Agilent High-Throughput Analysis of Epigenetic Targets with Agilent RapidFire/MS Systems: Sirtuin (SIRT) Enzymes Manual
Contents
1. S MS enabled the fast and direct measurement of multiple acetylated species Figure 2 Label Free Assays with Meaningful Data The RapidFire MS system measures native molecules directly thereby obviating the need for fluorescently tagged materials or secondary reactions which can be susceptible to compound interference Label free analysis enables substrate to product conversion measurements to be made directly facilitating the determination of accurate kinetic parameters for hit identification Figure 3 shows SIRT1 SIRT2 and SIRT3 inhibition curves with IC values that concur with those from previous reports 100 f Oac peptide lac peptide 2ac peptide 80 3ac peptide 60 Percent of control 40 20 0 100 200 300 400 Time minutes Figure 2 Sequential modification of a triply acetylated p53 peptide by SIRT1 over time 100 Nicotinamide IC values i SIRTI 62 uM E 4 SIRT2 10 uM 5 v SIRT3 49 uM 50 25 10 3 10 2 107 10 10 102 103 104 105 Nicotinamide uM Figure 3 SIRT1 SIRT2 and SIRT3 inhibition curves using the small molecule nicotinamide Conclusions RapidFire MS MS was used to analyze 1 SIRT1 SIRT2 and SIRT3 reactions at a sustained rate of approximately seven seconds per sample The label free method circumvented the need for non native surrogate substrates and radioactive methodologies RapidFire MS MS enabled direct detection of pe2. gle peptide which is common for sirtuins and other epigenetic targets MS detection enables every modification state to be measured discretely and concurrently 3 Figure 1 shows a triply acetylated 3ac p53 peptide which is a good candidate for RapidFire MS analysis because it has three possible deacetylation products 2ac lac and Qac 0 Il I H Il H I I H I H l H Il Il I H Il I H I H Il H Il H I H I H I HAN CH L N GHG N GHGS Ne GEO N GH G N GHG N GHG NS CHAGAS CHEER CH C N CH 6 N CH 0 N CH L N CH C N GHG N GH 6 N GH C N GHG 0H CH CH H CH CH CH CH CH OH CH CH cH b 0 CH CH NH NH NH CH OH 0 H H H CH CH CH CH GH CH OH CH ws CH CH CH CH I I CH Linu CH CH CH NH G C NH H NH CH CH CH GH OH CH h lae GH CH h i f f n CH CH OH Figure 1 The p53 peptide spanning amino acids 372 to 389 contains multiple lysine residues including 381 382 and 386 which are known deacetylation sites Fast and Direct Measurement of Multiple Reaction Products The RapidFire platform enables mass spectrometric analysis of native molecules by performing high throughput online desalting Following enzymatic reactions between SIRT1 enzyme and a multiply modified substrate RapidFire MS MS was used to monitor every possible acetylation state of the peptide with a sustained throughput of seven seconds per sample Analysis by RapidFire M
3. m High Throughput Analysis of Epigenetic Targets with Agilent RapidFire MS Systems of ae a Sirtuin SIRT Enzymes e e Application Note Author Introduction Peter Rye Lauren Frick Histones and other proteins are subject to a variety of posttranslational and William LaMarr Agilent Technologies Inc a ee oa Wakefield MA USA is the deacetylation of specific lysine residues by sirtuins whose activity has been modifications that regulate a host of biological processes One such modification associated with inflammatory cardiovascular proliferative neurodegenerative and metabolic disorders High throughput bioassays designed to identify sirtuin modulators are therefore of interest particularly when coupled to the highly sensitive and specific detection available with mass spectrometry MS oft Agilent Technologies Fast and Direct Measurement of Multiple Reaction Products Studying sirtuins by RapidFire MS offers numerous advantages over other existing assay formats First the system circumvents the costs and special handling procedures required when using radioactive substrates 0 Second RapidFire MS based assays do not involve secondary or coupled reactions that can complicate data interpretation Third because the peptide species are measured directly by MS there is no need for fluorescent tags which can cause data artifacts Finally in situations where multiple products can be formed on a sin
4. o guarantee as to the quality or suitability of this data for your specific application Information descriptions and specifications in this publication are subject to change without notice Agilent Technologies Inc 2011 Published in the USA November 21 2011 5990 9345EN Eo Agilent Technologies
5. ptide reactants and facilitated the identification of kinetic parameters for accurate 3 IC determinations Because RapidFire MS can measure multiple analytes from each sample quickly and directly the system is particularly well suited for studying epigenetic changes 4 which involve sequential modifications References Yamamoto H Schoonjans K Auwerx J Sirtuin functions in health and disease Mol Endocrinol 2007 21 8 1745 55 Rye PT et al Advances in Label Free Screening Approaches for Studying Sirtuin Mediated Deacetylation J Biomol Screen 2011 Sep 12 Epub ahead of print Rye PT et al Advances in Label Free Screening Approaches for Studying Histone Acetyltransferases J Biomol Screen 2011 Sep 9 Epub ahead of print Prives C and Manley J L Why is p53 acetylated Cell 2001 107 7 815 8 Porcu M and Chiarugi A The emerging therapeutic potential of sirtuin interacting drugs from cell death to lifespan extension Trends Pharmacol Sci 2005 26 2 94 103 Marcotte P A et al Fluorescence assay of SIRT protein deacetylases using an acetylated peptide substrate and a secondary trypsin reaction Anal Biochem 2004 332 1 90 9 www agilent com lifesciences rapidfire For research purposes only and not for use in diagnostic procedures The information described here is intended for reference and research purposes only Agilent Technologies offers n
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