Multiple Reaction Monitoring to Identify Sites of Protein Phosphorylation with High Sensitivity

Abstract

Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.

Global dynamics of Escherichia coli phosphoproteome in central carbon metabolism under changing culture conditions

“…Scheduled multiple reaction monitoring (sMRM) was used to quantify phosphorylated peptides using either a QTRAP 5500 or a QTRAP 4000 (Applied Biosystems, US) depending on instrument availability [54,55]. Peptides were separated on an analytical column (100mm × 2.1mm 2.6μm 100Å Kinetix C 18 , Phenomenex, US) using a 2 to 80% acetonitrile (in MilliQ water with 0.1% formic acid) gradient over 100 minutes.…”

“…In order to quantify both the extent of phosphorylation and any changes in response to medium changes, we developed targeted analysis using sMRM, the most sensitive MS technique for quantitative proteomics [54][55][56][57]. We first validated the technique using an sMRM protocol optimised for phosphorylated and unphosphorylated peptides of α-casein (Fig.…”

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

“…A cetylation©is©an©important©posttranslational modification© (PTM)© involved© in© the© regulation of© a© number© of© key© cellular© processes© including transcription© [1],©protein-protein©interactions© [2],©and protein© stability© [3].© The© importance© of© this© biologically relevant©modification©has©recently©been©compared©to that© of© phosphorylation© [4],© which© is© a© key© regulator© of biological© processes,© and© thus© there© is© a© growing© need for©the©sensitive©detection©and©location©of©sites©of acetylation© on© proteins.© A© number© of© mass© spectrometry (MS)-© based© methods© have© been© used© for© the© determination© of© protein© acetylation© [5,© 6].© One© such© approach relies© on© the© detection© of© a© characteristic© reporter© ion© at 126.1© Th,© corresponding© to© the© immonium© ion© of© acetyl lysine© ϪNH 3 ,© upon© collisionally© activated© dissociation (CAD)© [6].© 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.…”

“…The© MIDAS© protocol,© which© takes© full© advantage© of© the combined© functionality© of© the© 4000© QTRAP,© is© described in©detail©elsewhere© [7].©Briefly,©a©triple©quadrupole MRM© scan© is© used© to© sensitively© select© for© postulated acetylated© peptides.© Under© low-energy© collision© conditions,© acetyl© lysine© residues© have© been© shown© to© fragment,©producing©a©diagnostic©ion©at©m/z 126.1©corre-sponding©to©the©immoniumϪNH 3 ion© [6].©A©table©of potential©acetylated©peptides©for©the©protein©under study© is© first© generated© using© a© software© script© developed© by© Applied© Biosystems.© This© represents© the© set© of precursor© masses© to© be© sequentially© transmitted© by© Q1, held© at© low© resolution,© while© Q3© was© held© static© at© 126.1 m/z (unit© resolution).© Limited© studies© in© our© laboratory suggest© that© optimal© collision© energies© to© facilitate© these transitions©approximate©to©the©precursor©m/z ϫ©0.1 irrespective© of© charge© state.© Collision© energies© between 50©and©80©eV©(laboratory©frame©of©reference)©were assigned© to© each© transition© depending© on© the© precursor m/z such© that© m/z of© about© 400© -© 600,© CE© ϭ© 50© eV;© about 600 -700, CE ϭ 60 eV; about 700 -800, CE ϭ 70 eV; Ͼ800, CE ϭ 80 eV.…”

“…

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 .

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