 6 Vial 5 75mM salt 2DLCX M 1 Sample 75mM 20 7 Vial 6 90mM salt 2DLCX M 1 Sample 90mM 20 8 Vial 7 120mM salt 2DLCX M 1 Sample 120mM 20 9 Vial 8 300mM salt 2DLCX M 1 Sample 300mM 20 10 Vial 9 500mM salt 2DLCX M 1 Sample 500mM 20 11 Vial 10 1000mM salt 2DLCX M 1 Sample 1000mM 20 Protein ID Solutions LC MS
32. D Solutions LC MS App Guide 45 4 Setup of HPLC Chip MS lon Trap System To insert the HPLC Chip and start the spray Inserting the HPLC Chip 1 Insert the Protein ID Chip p n G4240 62001 into the slot on the HPLC Chip MS interface 2 In the ChemStation software right click the HPLC Chip icon and select Operate The automatic loading mechanism loads the HPLC Chip into the HPLC Chip MS interface and positions the needle tip 3 Check the video monitor to be sure the Protein ID Chip is in the correct position Starting the nanoflow pump Set the flow rate to 300 nL min 2 Set the solvent composition to 97 3 A1 B1 the starting composition for the methods 3 Turn the pump on Starting spray 1 Make sure the capillary voltage is set to 1800 V 2 Confirm that you have a stable current typically 40 to 50 nA The maximum voltage for the HPLC Chip MS orthogonal nanospray source is 2200 V and a typical voltage with a new chip is 1800 V 3 Save the method to store the new Vcap setting Increase the Vcap to the point where stable spray without spiking is achieved Splitting of the stream usually indicates that the voltage is too high Look for both a stable and reasonable MS spectrum as well as several minutes of stable chromatographic signal no spiking The capillary current is typically 40 to 50 nA at the starting composition of 97 3 A1 B1 and will drop as the percentage of B1 increases 46 Protein ID Solutions LC M
33. Go Highlight None lt b f tbH2o tbNHs ka Rank ll 0 50 100 150 200 250 300 350 80 Protein ID Solutions LC MS App Guide Data Analysis with Spectrum Mill MS Proteomics Workbench 7 Table 20 Guidelines for manual results validation Guideline Example Be suspicious of a clumped pattern that occurs frequently throughout A O 0030414004 0331 0331 0 pki MHa 1406 374 mie 400 13 2 3 Labels the LC run usually a background ieee Grow ion Shik FA Full 0 200 400 600 800 1000 1200 4400 Mass m z Ally My Me f bno yno a J bonis ynia V yet yetpH2z0 V be ws bts V benzo yHsroa y2HsPo4 258 93 LVGEDVHNImT 362 24 Go Highlight None bb kenzo kenis ka Rank gt Be suspicious if no ions appear E PP R TC E rons u E R oon above the precursor ion when QSCX24K21 COMPRESSED 992 0 2 pl MHs 987 631 mie 40492 2 2 Labels 4 80612 precursor charge state is gt 1 som a ma l Shrink Charge state 2 ai Mass m z gt awy My Mb M bomo Y yho Ma M boni V yni V y V yo V be V ys V bes V benzo V yarog V y2H3Po4 256 93 LVGEDVHNIMT 362 24 Go Highlight None f tb tbHzo tonks ba Rank l Remember When in doubt don t validate The goal is no false positives e Validating a questionable hit removes it from subsequent searches Protein ID Solutions LC MS App Guide 81 7 Data Analysis with Spectrum Mill MS P
34. MS analysis time 4 Transfer the well plates containing all collected fractions to the nanoflow LC MS MS system 5 Analyze the fractions using one of the following protocols Protocol for 1D enrichment LC short method for less complex samples on page 26 68 Protein ID Solutions LC MS App Guide Optional Peptide Fractionation with Offline SCX 6 Protocol for 1D enrichment LC long method for more complex samples on page 28 Protocol for 1D enrichment LC with Protein ID Chip short method for less complex samples on page 51 Protocol for 1D enrichment LC with Protein ID Chip long method for more complex samples on page 52 More details For more details see the application note entitled Tools and considerations to increase resolution of complex proteome samples by two dimensional offline LC MS Agilent publication number 5989 0213EN Additional materials required In addition to the 1100 Series modules you need the following e Water HPLC grade 18 megohm Agilent p n 8500 2236 or equivalent e Acetonitrile HPLC grade Agilent p n G2453 85050 or equivalent e Formic acid analytical grade Agilent p n G2453 85060 or equivalent e Ammonium formate reagent grade Protein ID Solutions LC MS App Guide 69 70 Optional Peptide Fractionation with Offline SCX Protocol for offline SCX Column Capillary pump Flow Gradient Choose one of the following e ZORBAX BioSCX Series II
35. Program Files MasCat E xample Data BSA00014 D 5Mix_200mM maf BSA00007 D xl mgf C Program Files MasCat Example Data BS400015 D 5Mix_300mM maf C Program Files MasCat Example Data BS400016 D 5Mix_400mM mat Output maf C Program Files MasCat Example Data BS400017 D 5SMix_500mM maf Add All gt gt C Program Files MasCat Example Data BS400018 D 5Mix_1000mM maf Add Selected gt Remove Selected Remove All tLe File Type MASCOT MGF files maf 7 m Calculate Number of Queries MASCOT Must Perform Global charge setting fis 2 and 34 z Ignore MS MS spectra with fewer than 5 fragment ions Cmpds with charge 1 Cmpds with charge 2 7 Total compounds 556 Output File Path and Name Cmpds with charge 3 o Total queries 3736 C Program Fies MasCat Evample Data Concatenatedma Bo Cmpds with charge gt 3 gt Cmpds with unknown charge Calculate Number of Queries Concatenate Status BEGIN MGF Profiling 11 8 2002 12 45 26 PM Profiling files Successfully processed file C Program Files MasCat E xample D ata BSA00001 D55Mix_00mM mgf Successfully processed file C Program Files MasCat E xample Data BSA00002 D 5Mix_10mM maf Successfully processed file C Program Files MasCat E xample Data BS400003 D 5Mix_20mM maf Successfully processed file C Program Files MasCat Example Data BSA00004 D 5Mix_30 mgf xl Figure 20 MASCAT concatenator to combine mgf files from multiple sampl
36. S App Guide Setup of HPLC Chip MS lonTrap System 4 To prepare the LC and MS just before an analysis After you have plumbed your HPLC Chip MS system you prepare it for stable analyses You purge the LC calibrate the MS if not already done and connect the two Repeat the LC purge any time the system has been shut down for more than a day To prepare the nanoflow pump Follow directions in To prepare the nanoflow pump on page 15 To prepare the capillary pump 1 Purge the capillary pump Purge the solvent channel s you will use at 2 5 mL min for four minutes each 2 Switch to your composition and flow rate for sample loading To prepare the well plate sampler If the well plate sampler has not been used for two days prime the needle wash for 120 seconds Go to Instrument gt More Injector gt Prime Flush Pump To prepare the Protein ID Chip The first time you use a new Protein ID Chip condition it as follows 1 Make two to three injections of a high level standard protein digest sample e g 100 fmol BSA digest using the short method on page 51 2 Inject a solvent blank To calibrate the MS 1 Just before use prepare a 1 5 dilution of the electrospray calibrant p n G2421A in acetonitrile 2 Install the MS Calibration and Diagnosis Chip p n G4240 61001 3 Connect the dedicated calibrant line from the HPLC Chip MS interface to the syringe pump 4 Load the syringe pump with diluted calibrant and start t
37. Switch to direct injection mode if that is an option Consider column mode is generally not as good Your injection volume as in direct injection mode Level of salts and other water soluble contaminants 36 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 If you have sample carryover Cause Solution 1 No needle wash Set up needle wash for well plate sampler 2 Inappropriate needle wash solvent e Switch to a solvent combination in which your sample is completely soluble 3 Residual hydrophobic peptides inthe Inject the following to remove hydrophobic peptides injection system First inject 50 TFE in water Then inject several solvent blanks with a high percentage of organic If you have poor sensitivity Cause Solution 1 Detector gain adjustment needs tobe See instructions for verifying detector setting in your LC MSD Trap redone documentation 2 Capillary voltage too high causing Reduce the capillary voltage corona which can destroy peptides 3 Sample degradation from sitting at Prepare fresh samples room temperature If you have the optional thermostat on the micro well plate sampler make sure it is turned on and set to 4 C 4 Peptides adsorbed on vial surface e Switch to a different vial material glass or plastic 5 Bad injection due to air bubble at Tap vial gently to dislodge air bubble bottom of vial 6 Unstable spray See If you h
38. ained within Protein ID Chip of DEJ HPLC Chip MS Protein Identification Solution Nanoflow pump ENS MS detection 1100 Micro well plate sampler 1100 Column compartment Figure 26 Enrichment column mode step 2 analyze sample 100 Protein ID Solutions LC MS App Guide Reference Sample Analysis Strategy 9 Two dimensional liquid chromatography 2D LC You can configure nanoflow LC MS MS for two dimensional liquid chromatography 2D LC for instances where separation in a single dimension is insufficient Two dimensional LC adds a separation based on a different physical property For example if one of the separations is reversed phase LC then the second could be ion exchange size exclusion affinity etc Use two dimensional LC for very complex protein digests or as a substitute for 2D gel electrophoresis A schematic of 2D LC is shown in Figure 27 In typical 2D LC for protein digests you first separate the peptides on a strong cation exchange SCX column You then further separate these fractions by reversed phase LC There are a number of configurations for the SCX separation e Offline SCX with fraction collection described in Chapter 6 e Online SCX with salt steps for sample elution described in detail here e Online SCX with a semi continuous salt gradient for sample elution described in Agilent publication number 5989 0212EN Figure 27 Two dimensional LC concept There are four steps for online two
39. ate the peptides in the second dimension with nanoflow LC MS MS as described in Chapter 3 or Chapter 5 1st dimension SCX c Capillary gt a ex 2 4 dimension Reversed Phase ZA im Cina ao e Figure 16 Overall schematic for offline 2D LC MS Protein ID Solutions LC MS App Guide 67 6 Optional Peptide Fractionation with Offline SCX Summary of steps 1 Load tryptic peptides onto the SCX column 2 Elute peptides with a linear salt gradient Some general guidelines are given in Table 17 More details for two of the gradients are given in Table 18 and Table 19 Table 17 Salt gradients for peptide mixtures of increasing complexity Sample complexity Goal SCX gradient min SCX gradient min Approximate number 1st part 2nd part of SCX fractions 0 100 mM 100 500 mM ammonium formate ammonium formate Simple mixture e g Maximum number of 15 3 7 highly resolved 2D gel proteins identified spot Subcellular fraction Reasonable number of 30 6 14 proteins identified with shorter analysis time Whole cell lysate Reasonable number of 60 12 28 proteins identified with shorter analysis time Whole cell lysate Maximum number of 120 24 45 proteins identified Assuming fractions collected as described on page 71 3 Collect fractions of about 15 uL each with the 1100 Series micro fraction collection system You can collect smaller fractions but this will increase LC
40. ave unstable spray on page 36 of this chapter 7 Other nanospray problems See the troubleshooting chapter in Agilent G1982B Orthogonal Nanospray lon Source User s Guide 8 Find Compounds settings not For low level samples reduce these thresholds in the data analysis method optimized for low level samples parameters giving the appearance of poor Find AutoMS n compound detection threshold sensitivity Mass List Apex absolute intensity threshold Note Because the Find Compounds function is so efficient you can often obtain a good Mascot search even when peaks are not evident in the base peak chromatogram Protein ID Solutions LC MS App Guide 37 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS If you have low database search scores or poor sequence coverage Cause Solution 1 Sample preparation or data acquisition problem 2 Number of precursors for data dependent MS set too low 3 Threshold for triggering MS set too high 4 SmartFrag turned off resulting in inadequate fragmentation 5 Poor sensitivity 6 Default settings for LC MSD Trap data analysis software not appropriate for sample applies only if you use Mascot search 7 Mascot parameters not optimized applies only if you use Mascot search 8 Spectrum Mill Data Extractor parameters not set properly applies only if you use Spectrum Mill workbench 9 Spectrum Mill Search parameters not set properly applies only if you
41. better the chance for improved sequence coverage Once you have located correlated MS and MS MS spectra using Find Compounds check to make sure the mass peaks are labeled If mass peaks are not labeled they will not be exported to Mascot Check a few of your lower intensity MS MS spectra Zoom in to see if the masses are labeled You need to zoom in to check because the software prevents the labels from overlapping on your screen If you observe good spectra many fragments at intensities greater than the parent ion but the mass peaks are not labeled even when you zoom in lower the threshold for Mass List Find Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide 2 ee 9 90 i Reference Sample Analysis Strategy Direct injection mode 1D LC 98 Enrichment column mode 1D LC 99 Two dimensional liquid chromatography 2D LC 101 This chapter provides an overview of three LC configurations for nanoflow LC MS MS and describes how they fit various sample analysis strategies You learn when to use each configuration and how to plumb each one You can configure nanoflow LC MS MS for either one dimensional liquid chromatography 1D LC or two dimensional liquid chromatography 2D LC Use 2D LC for very complex protein digests or as a substitute for 2D gel electrophoresis Otherwise use 1D LC You can perform conventional 1D LC either with or without
42. cle size The longer column gives better separations when there are more peaks 20 uL for salt steps must have 40 uL loop installed 0 15 30 45 60 75 90 120 300 500 mM ammonium formate prepared in 3 acetonitrile 0 1 formic acid in water Choose the number of salt steps based on sample complexity Prepare salt solutions from 5 M ammonium formate stock Agilent p n G1946 85021 Prepare the solutions directly into the 2 mL glass autosampler vials supplied with the Nanospray Protein Identification Solution Table9 Preparation of salt solutions for 2D LC with salt steps Concentration mM Volume of 5 M stock pL Volume of water pL 15 3 997 30 6 994 45 9 991 60 12 988 75 15 985 90 18 982 120 24 976 300 60 940 500 100 900 1000 100 400 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 Enrichment Position 1 at 0 min position 2 at 5 min then return to position 1 at 61 min if column switch you use the short LC method or 146 min if you use the long LC method Nanoflow pump Use the nanoflow pump settings from either the short or long enrichment methods See page 26 or page 28 Choose the appropriate method based on the complexity of the sample Second pump 3 acetonitrile 0 1 formic acid in water Isocratic quaternary or binary pumps use flow gradient given in Table 10 Capillary pumps use no flow gradient just set flow to 15 uL min Table 1
43. cot search page 90 If not go to step 6 Regenerate the individual Mascot generic files using a higher threshold for compound finding and or more stringent requirements for input spectral quality Repeat step 1 to step 5 Protein ID Solutions LC MS App Guide 89 8 Data Analysis with Mascot Protein Database Search Brat sax File Tools Help r Select Files Files Selected for Processing MAS C Program Files MasCat Example Data BSA00001 D 5Mix_0OmM maft Sc z C Program Files MasCat E xample Data BS400002 D 5Mix_10mM maf qc CAT C Program Files MasCat Example Data BSA00003 D 5Mix_20mM maft H r C Program Files MasCat Example Data BSA00004 D 5Mix_30 maf Program Files C Program Files MasCat E xample Data BSA00005 D 5Mix_40mM maf S MasCat MASCOT MGF File C Program Files MasCat Example Data BSAQ0006 D 5Mix_50mM mgf Concatenator v1 0 C Program Files MasCat E xample Data BSA00007 D 5Mix_60 mof C 85400001 D Agilent Technologies C Program Files MasCat Example Data BSA00008 D 5Mix_70mM mgf BSA00002 D 2002 C Program Files MasCat E xample Data BSA00009 D 5Mix_80mM maft C 8sA00003 D C Program Files MasCat E xample Data BSA0001 0 D 5Mix_90mM maf C esac 4 D z C Program Files MasCat E xample Data BS400011 D 5Mix_100mM maf l Add Allin Seq gt gt C Program Files MasCat E xample Data BSAQ0012 D 5Mix_125mM mat BSA00005 D F C Program Files MasCat E xample Data BS40001 3 D SMix_150mM maf BSAO0006 D Files ending with C
44. d date or use the real time monitor to run searches as Mascot generic files are written to a particular directory e g by use of a Visual Basic script Schedule score dependent follow up Search for Mascot generic format files mgf only Protein ID Solutions LC MS App Guide Data Analysis with Mascot Protein Database Search 8 Troubleshooting Problem Cause Solution Too few proteins Default settings for Check to see if the threshold for Find Compounds is set too high identified LC MSD Trap data a Inthe data analysis navigation tree scroll to the bottom of the analysis software not Compound Mass Spectra node You should observe a reasonable appropriate for sample number of compounds for your sample b If this is not the case select Method gt Parameters c Click the Find tab d Click the AutoMS n subtab e Enter a lower number for the Compound detection Intensity threshold Check to see if the retention time window for Find Compounds is set appropriately a Select Method gt Parameters b Click the Find tab c Click the AutoMS n subtab d Set Retention time window min to base width of the chromatographic peaks Check to see if the threshold for Mass List Find is set too high a Select Method gt Parameters b Click the Mass List tab c Make sure Apex is selected d Click the Apex subtab e Enter a smaller number for the Absolute intensity threshold Incorrect settings for See To use Mascot protein database searc
45. d oxidized methionine For Spectrum Mill version A 03 02 or later search in Variable mode for pyroglutamic acid and oxidized methionine For Spectrum Mill versions prior to A 03 02 search in Homology Multi mq mode b If you suspect phosphorylation For Spectrum Mill version A 03 02 or later search in Variable mode for the combination of phosphorylated S phosphorylated T and phosphorylated Y For Spectrum Mill versions prior to A 03 02 search in Homology Multi sty mode c Search for any other modifications you suspect for your sample This will increase sequence coverage 8 Search the previously validated results in no enzyme mode to find non specific cleavages for proteins you have already identified Set Digest to No enzyme Repeat autovalidation and manual validation 9 When you think you are done list sequence not validated spectra in Protein Details mode and look for proteins with multiple peptides These may represent legitimate proteins at low levels Re examine the spectra to confirm 10 Optional Search again using a larger database entire database or larger subset This is most important when the original subspecies is not well represented in the database Autovalidate and manually validate 11 Check statistics in Tool Belt If there is a significant number of unmatched filtered spectra continue searching Protein ID Solutions LC MS App Guide 71 7 Data Analysis with Spectrum Mill MS Proteomics Workb
46. de separation via a salt gradient and use of two enrichment columns for alternate trapping and sample elution For more details refer to the Agilent application note Improved 2D Nano LC MS for Proteomics Applications a Comparison on Yeast Agilent publication number 5989 0212EN 1st Dimension 2nd Eg SCX Column L gt ee Enrichment aL Enrichment Column Enrichment ae Column Cycle Column2 Capillary Pump q D Es facies Waste Figure 11 Online 2D LC with semi continuous salt gradient Additional materials required In addition to the nanoflow LC MS MS system you need the following solvents e Water HPLC grade 18 megohm Agilent p n 8500 2236 or equivalent e Acetonitrile HPLC grade Agilent p n G2453 85050 or equivalent e Formic acid analytical grade Agilent p n G2453 85060 or equivalent e Ammonium formate 5 M Agilent p n G1946 85021 provided with the Nanospray Protein Identification Solution Protein ID Solutions LC MS App Guide 23 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Protocol for 1D direct injection LC method Column Maximum injection volume Nanoflow pump Stop time Post time Injector program 24 ZORBAX 300SB C18 75 um x 150 mm 3 5 um particle size or ZORBAX 300SB C18 75 um x 50 mm 3 5 um particle size The longer column gives better separations when there are more peaks 1 uL maximum volume with this method A 0 1 formic acid in water B
47. e 98 Choosing the LC method Before you can plumb the system per instructions in this chapter you need to decide which LC configuration is most appropriate for your samples e For very complex samples e g whole cell lysates without prior protein or peptide separation choose 2D LC Within this category select one of the following If you want the maximum number of proteins identified and are willing to devote the longest analysis time choose offline SCX See Optional Peptide Fractionation with Offline SCX on page 65 Plumb the nanoflow LC MS MS for 1D LC with enrichment column If you want a moderate number of proteins identified and are willing to devote a moderate analysis time choose online SCX with semi continuous salt gradient To plumb and run this method refer to the Agilent application note Improved 2D Nano LC MS for Proteomics Applications a Comparison on Yeast Agilent publication number 5989 0212EN You must have the optional 10 port valve and additional tubing and enrichment column as described in the publication For a sample of only moderate complexity such as a gel band with less than 20 30 proteins choose online SCX with salt steps Plumb the nanoflow LC MS MS for 2D LC 8 Protein ID Solutions LC MS App Guide Setup of Conventional Nanoflow LC MS MS System 2 e For less complex samples choose 1D LC If samples contain salts or require concentration i e you want to do large volume in
48. e Protein ID Solutions Data analysis The final step in the workflow is data analysis A major challenge in proteomics research is analysis of the vast amounts of data that are generated Often thousands of MS MS spectra must be converted to protein identifications With the Nanospray and HPLC Chip MS Protein Identification Solutions two different data analysis paths are possible One path uses the Agilent LC MSD Trap DataAnalysis software to prepare and export the MS MS spectra to Mascot generic format The exported mass list files are then searched using the Mascot protein database search engine The searches are conducted sample by sample over the internet or are automated via licensed Mascot Daemon software The results are then tabulated manually or via software developed in the laboratory Another path uses the Spectrum Mill MS Proteomics Workbench to extract the MS MS spectra from the raw data files conduct the protein database searches validate the results via a combination of automated and manual validation and summarize the results in biologist friendly format The Spectrum Mill workbench allows data from the various LC fractions to be automatically consolidated and permits ready sample comparison Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide 2 ee 2 7 e Setup of Conventional Nanoflow LC MS MS System System components 6
49. e fractions To use Mascot protein database search 1 Decide how you will do the search The options are e Conduct an online search at www matrixscience com e Conduct an automated search using the optional Mascot Daemon software on an in house server 2 Set parameters for either type of search e See Figure 21 and the explanations that follow 90 Protein ID Solutions LC MS App Guide Data Analysis with Mascot Protein Database Search For more details see the Mascot online help 3 Click Start Search to initiate the Mascot search 4 View search results either immediately via your web browser or later via email MASCOT MS MS lons Search Your name la Scientist tist a Search title Digest of bovine proteins Database SwissProt 7 Taxonomy Enzyme Trypsin x Fixed SER K modifications Carbamyl N term Carboxymethyl C Deamidation NQ Guanidination K Protein mass kDa Peptide tol 2 0 Da 7 Peptide charge fit 2 and 3 7 a Variable 45 old_ICATdO C modifications ap old_ICATd8 C Acetyl K Acetyl N term Amide C term a cat 7 MS MS tol o s Da x Monoisotopic Average Data file D Trap Data MIX 55 D mix55 r Browse Data format Mascot generic Instrument ESI TRAP had Overview D Precursor o m z Report top 20 z hits Database Taxonomy Start Search Parameters for Mascot MS MS lons Search Reset Form Figure 21
50. e should be between 1600 and 2000 volts New needles usually require less voltage but need slightly more as the needle ages and the tip becomes eroded or enlarged 2 Nanospray needle not positioned Adjust needle position as described in the Agilent G1982B Orthogonal correctly Nanospray lon Source User s Guide 3 Flow path blockages See If you have flow path blockages on page 35 of this chapter 4 Nanospray needle tip is damaged or Replace the needle reinstall the needle column holder assembly in the partly blocked source and adjust needle position You observe sputtering or split spray 5 Ferrule not making good seal with e Replace the ferrule reinstall the needle column holder assembly in the needle source and adjust needle position 6 Flow too great or needle tip enlarged Reduce the LC flow You observe steady bowed stream of Replace the needle as above liquid If you have poor chromatography Cause Solution 1 Gaps at LC connections When you connect a capillary to a fitting or the column push the capillary into the fitting firmly and smoothly to avoid gaps When connections are leaking set column flow to zero loosen the fitting reinsert the fused silica and retighten the fitting If you tighten the fitting without re seating the fused silica tube you may allow a gap to remain between the fused silica and the fitting This will result in peak dispersion 2 Note Chromatography in enrichment
51. ench If the Tool Belt statistics show that only a small percentage of your collected MS MS spectra were filtered use the LC MSD Trap DataAnalysis to check your data file to see if you collected a large number of MS MS spectra on background If so your MS MS data acquisition threshold may be set too low To set a higher threshold go to the MS n tab on the Trap control screen and set Threshold Abs and or Threshold Rel to a larger number 12 Use Spectrum Summary to check for sequence not validated spectra with sequence tags greater than 6 or 7 Review these and mark as Good Spectrum as appropriate 13 Subject the good spectra to de novo sequencing 14 When you have gained enough information from your data summarize the results 78 Protein ID Solutions LC MS App Guide Data Analysis with Spectrum Mill MS Proteomics Workbench 7 To manually validate results See guidelines in Table 20 Table 20 Guidelines for manual results validation Guideline Example The best spectra will generally be a r MSTag 1 N 1 1 sai found by autovalidation so do not ee ee ee DERE a ee EA expect to see spectra like the one to the right Loss of NH3 occurs from R K Q and N residues Loss of H20 occurs from S T E and D R H Kand N are charge bearing residues and increase the maximum charge state allowed for a peptide fragment Enhanced fragmentation may be observed at the following bonds His Xaa Xaa Gly Xaa
52. entrate the sample an analytical column a nanoelectrospray tip and the microfluidic channels that connect them The chip design significantly reduces the number of capillaries and fittings you need for nanoflow HPLC MS It is reusable and is easier to set up and use The Protein ID Chip inserts into the Agilent HPLC Chip MS interface which couples the LC system and the LC MSD Trap The interface includes the valve to switch flows between the columns This chapter describes how to set up this system and how to prepare it for an analysis ee Agilent Technologies 41 4 Setup of HPLC Chip MS lon Trap System System components Protein ID Chip 2 Ff Sa HPLC Chip MS interface LC MSD Trap SSE Y Figure 13 HPLC Chip MS lon Trap system 42 The HPLC Chip MS system Figure 13 consists of e Agilent 1100 Series nanoflow pump with micro vacuum degasser e Agilent 1100 Series micro well plate autosampler with optional thermostat e Second pump for sample loading Agilent 1100 Series capillary pump e Agilent Protein ID Chip e Agilent HPLC Chip MS interface which includes gt HPLC Chip loading and ejection mechanism Microvalve for flow switching LC connections to the nanoflow LC pump and to the micro well plate autosampler with loading pump Orthogonal nanospray ion source with optics for spray visualization e Agilent ChemStation B 01 03 and LC MSD Trap software 6 0 e Agilent 1100 Series LC MSD Trap XCT Ultra Pr
53. f you are unsure read Choosing the LC method below Read and follow all instructions in the Agilent Nanoflow LC System for Mass Spectrometry MS G2229 Quick Start Guide Skip this step if you did it when the system was installed Arrange your LC modules as described in the Agilent 1100 Series G4240A HAPLC Chip MS Cube User s Guide Read step 1 of Plumbing overview on page 11 Also read step 2 of that section if you plan to do online 2D LC because you will need to install an SCX column The enrichment and analytical columns are incorporated into the Protein ID Chip Plumb the system For the capillaries to use refer to the Agilent 1100 Series G4240A HPLC Chip MS Cube User s Guide Choosing the LC method Before you can plumb the system per instructions in this chapter you need to decide which LC configuration is most appropriate for your samples e For very complex samples e g whole cell lysates without prior protein or peptide separation choose 2D LC Within this category select one of the following If you want the maximum number of proteins identified and are willing to devote the longest analysis time choose offline SCX See Optional Peptide Fractionation with Offline SCX on page 65 For a sample of only moderate complexity such as a gel band with less than 20 30 proteins choose online SCX with salt steps You will need to plumb the SCX column e For less complex samples choose 1D LC For
54. h on page 90 Mascot search Chosen database did Try searching a different database not give good results Default settings for See the data acquisition troubleshooting tips under If you have low LC MSD Trap data database search scores or poor sequence coverage on page 38 in acquisition softwarenot Chapter 3 appropriate for sample Protein ID Solutions LC MS App Guide 95 8 Data Analysis with Mascot Protein Database Search Problem Cause Solution Noisy spectra exported to Mascot Problem with sample See the troubleshooting information in the Sample Preparation Guide preparation In Trap DataAnalysis set a larger value for Abundance cutoff for deconvolution The refers to percent of the most intense peak in the mass spectrum a Select Method gt Parameters Click the Charge Deconvolution tab Click the Peptides Small Molecules subtab Set Abundance cutoff to a higher value ao s Tips Threshold for Find Compounds Threshold for Mass List Find The intensity threshold for Find Compounds determines how many mass spectra are exported to Mascot The appropriate threshold depends upon sample concentration and data acquisition parameters To evaluate where to set the threshold generate a total ion chromatogram containing all the MS data TIC All MSn Set the threshold to a level that includes at least half the MS data points The lower the threshold the slower the data processing but the
55. he flow at 18 uL hr about 300 nL min 5 Calibrate the ion trap mass spectrometer as directed in the Agilent Chip Cube Interface Assembly for LC MSD Trap User s Guide Protein ID Solutions LC MS App Guide 47 4 Setup of HPLC Chip MS lon Trap System CAUTION Be sure to check the ion trap mass axis calibrations scan isolation and fragmentation Calibrate the detector gain In MSD Trap Control click the Calibration tab then the Detector button The detector ages faster and must be calibrated more frequently when it is new It is a good idea to check the detector gain more frequently if the system is in constant use 48 You should calibrate the MS if e This is the first time you are using it e You observe mass shifts or other problems indicating the need to calibrate e Your background seems unusually low which indicates aging of the detector Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide 5 Peptide Separation and Detection with HPLC Chip MS Safety 50 Procedure summary 50 Additional materials required 50 Protocol for 1D enrichment LC with Protein ID Chip short method for less complex samples 51 Protocol for 1D enrichment LC with Protein ID Chip long method for more complex samples 52 MS MS method for LC MSD Trap XCT Ultra 56 To shut down the HPLC Chip MS system 58 Troubleshooting 59 Tips 63 This chapter p
56. ides inthe Analyze blanks with high organic until the peaks are gone injection system 60 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with HPLC Chip MS 5 If you have poor sensitivity Cause Solution 1 Detector gain adjustment needs tobe See instructions for verifying detector setting in your LC MSD Trap redone documentation 2 Capillary voltage too high causing Reduce the capillary voltage corona which can destroy peptides 3 Sample degradation from sitting at Prepare fresh samples room temperature If you have the optional thermostat on the micro well plate sampler make sure it is turned on and set to 4 C 4 Peptides adsorbed on vial surface e Switch to a different vial material glass or plastic 5 Bad injection due to air bubble at Tap vial gently to dislodge air bubble bottom of vial 6 Unstable spray See If you have unstable spray on page 59 of this chapter 7 Find Compounds settings not For low level samples reduce these thresholds in the data analysis method optimized for low level samples giving the appearance of poor sensitivity applies only if you use Mascot search Note Because the Find Compounds function is so efficient you can often obtain a good Mascot search even when peaks are not evident in the base peak chromatogram parameters Find AutoMS n compound detection threshold Mass List Apex absolute intensity threshold Protein ID Solutions LC MS App
57. into the Synapsia Import Data screen only the bold portion of the Upload Path from the Spectrum Mill screen To upload to LIMS see the Server Administration online help Protein ID Solutions LC MS App Guide Data Analysis with Spectrum Mill MS Proteomics Workbench 7 Tips Copying datato To make it easy to compare data sets set up the appropriate directory server structure on the Spectrum Mill server Whenever you want to compare samples in a set you need to set up a subdirectory for each sample This sample directory may contain data files from multiple sample fractions or gel slices Here are some examples e If you perform a two dimensional LC MS MS analysis of a single sample transfer all the files to a single directory on the server If you run the same two dimensional LC MS MS analysis on a second sample or if you repeat the run on the first sample transfer all these files to a second directory e If you conduct a differential expression study transfer samples from one cell state into one directory and the second cell state into a second directory Extracting When you extract your data be sure to remember the following spectra You extract only one directory at a time For Merge scans with same precursor m z if necessary change the time range to be compatible with your chromatographic data This option sets the time range for merging scans from the same peak A good starting point is 15 seconds with a 0 5
58. jections choose enrichment column mode If samples are clean and sufficiently concentrated so that a 1 uL injection is adequate for sensitivity choose direct injection mode For more details on 1D and 2D LC configurations see Chapter 9 Reference Sample Analysis Strategy starting on page 97 For the direct injection mode the column compartment and associated six port valve are unused The analytical column is housed in the nanospray needle holder Protein ID Solutions LC MS App Guide 9 2 Setup of Conventional Nanoflow LC MS MS System Stacking diagrams version Recommended for enrichment column version Recommended for enrichment column mode direct injection mode and 2D LC mode direct injection mode and 2D LC 1 Solvent compartment Micro degasser Nanoflow pump Thermostatted column compartment Micro well plate sampler Cooler optional ol amp o of A N Second pump lon trap version III Recommended for enrichment column mode and 2D LC only 1 4 Ble O i 8 u 2 3 10 Protein ID Solutions LC MS App Guide Setup of Conventional Nanoflow LC MS MS System 2 Plumbing overview With traditional LC plumbing you make all the system connections before you apply flow With the nanoflow system you apply flow after the addition of each capillary This allows you to systematically remove small particles before they mig
59. l workbench 79 to 81 reversed phase column 2 20 to 32 51 52 54 65 S salt gradient 68 salt steps 21 30 54 to 55 101 102 salts as contaminants 99 sample carryover 37 60 scores low 38 62 SCX 8 22 44 65 to 72 102 Protein ID Solutions LC MS App Guide second pump preparing 17 47 semi continuous salt gradient 23 32 101 sensitivity best 98 poor 37 61 sequence coverage better 98 poor 38 62 solvent naming of channels 7 43 preparation 7 43 stability 7 43 Spectrum Mill MS Proteomics Workbench 73 to 83 Spectrum Mill server 83 Spectrum Mill workbench manual results validation 79 to 81 overview 73 procedure summary 74 to 75 protocol 76 to 78 summarizing data 82 tips 83 stacking diagrams 10 strong cation exchange column 2 8 22 44 65 to 72 102 subset database 76 to 77 summarizing results with Spectrum Mill workbench 82 T taxonomy 91 tips HPLC Chip MS 63 Mascot 96 nanoflow LC MS MS 39 offline SCX 72 Spectrum Mill workbench 83 trapping column mode 99 to 100 troubleshooting Mascot 95 nanoflow LC MS MS 35 to 38 59 to 62 offline SCX 71 U unstable flow 35 59 spray 36 59 V Visual Basic script overview of processing steps 86 to export to Mascot 87 to 89 W well plate sampler preparing 17 47 Protein ID Solutions LC MS App Guide Index 107 Index 108 Protein ID Solutions LC MS App Guide www agilent com In this Book
60. lutions LC MS App Guide 25 26 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Protocol for 1D enrichment LC short method for less complex samples Columns Enrichment column ZORBAX 300SB C18 0 3 x 5 mm 5 um particle size e Reversed phase column ZORBAX 300SB C18 75 um x 150 mm 3 5 um or ZORBAX 300SB C18 75 um x 50 mm 3 5 um particle size The longer column gives better separations when there are more peaks Maximum 8 uL or 40 uL depending on loop installed njection volume Enrichment Position 1 at 0 min position 2 at 5 min then return to position 1 at 61 min column switch Nanoflow pump A 0 1 formic acid in water B 90 acetonitrile 0 1 formic acid in water Flow rate 300 nL min Primary flow 200 to 500 uL min From the Instrument menu choose More pumps gt Auxiliary Table3 Nanoflow pump solvent gradient Time min B 0 3 5 3 8 15 50 45 55 90 60 90 61 3 Stop time 75 min Post time 5min Second pump 3 acetonitrile 0 1 formic acid in water Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 Isocratic quaternary or binary pumps use flow gradient given in Table 4 on page 27 Capillary pumps use no flow gradient just set flow to 15 uL min Table4 Flow gradient for second non capillary pump Time min Flow mL min initial 0 005 0 01 0 1 0 50 0 1 1 0 0 05 1 01 0 01 8 0 0 01 8 01 0 005
61. ly understood and met A WARNING notice denotes a hazard It calls attention to an operating procedure practice or the like that if not correctly per formed or adhered to could result in personal injury or death Do not proceed beyond a WARNING notice until the indicated condi tions are fully understood and met Protein ID Solutions LC MS App Guide In This Guide The LC MS Application Guide presents the information you need to conduct the LC MS steps for the Nanospray and HPLC Chip MS Protein Identification Solutions In this guide you will learn How to fractionate peptides by liquid chromatography How to perform MS MS analyses of peptides e How to analyze the resulting data with the Spectrum Mill MS Proteomics Workbench or the Mascot protein database search software 1 Overview of LC MS Steps with the Protein ID Solutions Get an overview of the entire process for peptide separation MS MS analysis and translation of spectra to protein identifications 2 Setup of Conventional Nanoflow LC MS MS System Learn how to choose the appropriate 1D or 2D LC method how to plumb the system how to install the nanospray needle and how to prepare the LC and MS just before an analysis 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Follow the protocols to perform online 1D and 2D nanoflow LC MS MS analyses 4 Setup of HPLC Chip MS lon Trap System Learn how to plumb the system how
62. m z window If the time range or m z windows are too wide you may merge scans for more than one peptide If they are too narrow searches will take longer and you will observe redundant hits Searching Before you search a subset database review the Spectrum Mill species spectra definitions and modify if necessary For the default species definitions see the online help for the MS MS Search page Validating results For efficient validation of MS MS search results e Use the Autovalidation page to validate the highest scoring results those that do not require manual review e Use the Protein Peptide Summary page for manual review and validation of medium scoring results Protein ID Solutions LC MS App Guide 83 7 Data Analysis with Spectrum Mill MS Proteomics Workbench 84 Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide 8 Data Analysis with Mascot Protein Database Search Procedure summary 86 To use LC MSD Trap Visual Basic script to export data to Mascot 87 To use the MASCAT concatenator for multiple sample fractions 89 To use Mascot protein database search 90 To use Mascot Daemon 93 Troubleshooting 95 Tips 96 In this chapter you learn how to perform data analysis using the LC MSD Trap DataAnalysis software and Mascot database search Mascot from Matrix Science Limited is a search engine that uses mass spectra to identify proteins fr
63. mize the gradient for fraction collection for the complexity of your samples For moderately complex samples use a steeper gradient as outlined in Table 18 For more complex samples use a shallower gradient as outlined in Table 19 For all gradients described here you collect a fraction every three minutes To identify more proteins first try extending the gradient then try collecting fractions more frequently Note that each additional fraction adds to the LC MS analysis time Be sure the thermostat on the fraction collector is turned on and set to 4 C Since you do not want to hold samples too long at 4 C do not collect more fractions than you can analyze in a couple of days The capillary you plumb between the diode array detector and the well plate sampler depends on the flow rate you use for fraction collection For the 5 uL min flow rate you use with this application it is best to use the green 50 um ID capillary p n G1364 87305 Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide ee 7 e Data Analysis with Spectrum Mill MS e Proteomics Workbench Procedure summary 74 Protocol for data processing with Spectrum Mill workbench 76 To manually validate results 79 To summarize data 82 Tips 83 This chapter provides a general protocol and tips for processing your data with the Spectrum Mill MS Proteomics Workbench If you do
64. mple 2 Separate peptides on the nanoflow column 3 Perform MS and MS MS analysis of separated peptides to obtain molecular weight and sequence information Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 1D LC enrichment column mode This 1D LC method uses an enrichment pre column to remove salts and other water soluble contaminants from samples and to permit large volume injections several uL or greater that would take a long time to load using nanoflow rates A schematic is shown in Figure 9 For more detailed flow diagrams see Enrichment column mode 1D LC on page 99 Loading Pump Figure 9 1D LC with enrichment column 1 Using a higher flow rate load sample onto an enrichment column where the peptides are trapped and the samples are desalted 2 Switch enrichment column into the solvent path of the nanoflow pump and separate sample on the nanoflow column 3 Perform MS and MS MS analysis of separated peptides to obtain molecular weight and sequence information 2D LC with salt steps The 2D LC method shown in Figure 10 uses both SCX and reversed phase separations You load the sample onto an SCX column and then elute it in fractions by injecting sequential salt steps Each injected salt solution elutes a fraction of the peptides from the SCX column for analysis by nanoflow LC MS MS The result is an individual data file for each step of
65. n ID Chip of HPLC Chip MS Protein Identification Solution Nanoflow pum oa 1100 Micro well plate sampler 1100 Column compartment Figure 30 2D LC step 3 wash enrichment column Protein ID Solutions LC MS App Guide 103 9 Reference Sample Analysis Strategy In the fourth step you switch the nanoflow pump in line and backflush the peptide fraction off the enrichment column You then analyze the fraction using nanoflow LC MS MS See Figure 31 Second pum Samia ald Contained within Protein ID Chip of HPLC Chip MS Protein Identification Solution Nanoflow pump 1100 Micro well plate sampler 1100 Column compartment Figure 31 2D LC step 4 analyze sample The online and offline SCX protocols provided with the Nanospray and HPLC Chip MS Protein Identification Solutions are optimized for tryptic peptides Because trypsin cleaves on the carboxy side of lysine and arginine tryptic fragments most commonly have two charges one for the terminal amino group and one for the basic lysine or arginine residue With other enzymes the number of charges will be different Highly charged peptides may not elute from the SCX column with salt steps alone These may require that the pH be raised simultaneously to reduce the number of charges on these peptides For highly charged peptides run a combination salt and pH gradient 104 Protein ID Solutions LC MS App Guide Index Numerics 1DLC choice of 9 44 configuration 98 t
66. n by LC When MS MS analyses are performed to identify peptides the peptides are analyzed sequentially i e one peptide at a time To maximize the number of identified proteins it is important that peptides be well separated Liquid chromatography LC is commonly used to fractionate peptides Popular separation techniques include strong cation exchange SCX and reversed phase RP LC 2 Protein ID Solutions LC MS App Guide Overview of LC MS Steps with the Protein ID Solutions 1 RPLC can be performed alone or in combination with prior SCX RPLC can be the sole separation technique for simpler mixtures of peptides for samples where it is not as critical to identify as many proteins or where time constraints apply For the Nanospray Protein Identification Solution RPLC is done with a conventional nanoflow LC system For the HPLC Chip MS Protein Identification Solution RPLC is accomplished with a specialized HPLC Chip the Agilent Protein ID Chip The HPLC Chip is a microfluidic device that integrates sample preparation and analysis on a single chip The dedicated Protein ID Chip includes a sample enrichment column an analytical column a nanospray tip and all connections between them It inserts into the HPLC Chip MS interface which includes the nanospray source connections to the LC pumps and autosampler and the microvalve for column switching When both SCX and RP separations are performed they can be combined in a number of
67. n the Sample Preparation Guide The fundamental difference between the Nanospray Protein Identification Solution and the HPLC Chip MS Protein Identification Solution is that the former uses a conventional nanoflow HPLC system while the latter uses a microfluidic HPLC Chip This guide describes how peptides are fractionated by liquid chromatography LC in one or more dimensions An initial separation by strong cation exchange SCX chromatography may be done offline Chapter 6 online Chapter 3 and Chapter 5 or not at all For the final reversed phase separation the LC or HPLC Chip is interfaced to a nanospray ion trap mass spectrometer MS system for MS MS analysis of the eluted peptides The MS MS spectra are then searched against protein databases using either Mascot or the Spectrum Mill MS Proteomics Workbench Before you begin be sure you understand all safety considerations and have read all applicable material data safety sheets phe Agilent Technologies i 1 Overview of LC MS Steps with the Protein ID Solutions See Sample Preparation Guide From sample prep Choose peptide separation Offline SCX with fraction collection ae Spectrum Mill MS ae __ Data analysis Proteomics Workbench or Chapters 7 and 8 LC MSD Trap Mascot Figure 1 Workflow for LC MS steps of the Nanospray and HPLC Chip MS Protein Identification Solutions Peptide fractionatio
68. n the sep arate agreement shall control Technology Licenses The hardware and or software described in this document are furnished under a license and may be used or copied only in accor dance with the terms of such license Restricted Rights Legend If software is for use in the performance of a U S Government prime contract or subcon tract Software is delivered and licensed as Commercial computer software as defined in DFAR 252 227 7014 June 1995 oras a commercial item as defined in FAR 2 101 a or as Restricted computer soft ware as defined in FAR 52 227 19 June 1987 or any equivalent agency regulation or contract clause Use duplication or disclo sure of Software is subject to Agilent Tech nologies standard commercial license terms and non DOD Departments and Agencies of the U S Government will receive no greater than Restricted Rights as defined in FAR 52 227 19 c 1 2 June 1987 U S Government users will receive no greater than Limited Rights as defined in FAR 52 227 14 June 1987 or DFAR 252 227 7015 b 2 November 1995 as applicable in any technical data Safety Notices CAUTION A CAUTION notice denotes a haz ard It calls attention to an operat ing procedure practice or the like that if not correctly performed or adhered to could result in damage to the product or loss of important data Do not proceed beyond a CAUTION notice until the indicated conditions are ful
69. ncentration and how important it is to identify as many proteins as possible for your study In general the 2D methods provide more identifications but at the cost of additional analysis time For guidelines on protocol selection see Choosing the LC method on page 8 Before you run any of the protocols prepare the system as described in To prepare the LC and MS just before an analysis on page 15 If you plan to use the HPLC Chip MS Ion Trap system skip to Chapter 5 ee Agilent Technologies 19 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Safety Always take proper precautions for handling and disposing of solvents and other chemicals Consult the material data safety sheets supplied by the vendors For additional safety precautions see the manuals you received with the individual system components Procedure summary 20 The following summarizes the various modes of nanoflow LC MS MS analysis If you have not yet chosen your operating mode see Choosing the LC method on page 8 1D LC direct injection mode A schematic 1D LC method with direct injection is shown in Figure 8 and a more detailed flow diagram is given in Direct injection mode 1D LC on page 98 For amenable samples this mode gives the best chromatography the best sequence coverage and the best sensitivity cis a Figure 8 ID LC with direct injection 1 Inject no more than 1 uL sa
70. nch Try searching another database 38 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 Tips See the tips in the Agilent Nanoflow LC System for Mass Spectrometry MS G2229 Quick Start Guide and the Agilent Nanoflow Proteomics Solution Quick Reference Guide Protein ID Solutions LC MS App Guide 39 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS 40 Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide ee 4 7 e Setup of HPLC Chip MS lon Trap 7 System System components 42 Safety 43 Additional materials required 43 To prepare solvents 43 To plumb the system 44 To configure the Chip Cube 45 To insert the HPLC Chip and start the spray 46 To prepare the LC and MS just before an analysis 47 The HPLC Chip MS Ion Trap system used for the HPLC Chip MS Protein Identification Solution consists of Agilent nanoflow LC modules the HPLC Chip MS interface the Protein ID Chip and the LC MSD Trap XCT Ultra The LC MSD Trap software is used for complete system control and automation The Agilent HPLC Chip is a polyimide based chip with microfluidic channels The Protein ID Chip you use for the HPLC Chip MS Protein Identification Solution is fabricated in a one dimensional LC configuration It incorporates an enrichment column to remove salts and conc
71. nd fragmentation e Calibrate the detector gain In MSD Trap Control click the Calibration tab then the Detector button Protein ID Solutions LC MS App Guide 17 18 2 Setup of Conventional Nanoflow LC MS MS System CAUTION The detector ages faster and must be calibrated more frequently when it is new You should calibrate the MS if e This is the first time you are using it e You observe mass shifts or other problems indicating the need to calibrate e Your background seems unusually low which indicates aging of the detector Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Safety 20 Procedure summary 20 Additional materials required 23 Protocol for 1D direct injection LC method 24 Protocol for 1D enrichment LC short method for less complex samples 26 Protocol for 1D enrichment LC long method for more complex samples 28 Protocol for online 2D LC method with salt steps 30 Protocol for 2D LC method with semi continuous salt gradient 32 MS MS method for LC MSD Trap XCT Plus or XCT Ultra 33 To shut down the nanoflow LC MS MS system 34 Troubleshooting 35 Tips 39 This chapter presents protocols for conventional 1D and 2D nanoflow LC MS analyses Choice of protocol depends on sample complexity sample cleanliness presence or absence of salts peptide co
72. ng HPLC Chip 46 modules 42 overview 3 plumbing 44 procedure summary 50 shutdown 58 starting spray 46 tips 63 injections large volume 99 installing capillaries 11 column 11 nanospray needle 12 to 14 iontrap 3 17 33 to 34 47 56 to 57 L large volume injections 99 LC columns See column modules for fraction collection 66 modules for HPLC Chip MS 42 modules for nanoflow LC MS MS 6 stacking modules 10 M MASCAT concatenator 89 Mascot procedure summary 86 protocol 87 to 94 tips 96 troubleshooting 95 Mascot Daemon 93 to 94 105 Index Mascot database search data analysis steps 86 overview 85 procedure 90 to 94 setting parameters 91 to 92 Mass List Find 86 96 mass spectrometry 3 33 to 34 56 to 57 missed cleavages 92 modifications fixed and variable 92 S calibration 17 47 S MS 3 S MS lons Search 91 S MS method 33 to 34 56 to 57 nanoflow LC MS MS system 1D vs 2D 19 49 capillary installation 11 column installation 11 modules 6 plumbing 8 procedure summary 20 to 23 protocols 24 to 32 sample analysis strategy 97 shutdown 34 stacking diagrams 10 tips 39 nanoflow pump preparing 15 47 unstable flow 35 59 nanospray needle blockage 12 installation 12 to 14 part number 12 positioning 13 rotating 14 starting spray 13 46 unstable spray 36 59 106 0 offline SCX advantages 8 44 overview 3 65 procedure summary 67 to 69 prot
73. not have the Spectrum Mill workbench proceed to Chapter 8 The Spectrum Mill workbench is a comprehensive software package that starts with raw mass spectral data files and provides tools to rapidly convert these to tables of protein identities This software includes modules for extraction of high quality spectra from raw data files rapid protein database search of MS or MS MS spectra data review and validation and results summary for single samples or groups of samples that span complex studies It also includes capabilities for identification of post translational modifications for quantitative analysis including but not limited to ICAT quantitation and for de novo sequencing The software accepts data from multiple vendors instrument types in multiple file formats Both public and proprietary databases are supported The Spectrum Mill workbench provides a means to segregate search results that contain a valid interpretation of an MS MS spectrum from those that do not Results that are validated can be summarized in a results table Results that are not validated can then be subjected to subsequent rounds of searches against other databases or in homology mode for example This iterative approach allows for efficient processing that can be customized to the needs of the study Apg Agilent Technologies 73 7 Data Analysis with Spectrum Mill MS Proteomics Workbench Procedure summary Spectrum Mill Data ma z gt Extractor
74. o 100 procedure summaries 20 to 21 protocols 24 to 29 51 to 53 1D vs 2D LC 8 19 44 49 97 2D LC choice of 8 44 configuration 101 to 104 procedure summaries 21 to 23 67 to 69 protocols 30 to 32 54 to 55 70 to 71 A application note 8 23 69 101 C C18 particles 12 capillaries installing 11 part numbers 8 capillary voltage 14 carryover problems 37 60 cation exchange column 2 8 22 44 65 to 72 102 charge deconvolution 86 ChemStation 6 42 66 chip See HPLC Chip MS system chromatography best 98 poor 36 60 column enrichment 9 21 22 26 to 29 51 to 53 99 to 100 104 installing 11 preparing 17 reversed phase 2 20 to 32 51 52 54 65 SCX 2 8 22 44 65 to 72 102 configuration 1DLC 98 to 100 2D LC 101 to 104 D data analysis Mascot 86 overview 4 Spectrum Mill workbench 76 to 78 database search 75 86 90 to 94 database search scores low 38 62 databases 91 de novo 78 direct injection mode 20 24 to 25 98 droplet noise 14 E enrichment column 9 21 22 26 to 29 51 to 53 99 to 100 104 enzyme 75 92 104 evaluating results Spectrum Mill workbench 79 to 81 export to Mascot 86 F Find Compounds 86 96 flow stable 16 unstable 35 59 fraction collection 3 65 66 homology search 77 HPLC Chip preparing 47 Protein ID Solutions LC MS App Guide Index HPLC Chip MS system 1D protocol 51 to 53 2D protocol 54 to 55 inserti
75. ocol 70 to 71 tips 72 troubleshooting 71 offline vs online SCX 65 101 P part numbers 2L solvent bottles 7 43 C18 particles 12 capillary for fraction collection 72 Eppendorf well plates 25 27 29 52 53 LC solvents 7 23 43 50 69 micro vials 25 27 29 52 53 nanospray needle 12 peptide charge 79 81 92 104 fractionation 2 8 21 to 23 44 65 recovery 98 plumbing capillary installation 11 column installation 11 HPLC Chip MS 44 nanoflow LC MS MS 8 stacking diagrams 10 stacking LC modules 10 poor sensitivity 37 61 poor sequence coverage 38 62 preparing HPLC Chip 47 LC columns 17 MS 17 47 nanoflow pump 15 47 second pump 17 47 solvents 7 43 well plate sampler 17 47 procedure summary HPLC Chip MS 50 Mascot 86 nanoflow LC MS MS system 20 to 23 Spectrum Mill workbench 74 to 75 processing method 87 protein database search 75 86 90 to 94 Protein ID Solutions overview 1 workflow 2 protocol 1D direct injection LC method 24 to 25 1D enrichment for HPLC Chip MS 51 to 53 1D enrichment long LC method 28 to 29 1D enrichment short LC method 26 to 27 2D for HPLC Chip MS 54 to 55 2D LC offline 70 to 71 2D LC with salt steps 30 to 32 54 to 55 2D LC with semi continuous salt gradient 32 Mascot 87 to 94 MS MS analysis 33 to 34 56 to 57 nanoflow LC MS MS system 24 to 32 offline SCX 70 to 71 Spectrum Mill workbench 76 to 78 results validation Spectrum Mil
76. offline SCX 70 Troubleshooting 71 Tips 72 vi Protein ID Solutions LC MS App Guide Contents 7 Data Analysis with Spectrum Mill MS Proteomics Workbench 73 Procedure summary 74 Protocol for data processing with Spectrum Mill workbench 76 To manually validate results 79 To summarize data 82 Tips 83 8 Data Analysis with Mascot Protein Database Search 85 Procedure summary 86 To use LC MSD Trap Visual Basic script to export data to Mascot 87 To use the MASCAT concatenator for multiple sample fractions 89 To use Mascot protein database search 90 To use Mascot Daemon 93 Troubleshooting 95 Tips 96 9 Reference Sample Analysis Strategy 97 Direct injection mode 1D LC 98 Enrichment column mode 1D LC 99 Two dimensional liquid chromatography 2D LC 101 Index 105 Protein ID Solutions LC MS App Guide vii Contents viii Protein ID Solutions LC MS App Guide Agilent Nanospray and HPLC Chip MS Protein Identification Solutions LC MS Application Guide ee ece 1 90 R Overview of LC MS Steps with the 6 e Protein ID Solutions Peptide fractionation byLC 2 Mass spectrometry 3 Data analysis 4 Figure 1 gives an overview of the LC fractionation and MS analysis portion of the Agilent Nanospray and HPLC Chip MS Protein Identification Solutions The applicable chapters from this LC MS Application Guide are indicated adjacent to the workflow You do the steps in this manual after you complete the sample preparation steps covered i
77. oftware on an in house server Protein ID Solutions LC MS App Guide Data Analysis with Mascot Protein Database Search 8 To use LC MSD Trap Visual Basic script to export data to Mascot To activate the Visual Basic script 1 In MSD Trap Control open the LC MSD Trap method you created to do the 1D or 2D LC MS MS analysis of peptides 2 Select Method gt Add DataAnalysis Part 3 Select the DataAnalysis method Example DA Methods with Scripts Auto_MIS_MgfExport ms 4 Select Method gt Save entire Method As to save the LC MS MS method with the DataAnalysis part attached To change processing method parameters The method Auto_MIS_MgfExport ms includes e The Visual Basic script e Starting parameters for Find Compounds AutoMS n Mass List Find Deconvolution and Mascot Export Options The script does not change or optimize the starting method parameters If you change method parameters i e based on recommendations from Troubleshooting on page 95 test and save your changes as follows 1 Select Method gt Run to execute the script again on your test data file 2 Select Method gt Save As to save a new copy of the processing method To change the Visual Basic script The Visual Basic script is shown in Figure 19 To change the script 1 Select Method gt Script 2 Make the desired change 3 Save a new copy of the processing method by selecting Method gt Save As Protein ID Solutions LC MS App Guide 87 Data
78. om sequence databases Mascot searches can be performed free of charge at www matrixscience com Optional Mascot Daemon software automates the searches but can only be used with an in house Mascot Server license If you have the Spectrum Mill MS Proteomics Workbench disregard this chapter and read Chapter 7 instead ee Agilent Technologies 85 Data Analysis with Mascot Protein Database Search Procedure summary Locate spectra with Find Compounds J Label spectra with Mass List Find Automate via Trap J DataAnalysis method Deconvolute spectra I Export spectra to Mascot format Automate via Mascot Perform Mascot Daemon database search Figure 18 Data analysis with the LC MSD Trap software and Mascot search When you use Mascot data analysis for protein digest samples consists of five steps 1 2 3 4 5 Use Find Compounds to locate and hierarchically organize related MS and MS MS spectra Use Mass List Find to label spectra with masses Perform optional charge deconvolution Export spectra to Mascot format Perform Mascot database search The first four steps are automated via a Visual Basic script that you add to your LC MSD Trap methods Once you have generated a Mascot generic format mgf file from your mass spectral data you conduct an online search on a single file at www matrixscience com or an automated search using the optional Mascot Daemon s
79. or the HPLC Chip Note that the Protein ID Chip contains both the enrichment column and the analytical column C18 nanocolumn The Protein ID Chip does not contain an SCX column Additional materials required 50 In addition to the HPLC Chip MS system you need the following solvents e Water HPLC grade 18 megohm Agilent p n 8500 2236 or equivalent e Acetonitrile HPLC grade Agilent p n G2453 85050 or equivalent e Formic acid analytical grade Agilent p n G2453 85060 or equivalent e Ammonium formate 5 M Agilent p n G1946 85021 provided with the HPLC Chip MS Protein Identification Solution Protein ID Solutions LC MS App Guide Peptide Separation and Detection with HPLC Chip MS 5 Protocol for 1D enrichment LC with Protein ID Chip short method for less complex samples Configuration See To configure the Chip Cube on page 45 HPLC Chip Use the Protein ID Chip p n G4240 62001 The chip integrates the following columns e Enrichment column ZORBAX 300SB C18 40 nL 5 um particle size e Reversed phase column ZORBAX 300SB C18 75 um x 43 mm 5 um particle size Maximum 8 uL or 40 uL depending on loop installed injection volume Chip Cube Inner valve Enrichment e Valve timetable Enrichment at 7 min Nanoflow pump A 0 1 formic acid in water B 90 acetonitrile 0 1 formic acid in water Flow rate 300 nL min Primary flow 500 to 800 uL min From the Instrument menu choose More pumps gt Au
80. or the conventional nanoflow LC MS MS system in enrichment column mode are shown in Figure 25 and Figure 26 Flow diagrams for the HPLC Chip MS system are similar but some components are contained within the Protein ID Chip For details on these microfluidic components see the flow diagrams in the Agilent 1100 Series G4240A HAPLC Chip MS Cube User s Guide You use the enrichment column mode to remove salts and other water soluble contaminants from your sample These are removed before the sample is backflushed from the enrichment column onto the analytical column By removing these contaminants the enrichment column helps to keep the system free of materials which can block narrow flow pathways and contribute to chemical background You also use the enrichment column mode for large volume injections several uL or greater that would take a long time to load using nanoflow rates The enrichment column has a larger internal diameter id than the analytical column and permits sample loading with a standard or capillary flow pump rather than a nanoflow pump Second pump 1100 Micro well plate sampler Figure 25 Contained within Protein ID Chip of HPLC Chip MS Protein Identification Solution Nanoflow pump ks H 5 x eee MS detection 1100 Column compartment Enrichment column mode step 1 load sample on enrichment column Protein ID Solutions LC MS App Guide 99 9 Reference Sample Analysis Strategy Second a Cont
81. otein ID Solutions LC MS App Guide Setup of HPLC Chip MS lon Trap System Safety WARNING Always take proper precautions for handling and disposing of solvents and other chemicals Consult the material data safety sheets supplied by the vendors 4 For additional safety precautions see the manuals you received with the individual system components Additional materials required In addition to the HPLC Chip MS Ion Trap system you need the following solvents e Water HPLC grade 18 megohm Agilent p n 8500 2236 or equivalent e Acetonitrile HPLC grade Agilent p n G2453 85050 or equivalent e Formic acid analytical grade Agilent p n G2453 85060 or equivalent To prepare solvents 1 To avoid bacterial growth use fresh clean solvent bottles If you plan to analyze a lot of samples use a 2 L bottle Agilent p n 9301 6342 for the aqueous solvent The solvent channels used for the nanoflow pump are Al and B1 2 Prepare 0 1 formic acid in water for solvent Al 3 Prepare 90 acetonitrile 0 1 formic acid in water for solvent B1 CAUTION Replace the solvent for the B1 channel at least weekly The A1 solvent is stable longer Remember to check your waste bottle frequently The bottle fills more quickly because you split your flow Protein ID Solutions LC MS App Guide 43 4 Setup of HPLC Chip MS lon Trap System To plumb the system 1 Decide which LC configuration is most appropriate for your samples I
82. r more information on modifying this script or writing new ones see the following help e DataAnalysis Online Help Help gt Help Topics gt Using processing methods and scripting e Visual Basic Script Language Reference Help gt VBScript Language Reference Protein ID Solutions LC MS App Guide Data Analysis with Mascot Protein Database Search 8 To deactivate the script If you would prefer not to run this script automatically as part of your sequence do one of the following To deactivate in ChemStation 1 2 3 Go to the Method and Run Control view of the ChemStation software Select Method gt Do MS Post Run Processing to remove the check mark Select Method again to verify that the check mark is removed from Do MS Post Run Processing To deactivate in LC MSD Trap software 1 2 3 Go to the MSD Trap Control screen Select Method Clear the Run Processing Script check box To use the MASCAT concatenator for multiple sample fractions MASCAT concatenates multiple mgf files for Mascot data base search enabling consolidated search of spectra from two dimensional LC MS MS analyses To use MASCAT 1 2 3 4 5 Click the MASCAT icon Select data files you wish to combine See Figure 20 as an example Enter an output file path and name Click Concatenate Click Calculate Number of Queries and decide if the number fits within your time and computer memory constraints If so proceed to do the Mas
83. rate through the system and cause blockages 1 Connect capillaries Start installing capillaries at the pump Follow this procedure a Before connecting each capillary tube set the LC flow to zero b Use a wash bottle of isopropyl alcohol or acetone to lightly rinse the outside of the capillary and fitting This will remove any particles before you insert the capillary into the fitting or valve c Install the capillary Tighten the fitting at the flow sensor if this is the first capillary and set the LC to pump 50 50 A1 B1 at 0 5 uL min d Monitor flow and pressure at the LC control panel Make sure these stabilize before you make the next connection If they do not stabilize there are likely particles at the front end of the capillary Backflush the capillary to remove them e After you observe liquid at the outlet of the capillary and the flow and pressure have stabilized proceed to the next step f Repeat step a through step e until all the capillaries have been installed 2 Connect columns Depending on your configuration you will need to connect one to three columns Connect columns in the same manner as the capillaries Allow each column to purge to waste before you connect the outlet Note that you may need to activate the column switching valve to establish flow to the various columns If you have not yet calibrated the MS do not connect the outlet of the analytical column to the nanospray needle First follow the instruc
84. ray away from entrance to MS To prepare the LC and MS just before an analysis After you have plumbed your nanoflow LC MS system you prepare it for stable analyses You purge the LC calibrate the MS if not already done and connect the two Repeat the LC purge any time the system has been shut down for more than a day To prepare the nanoflow pump 1 Activate the purge mode by selecting Instrument gt More Pump gt Purge Purge the nanoflow pump A1 and B1 solvent channels at 2 5 mL min for two minutes each If the system has been shut down for more than a few days extend the purge time to five to ten minutes 2 Switch to 97 3 A1 B1 the starting composition for the methods and purge for one additional minute In the purge mode the flow goes to waste rather than through the analytical system You will not damage your system by using the purge mode at 2 5 mL min Protein ID Solutions LC MS App Guide 15 2 16 Setup of Conventional Nanoflow LC MS MS System 3 Switch to the starting composition for your analysis Pump solvent through the analytical flow path at 0 3 uL min 4 Continue pumping at 0 3 uL min and monitor both the flow and pressure For stable nanospray conditions it is very important that the flow be stable Ripple should be less than 3 The flow and pressure plots should look similar to those in Figure 6 and Figure 7 5 While you wait for the system to stabilize prepare the rest of the LC and the MS
85. resents protocols for 1D enrichment mode and 2D nanoflow HPLC Chip MS analyses Choice of the 1D or 2D protocol depends on sample complexity peptide concentration and how important it is to identify as many proteins as possible for your study In general the 2D methods provide more identifications but at the cost of additional analysis time For guidelines on protocol selection see Choosing the LC method on page 44 Before you run any of the protocols prepare the system as described in To prepare the LC and MS just before an analysis on page 47 Ag Agilent Technologies 49 5 Peptide Separation and Detection with HPLC Chip MS Safety Always take proper precautions for handling and disposing of solvents and other chemicals Consult the material data safety sheets supplied by the vendors For additional safety precautions see the manuals you received with the individual system components Procedure summary For a summary of the 1D LC procedure for the HPLC Chip MS see 1D LC enrichment column mode on page 21 For more detailed flow diagrams see Enrichment column mode 1D LC on page 99 For a summary of the 2D LC procedure for the HPLC Chip MS see 2D LC with salt steps on page 21 For more detailed flow diagrams see Two dimensional liquid chromatography 2D LC on page 101 While some of the figures in the referenced sections depict a conventional nanoflow system the same principles apply f
86. rocedure summary 20 Additional materials required 23 Protocol for 1D direct injection LC method 24 Protocol for 1D enrichment LC short method for less complex samples 26 Protocol for 1D enrichment LC long method for more complex samples 28 Protocol for online 2D LC method with salt steps 30 Protocol for 2D LC method with semi continuous salt gradient 32 MS MS method for LC MSD Trap XCT Plus or XCT Ultra 33 To shut down the nanoflow LC MS MS system 34 Troubleshooting 35 Tips 39 Protein ID Solutions LC MS App Guide v Contents 4 Setup of HPLC Chip MS lon Trap System 41 System components 42 Safety 43 Additional materials required 43 To prepare solvents 43 To plumb the system 44 To configure the Chip Cube 45 To insert the HPLC Chip and start the spray 46 To prepare the LC and MS just before an analysis 47 5 Peptide Separation and Detection with HPLC Chip MS 49 Safety 50 Procedure summary 50 Additional materials required 50 Protocol for 1D enrichment LC with Protein ID Chip short method for less complex samples 51 Protocol for 1D enrichment LC with Protein ID Chip long method for more complex samples 52 Protocol for online 2D LC method with Protein ID Chip 54 MS MS method for LC MSD Trap XCT Ultra 56 To shut down the HPLC Chip MS system 58 Troubleshooting 59 Tips 63 6 Optional Peptide Fractionation with Offline SCX 65 System components 66 Safety 67 Procedure summary 67 Additional materials required 69 Protocol for
87. rocedure summary 6 7 Additional materials required 69 Protocol for offline SCX 70 Troubleshooting 71 Tips 72 This chapter describes an optional peptide fractionation by offline strong cation exchange SCX chromatography This technique can be used to fractionate complex samples such as whole cell lysates and biological fluids The collected fractions are later analyzed by LC MS MS When samples are less complex or there is a need for greater automation SCX can be accomplished online as the first step in online two dimensional 2D LC MS MS With the online technique described in Chapter 3 peptides are eluted in fractions from the SCX column by injection of salt solutions of increasing ionic strength and then are analyzed by reversed phase LC MS MS With the offline technique described in this chapter peptides are eluted continuously from the SCX column by use of a salt gradient are collected in fractions and then are analyzed by reversed phase LC MS MS The advantage of doing the SCX offline is increased resolution and fewer peptides per fraction which enables identification of more of the low abundance proteins The disadvantage is that there are more fractions and this requires more LC MS MS and data reduction time Apg Agilent Technologies 65 6 Optional Peptide Fractionation with Offline SCX System components Fraction collection system Solvent tray Degasser Tec u6 port valve DAD I I r
88. roteomics Workbench To summarize data Validate results Compare results across samples Evaluate fractionation Do light heavy calculations Print results Import data into Excel Synapsia Informatics Workbench or LIMS 82 When you summarize data with the Protein Peptide Summary page remember that you can For Mode select Peptide Protein Details Protein Single Peptide ID or Protein Sample Centric Rows Details For Data Directory select more than one sample folder For Mode select Protein Peptide Distribution Columns Under Review Fields mark the L H check box and select the site of the light heavy modification ao oo A Ww N Enable Print background colors and images in Internet Explorer Select Tools gt Internet Options Click the Advanced tab and mark the check box for Print background colors and images Select File gt Page setup to set the page to landscape mode Click in the frame you wish to print Select File gt Print Preview At the top of the Print Preview window select Only the selected frame Click the Print button Mark the Excel Export check box 2 Click the Summarize button 3 Check that you see a display with two buttons one to upload to LIMS and another to display the file Do one of the following To import the data into Excel import as semicolon delimited data To import the data into Synapsia see the Synapsia online help Be sure to copy and paste
89. spect other modifications search for them d Search in no enzyme mode against validated protein hits e Search in identity mode against a larger database 8 Continue to perform iterative cycles of database search and validation to identify as many proteins as desired 9 Use Spectrum Summary to examine the remaining unmatched spectra to determine if there are high quality spectra 10 Perform de novo sequencing on high quality spectra Protein ID Solutions LC MS App Guide 75 7 Data Analysis with Spectrum Mill MS Proteomics Workbench Protocol for data processing with Spectrum Mill workbench The following describes in detail the iterative search strategy to process LC MS MS data with the Spectrum Mill workbench The strategy assumes that the goal is to identify as many proteins as possible If your study does not require you to identify so many proteins you may omit step 5 through step 13 For details on use of the Spectrum Mill workbench see the manuals and online help you received with the product 1 Copy or move the raw LC MS MS data files to the Spectrum Mill server Be sure to set up a directory structure on the server that makes it easy to summarize and compare your results See Copying data to server on page 83 2 Extract the data Set the correct cysteine modification For Merge scans with same precursor m z if necessary change the time range to be compatible with your chromatographic data A good starting point is
90. ssolve your peptides For complex samples inject 50 TFE in 0 1 formic acid in water to remove hydrophobic peptides 2 In the Sequence Parameters set the post sequence command macro to macro SHUTDOWN MAC go Note that the Standby command stops the LC pumps but does not put the ion trap into standby mode The Shutdown macro stops the LC pumps and puts the ion trap into standby mode 3 Before you run your next sequence open the data files for the blanks and check for peptide peaks If there were substantial peaks in the last injection inject more blanks If you do not plan to use the system for several days remove the nanospray needle column holder assembly and store in protective plastic sleeve This will extend the life of the needle 34 Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 Troubleshooting If you have flow path blockages Cause Solution 1 Fused silica particles in system 2 Particles from solvent or sample 3 Nanospray needle blocked due to heat without flow Solvent rinse capillaries and fittings prior to installation Avoid overtightening fittings Avoid excessive bending or coiling of the capillaries If you coil to a radius of less than 40 mm you will damage the capillaries Avoid kinking bending or crushing capillaries with LC doors or cover panels Backflush capillaries if they are already blocked
91. t pump 500 nL min of 50 B for 20 min c lf bubbles still persist disconnect first capillary at pump end of system and purge at higher flow rate Reconnect first capillary and disconnect next one Purge again Continue disconnecting capillaries sequentially and purging until bubbles are gone If you have unstable spray Cause Solution 1 Unstable flow See If you have unstable flow above 2 Capillary voltage not set correctly Adjust the capillary voltage You observe sputtering or split With a flow of 300 nL min the capillary voltage should be 1800 volts spray If you observe a split stream this typically means Vcap is too high 3 Flow path blockages See If you have flow path blockages above Protein ID Solutions LC MS App Guide 59 5 Peptide Separation and Detection with HPLC Chip MS If you have poor chromatography Cause Solution 1 New chip not conditioned Before you use a new Protein ID Chip for analyses condition it as described in To prepare the Protein ID Chip on page 47 2 Sample overloaded the chip e Inject 1 10 the sample volume or dilute the sample 1 10 and inject again Observe whether the chromatography improves If you have sample carryover Cause Solution 1 No needle wash Set up needle wash for well plate sampler 2 Inappropriate needle wash solvent e Switch to a solvent combination in which your sample is completely soluble 3 Residual hydrophobic pept
92. tem The nanoflow LC MS MS system Figure 2 consists of Agilent 1100 Series nanoflow pump with micro vacuum degasser Agilent 1100 Series micro well plate autosampler with optional thermostat Agilent 1100 Series thermostatted column compartment with 2 position 6 port micro valve or the optional Agilent 10 port micro valve and holder required only for online SCX with semi continuous salt gradient Agilent 1100 Series LC MSD Trap XCT Plus or XCT Ultra Agilent orthogonal nanospray ion source Agilent ChemStation B 01 03 and LC MSD Trap software 6 0 Optional second pump Agilent 1100 Series quaternary binary isocratic or capillary pump Protein ID Solutions LC MS App Guide Setup of Conventional Nanoflow LC MS MS System Safety WARNING Always take proper precautions for handling and disposing of solvents and other chemicals Consult the material data safety sheets supplied by the vendors 2 For additional safety precautions see the manuals you received with the individual system components Additional materials required In addition to the nanoflow LC MS MS system you need the following solvents e Water HPLC grade 18 megohm Agilent p n 8500 2236 or equivalent e Acetonitrile HPLC grade Agilent p n G2453 85050 or equivalent e Formic acid analytical grade Agilent p n G2453 85060 or equivalent To prepare solvents 1 To avoid bacterial growth use fresh clean solvent bottles If you plan to analyze
93. tem 2 Start the spray with caution a Verify that the flow rate is 300 nL min and the solvent composition is 97 3 A1 B1 the starting composition for the methods b Be sure that a droplet has formed at the needle tip or that a stream has started c Verify that the dry gas setting is 300 C d Gradually increase the capillary voltage to achieve good spray View the spray through the ocular and if necessary slightly rotate the needle column assembly so the spray bends away from the entrance to the MS See Figure 5 This will reduce droplet noise e Adjust the capillary voltage until a stable current is reached typically 80 to 100 nA The maximum voltage for the G1982B orthogonal nanospray source is 2200 V and a typical voltage with a new needle is 1600 to 1700 V 3 Save the method to store the new Vcap setting Increase the Vcap only to the point where stable spray without spiking is achieved as the lifetime of the needle is usually longer at lower Veap Look for both a stable and reasonable MS spectrum as well as several minutes of stable chromatographic signal no spiking The capillary current should be around 80 to 100 nA at the starting composition of 97 3 A1 B1 and will drop as the percentage of B1 increases Protein ID Solutions LC MS App Guide Setup of Conventional Nanoflow LC MS MS System 2 Tip of needle Spray bends away from MS entrance towards 2nd electrode Entrance to MS Figure 5 Bending of sp
94. the default of 15 seconds with a 1 4 m z window 3 Do the first database search Use a database subset e g mammals Search in identity mode For Spectrum Mill version A 03 02 or later mark the check box to Calculate reversed database scores 4 Autovalidate in Protein Details mode then optionally in Peptide mode using default settings 5 Manually validate down to score of 6 SPI 70 for simple sets Depending on the size of the data set it may be easiest to do this in batches i e first score gt 8 then score gt 6 For guidelines see To manually validate results on page 79 If your goal is to maximize the number of identified proteins do a thorough job at this step because your validated results will be the basis for further searches If you do not need to maximize the number of identified proteins skip this step because it can be time consuming and may not produce a large number of identifications 6 Use Tool Belt to create a saved results file of validated protein hits res file 76 Protein ID Solutions LC MS App Guide Data Analysis with Spectrum Mill MS Proteomics Workbench 7 Be sure to indicate the database you searched since this maps to accession numbers 7 Perform database searches for suspected modifications Search against previously validated protein hits Search as follows with autovalidation and manual validation between each set of conditions a Search for pyroglutamic acid an
95. the salt gradient For more detailed flow diagrams see Two dimensional liquid chromatography 2D LC on page 101 Protein ID Solutions LC MS App Guide 21 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS Pump 24 dimension reversed phase Salt gradient as steps P Column t l MN 15t dimension SCX Figure 10 Online 2D LC with salt steps 1 22 Load sample onto a strong cation exchange column Non bound peptides flow through to the enrichment column where the peptides are trapped and the samples are desalted Switch enrichment column into the solvent path of the nanoflow pump and separate sample on the nanoflow column Perform MS and MS MS analysis of separated peptides to obtain molecular weight and sequence information After the first analysis is complete switch enrichment column back into the path of the cation exchange column Inject salt solution to elute peptides onto enrichment column In this step you inject the lowest concentration first and increase salt concentration each time you perform the step Repeat steps 2 to 5 until you finish the highest salt concentration and all peptides have been analyzed Protein ID Solutions LC MS App Guide Peptide Separation and Detection with Conventional Nanoflow LC MS MS 3 2D LC with semi continuous salt gradient This 2D LC method shown in Figure 11 is similar to 2D LC with salt steps but provides better pepti
96. tions under To calibrate the MS on page 17 Protein ID Solutions LC MS App Guide 11 2 Setup of Conventional Nanoflow LC MS MS System To prepare and install the nanospray needle CAUTION Insertion of C18 particles to prevent needle blockage 1 Assemble the following materials 30 um C18 particles Agilent p n 79903 85031 included with the Nanospray Protein Identification Solution 1 mL methanol or acetonitrile Microcentrifuge tube 1 5 mL capacity 2 Place avery small amount less than 1 mg of the C18 particles into the vial There should be only enough to see at the bottom of the vial 3 Add 1 mL of the organic solvent close lid and shake rapidly for about five seconds 4 Dip the blunt end of a nanospray needle Agilent 9301 6378 or New Objective FS360 50 8 D into the suspension of C18 particles for only one second Do this step quickly because it is easy to get too many of the C18 particles into the needle which could result in unwanted chromatography 5 Recap the vial and store Needle installation 1 Insert the blunt end of the needle through the nanospray needle nut and conductive ferrule It is not necessary to rinse the blunt end prior to installation 2 Connect the fitting directly to the outlet of the LC column as shown in Figure 3 3 Tighten the needle very gently It should seem as if the needle is not tightened enough Over tightening may break off glass particles that plug the needle If
97. to insert the HPLC Chip and how to prepare the LC and MS just before an analysis 5 Peptide Separation and Detection with HPLC Chip MS Follow the protocols to perform online 1D LC MS MS analyses with an enrichment column or 2D LC MS MS analyses Protein ID Solutions LC MS App Guide iii Optional Peptide Fractionation with Offline SCX Learn how to fractionate peptides by offline LC with a strong cation exchange column and micro fraction collection Data Analysis with Spectrum Mill MS Proteomics Workbench Follow a general Spectrum Mill workbench protocol to process MS MS data to generate tables of protein identifications Data Analysis with Mascot Protein Database Search Learn how to use a Visual Basic script to export LC MSD Trap MS MS data to Mascot format and how to use Mascot protein database search Reference Sample Analysis Strategy Read details about 1D and 2D nanoflow LC MS MS sample analysis strategy Protein ID Solutions LC MS App Guide Contents 1 Overview of LC MS Steps with the Protein ID Solutions 1 Peptide fractionation by LC 2 Mass spectrometry 3 Dataanalysis 4 2 Setup of Conventional Nanoflow LC MS MS System 5 System components 6 Safety 7 Additional materials required 7 To prepare solvents 7 To plumb the system 8 To prepare and install the nanospray needle 12 To prepare the LC and MS just before an analysis 15 3 Peptide Separation and Detection with Conventional Nanoflow LC MS MS 19 Safety 20 P
98. xiliary Table 13 Nanoflow pump solvent gradient Time min B 0 3 7 80 7 01 30 Stop time 9 min Post time none Capillary pump 3 acetonitrile 0 1 formic acid in water Capillary pumps use no flow gradient just set flow to 4 uL min Needle flush 20 methanol 0 1 formic acid in water solvent Protein ID Solutions LC MS App Guide 51 5 Peptide Separation and Detection with HPLC Chip MS Bottom sensing On 0 9 mm offset for plastic conical micro vials Agilent p n 9301 0978 or 0 2 mm for 96 well Eppendorf well plates Agilent p n 5042 8502 Protocol for 1D enrichment LC with Protein ID Chip long method for more complex samples Configuration HPLC Chip Maximum injection volume Chip Cube Nanoflow pump 52 Stop time See To configure the Chip Cube on page 45 Use the Protein ID Chip p n G4240 62001 The chip integrates the following columns e Enrichment column ZORBAX 300SB C18 40 nL 5 um particle size e Reversed phase column ZORBAX 300SB C18 75 um x 43 mm 5 um particle size 8 uL or 40 uL depending on loop installed e Inner valve Enrichment e Valve timetable Enrichment at 37 min A 0 1 formic acid in water B 90 acetonitrile 0 1 formic acid in water Primary flow 200 to 500 uL min From the Instrument menu choose More pumps gt Auxiliary Table 14 Nanoflow pump solvent and flow gradient Time min B Flow pL min 0 3 0 30 30 45 0 3
99. y define the list of possible charge states For example if you have excluded singly charged ions when setting up your data dependent acquisition parameters you can omit the 1 possibility Select your mgf file from the folder where you saved the exported file Select ESI Trap Protein ID Solutions LC MS App Guide Data Analysis with Mascot Protein Database Search 8 To use Mascot Daemon The optional Mascot Daemon software on an in house server automates database searching You set search parameters using the Mascot Daemon Parameter Editor shown in Figure 22 Parameter entry is similar to that discussed in To use Mascot protein database search on page 90 Mascot Daemon lt taskname gt lt parameters gt submitted from lt localhost gt Typsin E NIPCAM C E term Z 018 C term Carboxymethy C ie Oxidation Hw Deamidation NO 7 Oxidation M hd fs ESI TRAP ha Figure 22 Mascot Daemon Parameter Editor You set automation parameters using the Mascot Daemon Task Editor shown in Figure 23 Some of these fields are discussed briefly below Additional information is available via the Mascot Daemon online help Protein ID Solutions LC MS App Guide 93 8 Data Analysis with Mascot Protein Database Search Schedule Follow up Optional wild card filename 94 Mascot Daemon ea de Figure 23 Mascot Daemon Task Editor Schedule searches by time an
100. you suspect you have glass particles use a can of pressurized air to blow out the fitting and ferrule If you fail to do this the particles may become lodged in subsequent replacement needles 12 Protein ID Solutions LC MS App Guide Setup of Conventional Nanoflow LC MS MS System 2 Figure 3 Nanospray needle installation 4 Verify that the LC solvent continues to flow out of the needle A very small drop will form within 10 to 20 seconds Hold the needle assembly under the nanospray lamp for best illumination CAUTION Do not wipe this drop away Do not touch the tip of the needle to anything because it is very easily damaged Installation of assembly into nanospray source 1 Make sure the capillary voltage is set to 1400 V 2 Insert the needle column assembly into the needle holder assembly mount on the nanospray source See Figure 4 Figure 4 Needle holder in needle holder assembly Needle positioning and starting of spray 1 With the needle assembly installed use the microscope to position the needle within 3 mm of the flat electrode modified capillary cap e The microscope field of view is 3 5 mm so if the needle tip is just seen at one end the capillary cap will be just seen at the opposite end e For more information on proper alignment of the needle tip see the Orthogonal Nanospray Ion Source User s Guide Protein ID Solutions LC MS App Guide 13 2 14 Setup of Conventional Nanoflow LC MS MS Sys

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