The application of a hypothesis-driven strategy to the sensitive detection and location of acetylated lysine residues

Abstract

The©application©of©a©hypothesis-driven©method©for©the©sensitive©determination©of©lysine acetylation© sites© on© enzymatically© digested© proteins© is© described.© Comparative© sensitivity© tests were© carried© out© using© serial© dilution© of© an© acetylated© bovine© serum© albumin© (AcBSA)© digest to©assess©the©performance©of©a©multiple©reaction©monitoring©(MRM)-© based©approach©as compared© to© a© more© conventional© precursor© scanning© (PS)© method.© Both© methods© were© capable of© selectively© detecting© an© acetylated© peptide© at© the© low© femtomole© level© when© spiked© into© a background© of© 500© fmol© six-protein© tryptic© digest.© The© MRM© approach© was© roughly© tenfold more© sensitive© than© precursor© scanning© with© one© acetylated© peptide© detected© and© sequenced at© the© level© of© 2© fmol© on-column.© The© technique© was© subsequently© applied© to© a© gel-derived sample© of© cytokeratin-8© (CK8)© shown© to© contain© acetylated© lysine© residues© by© Western© blot analysis.©The©strategy©applied©herein,©termed©MRM-initiated©detection©and©sequencing .© Although© this© is© indeed© a© sensitive© approach, and© has© the© added© advantage© of© being© compatible© with additional© protein/peptide© modifications,© a© more promising© strategy© with© regard© to© sensitivity© would© be to© use© a© multiple© reaction© monitoring© (MRM)© transition as© the© trigger© for© an© information-dependent© acquisition (IDA)©experiment©and©generate©sequence©information from©a©subsequent©product©ion©scan.©This©technique, termed MIDAS-for MRM-initiated detection and sequencing-has been previously applied to phosphorylation studies and was shown to be on the order of ten times more sensitive than the commonly used Ϫ79 precursor© ion© scan© [7].© In© this© work,© for© the© first© time,© we demonstrate a similar improvement in sensitivity of a MIDAS-based acetylation method over an existing precursor scanning approach. Experimental Sensitivity ComparisonAcetylated bovine serum albumin (AcBSA, Invitrogen, Paisley, UK) was diluted to 15 pmol L Ϫ1 in 100 L of 25 mM ammonium bicarbonate and digested at 37°C overnight by addition of modified porcine trypsin (Promega, Southampton, UK) at a 50:1 total protein:enzyme ratio. The digest was dried and resuspended in 150 L of 2% (vol/vol) acetonitrile/0.1% (vol/vol) formic acid, providing a stock concentration of 10 pmol L Ϫ1 . The stock sample was further diluted in the same solvent containing a six-protein mix tryptic digest, to generate standard solutions. A single preparation of this AcBSA Address reprint requests to Prof.

Current affairs in quantitative targeted proteomics: multiple reaction monitoring-mass spectrometry

“…This technique has been recently utilized to quantify putative peptide and protein biomarkers where a proteotypic peptide is selected as a surrogate for the protein of interest [1][2][3][4][5][6][7]. Aptly suited to the multiplex capabilities of this MRM strategy, multiple signature peptides representing a particular protein can then be examined in one experiment allowing for the development of a highly selective, sensitive and high throughput quantitative methodology [1,8].…”

“…This however greatly increases the search time and cost. While MRM-MS has not been commonly used to focus on acetylation as a PTM like phosphorylation, Griffiths et al, demonstrated an interesting approach [8]. Since acetylation can be detected by the characteristic reporter ion of 126.1 m/z after CID, corresponding to the immonium ion of the acetyl-lysine, the authors utilized a MRM-MS approach to detect acetylation in a test case proteins.…”

“…Growing interest in using this technology to quantify proteins has led to a number of new software developments over the last few years. As state above, the MIDAS and TIQAM workflows were developed for targeted detection of proteins [21,44], their respective PTM [8,41] and for the rapid development of MRM assays [1,23,53]. Informatic strategies for the prediction of the most likely peptides (proteotypic peptides) that will be observed for proteins of interest are emerging [14,15,23,54,55].…”

Multiple reaction monitoring for quantitative biomarker analysis in proteomics and metabolomics☆

“…Detection was achieved within the low fmol (on-column) range and the utility of the application was confirmed by analysing naturally acetylated peptides derived from cytokeratin 8 isolated from a cell lysate by 2D-gel electrophoresis. Using a total of 84 MRM transitions for potential acetylation sites, the authors were able to identify five novel acetylated peptides [113], confirming that the method has the selectivity and sensitivity to identify naturally modified proteins present within a biological matrix.…”

“…Griffiths and co-workers applied a MIDAS-based approach similar to the one developed by the same group for phosphorylation analysis [51,113] to determine the sensitivity of detection of acetylated peptides derived from bovine serum albumin. Once again they found that the MRM method offered >10-fold enhanced sensitivity compared with precursor ion scanning for the immonium ion of acetyl lysine minus NH 3 (mass 126.1 Da).…”