Agilent An Integrated Approach to Improve Sequence Coverage Protein Identification by Combining LC-MALDI
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1. Human serum sample Denature reduce Alkylate digest Direct MALDI LC MALDI Nano LC MS MS Figure 1 Experimental design LC MALDI MS MS Samples were separated using a capillary HPLC equipped with a thermostatted micro fraction collector spotter Agilent Technologies The flow rate was 1 uL min The matrix CHCA 2 mg mL diluted with 50 2 propanol 1 acetic acid was added to the sample at 1 uL min using a syringe pump connected to a T connector The spotter spotted every minute with 2 uL of total volume on the MALDI target The target was then analyzed using the LC MSD Trap XCT Plus ion trap mass spectrometer with a PDF MALDI source Nano LC MS MS All LC MS analyses were done using the Agilent Protein Identification Solution which consisted of an Agilent 1100 Series nanoflow liquid chromatograph LC system coupled to an Agilent 1100 Series LC MSD Trap XCT Plus Database search Data processing was done using Spectrum Mill MS pro teomics workbench software The spectra were searched against the PI human database using carbamidomethy lation as the Cys modification The search was done with three iterative cycles The first search was done in the identity mode against the IPI human database with trypsin cleavage specified and 2 missed cleavages allowed The validated search results were saved as a new database for the next search cycle The second search cycle was done against the smaller saved results databa2. on a MALDI plate M Rapid Commun Mass spectrum 2004 18 3008 2 Anderson N L et al The human plasma proteome Mol Cell Proteomics 2004 3 4 311 326 www agilent com chem Agilent Technologies Inc 2005 Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies shall not be liable for errors contained herein or for incidental or consequential damages in connection with the furnishing performance or use of this material Printed in the U S A July 29 2005 5989 3500EN
3. AQOR 1468 8025 3 23e 007 M 3 R FLHVPDTFEGHFDGVPVISKG 2298 1559 1 35e 007 4 _ MFVHFFAPDNLDPIPK N 1756 9063 4 32e 007 5 K FYNQVSTPLLRIN 1337 7218 L 1 90e 009 6 _ 0QIQPSGGTNINEALLR A 1582 8553 Lo 243e 009 7 QKFYNQVSTPLLRN 1465 8167 6 93e 007 8 RYKLWAYLO 793 4612 o 7 86e 008 9 R LSNENHGIAQR 1238 6242 1 46e 006 10 M SLDHHIVTPLTSLVIENEAGDER M 2545 2898 sg O7e 007 11 F VHFFAPDNLDPIPK N 1609 8379 7 15e 007 12 VOFELHYQEVKiW 1419 7272 6 92e 005 NE aSer008 13 QWNNSPOPONWFDVOIPK G 2122 1297 14 OYDVKREEKAGELEYF N 1811 9180 Figure 3 Spectrum Mill display of all the peptides identified by LC MALDI MS MS and nano LC MS MS Discussion We compared our results with the nonredundant list developed by Anderson et al from a combination of four separate sources see Table 3 Thirty seven of the pro teins we identified were also identified by those other 4 sources These proteins we have identified with rela tively high sequence coverage and Spectrum Mill data base search scores They represented the high abundant proteins and are more likely to be identified most of the time regardless of the different separation techniques and mass spectrometry that was used Twenty one pro teins we identified were also identified by three other sources They have lower sequence coverage and Spec trum Mill scores Twelve proteins were identified by two other sources and
4. An Integrated Approach to Improve Sequence Coverage and Protein Identification by Combining LC MALDI MS MS and Nano LC MS MS This material was presented in a poster at the 53rd ASMS Conference on Mass Spectrometry in 2005 Research presented in posters at scientific conferences may include results from instruments or products that are not yet commercially o o Application Note s Ning Tang and Christine Miller Agilent Technologies available Introduction Electrospray ionization ESI and matrix assisted laser desorption ionization MALDI have proven to be com plementary as different peptides are typically seen with the two ionization techniques however very little was known about the characteristics of the peptides that prefers each ionization technique Coupling of the RP HPLC with MALDI via a MALDI spotting device has provided an automated and robust solution for analyzing medium complexity samples using MALDI In this study we have investigated the improvements in proteome coverage for complex protein samples by combining nano LC MS MS with LC MALDI MS MS The integrated approach with better sequence coverage facilitated pro tein identification Experimental Three dimensions of separations were employed to sim plify the human serum sample before it was analyzed by the mass spectrometry see Figure 1 The first dimen sion of separation was immunodepletion of six of the most abundant proteins in human serum usin
5. fifty nine proteins were identified by less than two other sources They have relatively low sequence coverage and low spectrum mill scores An Integrated Approach to Improve Sequence Coverage and Protein Identification Agilent Technologies by Combining LC MALDI MS MS and Nano LC MS MS Table 3 Correlation of Agilent results with nonredundant list comprised of data from four separate sources Average Average Average ID by other of of peptides sequence Spectrum sources proteins per protein coverage Mill scores 4 37 15 26 210 3 21 11 12 143 2 12 3 11 36 lt 2 59 4 7 40 Conclusions References LC separation coupled with MALDI has significantly increased the number of proteins identified LC MALDI MS MS and nano LC MS MS have each identified unique peptides among the proteins found by both techniques and combining the results gives increased sequence coverage for most of the proteins Each technique has identified unique proteins LC MALDI MS MS has identified 40 unique proteins and nano LC MS MS has identified 49 unique proteins Combining both techniques 129 proteins were iden tified Combining both techniques increased the confidence in the protein identification and gave the optimum sequence coverage for the proteins Ai Agilent Technologies 1 Nagele E and Vollmer Coupling of nanoflow liquid chromatography to matrix assisted laser desorption ionization mass spectrometry real time liquid chromatography run mapping
6. g an anti body column The second dimension was strong cation exchange SCX fractionation of the tryptic peptides The third dimension was reversed phase chromatogra phy of the SCX fractions before mass spectrometry analysis Materials HPLC grade solvents were obtained from Burdick amp Jackson Human serum was purchased from Genomics Collaborative Cambridge MA It was from a healthy anonymous male donor The immunodepletion column and reagents the o cyano 4 hydroxy cinnamic acid CHCA matrix solution and the in gel digestion kit were from Agilent Technologies Immunodepletion Human serum was immunodepleted using the Agilent Multiple Affinity Removal system MARS which removes six of the highest abundance proteins in serum The depleted serum was then denatured reduced alky lated and digested with trypsin Offline SCX separation Offline capillary LC prefractionation was done using a Zorbax Bio SCX Series II column 50 x 0 8 mm with an ammonium formate gradient The gradient went from 0 100 mM in 120 minutes then at 500 mM at 134 min utes and finally to 1 M at 140 minutes Fifty fractions were collected Fractions were dried washed twice with H 0 to remove the ammonium bicarbonate and then dried before LC MALDI and direct MALDI analysis Agilent Technologies An Integrated Approach to Improve Sequence Coverage and Protein Identification Agilent Technologies by Combining LC MALDI MS MS and Nano LC MS MS
7. ing the results from nano LC MS MS and LC MALDI MS MS Comparison of LC MALDI MS MS with nano LC MS MS The number of proteins and peptides identified by each technique were summarized in Table 2 LC MALDI MS MS and nano LC MS MS identified similar number of proteins However nano LC MS MS identified 2 3 fold more peptides therefore it provided higher sequence coverage and more confident protein ID Combining the two techniques we have identified more proteins with more peptides Agilent Technologies Table 2 Comparison of the number of proteins and peptides identified by LC MALDI MS MS and nano LC MS MS of proteins of peptides Nano LC MS MS 89 876 LC MALDI 76 ml Fe Combination 129 1044 Each technique identified unique peptides among the proteins found by both methods Combining the results increased the sequence coverage of the proteins Figure 3 is an example of Spectrum Mill database search results of one of the protein that was identified as inter alpha trypsin inhibitor heavy chain H2 precursor Distinct lean Summed AA Peptide Database MS MS Search Coverage Spectral Accession Sco Intensity Distinct Group Spectra Peptides ee Protein Name u4 n B 192 93 18 EE 1100305461 Inter alpha trypsin inhibitor heavy chain H2 precursor Sequence oll d papon iaa CM ASMSO0SWitra nx scx2 Da 1 RAEDHFSVIDFNANIRT 1804 8618 2 47e 006 2 QAHVSFKPTV
8. se to search for single KQMSTY modifications The results were then validated and saved The third search cycle was done against the saved results database with the enzyme selection set to none Results Comparison of LC MALDI MS MS with direct MALDI MS MS The SCX fractions were run by both MALDI MS MS and LC MALDI MS MS The number of proteins and pep tides identified by each technique were summarized in Table 1 LC MALDI identified 3 4 fold more proteins and 5 6 fold more peptides compared to direct MALDI Sequence coverage of identified proteins increased by LC MALDI which also provided better confidence in the protein ID An Integrated Approach to Improve Sequence Coverage and Protein Identification by Combining LC MALDI MS MS and Nano LC MS MS Table 1 Comparison of the number of proteins and peptides identified by LC MALDI MS MS and direct MALDI MS MS of proteins of peptides Direct MALDI 23 56 LC MALDI 76 313 Combine results from LC MALDI MS MS and nano LC MS MS We have identified 129 proteins in total combining the results from both techniques Out of the 129 proteins 40 of them were identified by both techniques Forty pro teins were only identified by LC MALDI MS MS and 49 proteins were uniquely identified by nano LC MS MS Combining the two techniques has increased the number of proteins identified as well as sequence coverage and database search scores LC MALDI MS MS Nano LC MS MS Figure 2 Combin
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