SIMAC (Sequential Elution from IMAC), a Phosphoproteomics Strategy for the Rapid Separation of Monophosphorylated from Multiply Phosphorylated Peptides

Abstract

The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 μg of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides. The complete analysis of phosphoproteomes has been hampered by the lack of methods for efficient purification, detection, and characterization of phosphorylated peptides from complex biological samples. Despite several strategies for affinity enrichment of phosphorylated peptides prior to mass spectrometric analysis, such as immobilized metal affinity chromatography or titanium dioxide, the coverage of the phosphoproteome of a given sample is limited. Here we report a simple and rapid strategy, SIMAC (sequential elution from IMAC), for sequential separation of monophosphorylated peptides and multiply phosphorylated peptides from highly complex biological samples. This allows individual analysis of the two pools of phosphorylated peptides using mass spectrometric parameters differentially optimized for their unique properties. We compared the phosphoproteome identified from 120 μg of human mesenchymal stem cells using SIMAC and an optimized titanium dioxide chromatographic method. More than double the total number of identified phosphorylation sites was obtained with SIMAC, primarily from a 3-fold increase in recovery of multiply phosphorylated peptides. Reversible protein phosphorylation is an important post-translational modification in most intracellular biological processes (1Graves J.D. Krebs E.G. Protein phosphorylation and signal transduction.Pharmacol. Ther. 1999; 82: 111-121Crossref PubMed Scopus (350) Google Scholar) because it can increase or decrease a regulatory response to external stimulation or an affinity toward other proteins or nucleic acids. Often multiphosphorylation on adjacent amino acids can have a large impact on the activity of regulatory proteins (2McDonald B.J. Amato A. Connolly C.N. Benke D. Moss S.J. Smart T.G. Adjacent phosphorylation sites on GABAA receptor β subunits determine regulation by cAMP-dependent protein kinase.Nat. Neurosci. 1998; 1: 23-28Crossref PubMed Scopus (204) Google Scholar, 3Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase).EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (841) Google Scholar, 4Rabinovitz I. Tsomo L. Mercurio A.M. Protein kinase C-α phosphorylation of specific serines in the connecting segment of the β4 integrin regulates the dynamics of type II hemidesmosomes.Mol. Cell. Biol. 2004; 24: 4351-4360Crossref PubMed Scopus (83) Google Scholar). One of the challenges in large scale phosphoproteomics is the analysis of multiply phosphorylated peptides. The presence of mono- or non-phosphorylated peptides in samples for MS suppresses the ionization of multiple phosphorylated peptides and thereby decreases the chance to detect them. Therefore new phosphoproteomics tools are required to study multiple phosphorylation of proteins. Phosphopeptide enrichment prior to MS analysis is essential for large scale phosphoproteomics studies because phosphorylated peptides are rarely detected in “shotgun” MS analysis. A widely used enrichment technique for phosphorylated peptides is the use of metal ions for the binding of the negatively charged phosphopeptides, i.e. IMAC. IMAC was introduced to the characterization of phosphorylated proteins by Andersson and Porath (5Andersson L. Porath J. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography.Anal. Biochem. 1986; 154: 250-254Crossref PubMed Scopus (647) Google Scholar) and was later extensively adapted for enrichment of phosphorylated peptides prior to mass spectrometric analysis (6Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 7Figeys D. Gygi S.P. Zhang Y. Watts J. Gu M. Aebersold R. Electrophoresis combined with novel mass spectrometry techniques: powerful tools for the analysis of proteins and proteomes.Electrophoresis. 1998; 19: 1811-1818Crossref PubMed Scopus (90) Google Scholar, 8Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. 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Immobilized gallium(III) affinity chromatography of phosphopeptides.Anal. Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (788) Google Scholar). The IMAC technique improves identification of phosphopeptides from complex biological mixtures (6Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 8Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 11Nuhse T.S. Stensballe A. Jensen O.N. Peck S.C. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2003; 2: 1234-1243Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar). However, non-phosphorylated peptides containing multiple acidic amino acid residues co-purify with the phosphorylated peptides in IMAC. O-Methyl esterification of the acidic residues has been suggested as a means to prevent this, but this step may introduce unwanted side reactions and loss of peptides due to extensive lyophilization (13Stewart I.I. Thomson T. Figeys D. 18O labeling: a tool for proteomics.Rapid Commun. Mass Spectrom. 2001; 15: 2456-2465Crossref PubMed Scopus (310) Google Scholar). IMAC appears to have a stronger selectivity for multiply phosphorylated peptides in biological buffers (6Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 14Jensen S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 15: 21, 3635-3645Google Scholar). To counter this, buffer exchange using reversed phase chromatography prior to IMAC has been used in many studies (8Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 11Nuhse T.S. Stensballe A. Jensen O.N. Peck S.C. Large-scale analysis of in vivo phosphorylated membrane proteins by immobilized metal ion affinity chromatography and mass spectrometry.Mol. Cell. Proteomics. 2003; 2: 1234-1243Abstract Full Text Full Text PDF PubMed Scopus (523) Google Scholar) with a high risk of losing phosphorylated peptides (15Larsen M.R. Graham M.E. Robinson P.J. Roepstorff P. Improved detection of hydrophilic phosphopeptides using graphite powder microcolumns and mass spectrometry. Evidence for in vivo doubly phosphorylated dynamin and dynamin Cell. Proteomics. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). dioxide used titanium SIMAC, sequential elution from human mesenchymal stem protein chromatography has to an efficient to IMAC M.J. the of phosphopeptides from using and titanium Chem. 2004; PubMed Scopus Google Scholar, M.R. Jensen O.N. Roepstorff P. enrichment of phosphorylated peptides from mixtures using titanium dioxide Cell. Proteomics. 2005; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). has a selectivity for phosphorylated peptides than and binding from non-phosphorylated peptides can by acid or acid and high of in the buffer S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 15: 21, 3635-3645Google Scholar, M.R. Jensen O.N. Roepstorff P. enrichment of phosphorylated peptides from mixtures using titanium dioxide Cell. Proteomics. 2005; 4: Full Text Full Text PDF PubMed Scopus Google Scholar, Jensen O.N. Larsen M.R. enrichment of phosphorylated peptides using titanium 1: PubMed Scopus Google Scholar). chromatography of phosphorylated peptides is of most biological buffers S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 15: 21, 3635-3645Google Scholar). A of different phosphopeptide enrichment methods Watts J.D. Aebersold R. A to the analysis of protein Biotechnol. 2001; 19: PubMed Scopus Google and chromatography has of a is of a phosphoproteome M. Aebersold R. of of the 2007; 4: PubMed Scopus Google Scholar). to IMAC a toward monophosphorylated peptides has been for We is to multiply phosphorylated peptides as as monophosphorylated peptides but the multiply phosphorylated peptides are to from the due to a high binding multiply phosphorylated peptides are in the ionization of MS in the presence of monophosphorylated peptides and monophosphorylated peptides are identified in large scale phosphoproteomics using most mass are to a number of MS in a given and in most the monophosphorylated peptides in a complex the multiply phosphorylated peptides. the of phosphorylated peptides in MS is than of non-phosphorylated peptides because the loss of the is the This in of are the of identification and phosphorylation The of phosphorylated peptides can by using MS M. D. J. Gygi S.P. Large-scale characterization of Sci. S. A. 2004; PubMed Scopus Google Scholar) an ion from a loss signal detected in the is for a of to this the analysis of monophosphorylated peptides because multiply phosphorylated peptides is an can applied to multiply phosphorylated peptides because this primarily in loss of the M.J. R. R. M.R. Protein kinase A phosphorylation by ion mass 2004; 4: PubMed Scopus Google Scholar, M.J. D.J. Shabanowitz J. D.F. for the detection of post-translational and proteins by mass 2005; 4: PubMed Scopus Google Scholar). However, is in most Here we a rapid and simple in recovery of of phosphorylated peptides from of complex biological samples. The new or and has The of the is sequential elution from IMAC we and is on acidic primarily monophosphorylated peptides from IMAC elution the multiply phosphorylated peptides are A of the IMAC chromatography to most of the non-phosphorylated peptides from the of monophosphorylated peptides in a complex the two phosphopeptide pools can using mass spectrometry parameters are optimized for type of The SIMAC was applied to a phosphoproteomics study of human mesenchymal stem cells in 120 μg of total protein was used as the the SIMAC than the total number of phosphorylation sites from a of in with chromatography using simple and and the for was from was from reversed phase was from from and acid was from J. T. was from was from used in the and the was from a The and of the using a from of proteins. was a from and from protein was in and for was and the sample was for The was with and the proteins using for mesenchymal stem cells in in with containing and The cells with buffer and of was to the mixtures and and of to the The cells with the for with the cells using the the was in of buffer of The cells with of on to the cells and for was The sample was for was by for in the protein of was to the by for The proteins by for and the was and A total of of the and protein from human mesenchymal stem cells was in to a of The proteins with μg of μg of protein for The sample was with and μg of μg of protein was The sample was for The is for the using of The in the the used for 120 μg of of metal was The in buffer as M. Y. T. T. Y. of immobilized metal enrichment of phosphopeptides for protein phosphorylation Chem. 2005; PubMed Scopus Google Scholar). The with of buffer and of μg of The in a for the in the of a by application of an IMAC the complex the IMAC in The IMAC was in an for analysis by chromatography The IMAC was using of was with the IMAC The monophosphorylated peptides and non-phosphorylated peptides from the IMAC using of and the multiply phosphorylated peptides from the IMAC using of of in of The IMAC and the IMAC by the and from the IMAC was for phosphopeptides using the complex the monophosphorylated was to chromatography as A was by a of from a and the in the of a M.R. Jensen O.N. Roepstorff P. enrichment of phosphorylated peptides from mixtures using titanium dioxide Cell. Proteomics. 2005; 4: Full Text Full Text PDF PubMed Scopus Google Scholar, Jensen O.N. Larsen M.R. enrichment of phosphorylated peptides using titanium 1: PubMed Scopus Google Scholar). The in in the the the from The was by the application of used for or of the to prevent binding to the membrane and the The sample was in of and of S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 15: 21, 3635-3645Google Scholar) and in buffer acid in and a of complex samples two microcolumns used The was with of buffer and with of buffer The phosphopeptides to the microcolumns using of by elution using of to phosphopeptide to the The was by of acid prior to the The reversed phase resin was in The and was used to the to microcolumns of J. R. Roepstorff P. purification and technique on for the analysis of complex mixtures by matrix-assisted laser mass Mass Spectrom. 1999; PubMed Scopus Google Scholar). sample was a The microcolumns with of and the phosphopeptides the using of analysis of the monophosphorylated peptides from the complex the phosphopeptides in a the phosphorylated peptides from the using of by The phosphopeptides in of acid and of A prior to analysis. MS was on a obtained in ion The used was in acid S. Jensen O.N. acid as a for MS analysis of phosphopeptides and Chem. 2004; PubMed Scopus Google Scholar). The using the The using a mass The sample was applied an The peptides on a The peptides from the using a from phase A acid to phase in an The was in a and M. D. J. Gygi S.P. Large-scale characterization of Sci. S. A. 2004; PubMed Scopus Google Scholar). The was to and the ion from the loss of acid from the ions the monophosphorylated from the SIMAC or the phosphorylated peptides by the analysis of multiply phosphorylated peptides from the SIMAC the was to and the ion from the loss of a of two from the ion The and using the The the human in the Protein protein human using an The was as the with was as the phosphorylation and phosphorylation The with a mass of and a mass of A was in a human total number of from the human for of the A identified by was it a in the The selectivity of IMAC is toward of multiply phosphorylated peptides (6Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 8Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 14Jensen S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 15: 21, 3635-3645Google Scholar). of monophosphorylated peptides using IMAC is by the buffers multiply phosphorylated peptides to have a affinity toward the IMAC in such buffers S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 15: 21, 3635-3645Google Scholar). a step is required prior to phosphopeptide enrichment with IMAC. This multiply phosphorylated peptides may to the IMAC resin than monophosphorylated peptides. this is due to binding is to We monophosphorylated peptides from the IMAC resin multiply phosphorylated peptides separation of phosphopeptides two This was and optimized using a of peptides from of different A of the phosphorylated peptides from the proteins and their is in of phosphorylated peptides identified from the using of was by of the by was by of the by in a new phosphorylated peptides from the IMAC resin using a of This in a separation of the monophosphorylated from the multiply phosphorylated peptides but the phosphopeptides several we used a This was on the use of in the buffer M. Y. T. T. Y. of immobilized metal enrichment of phosphopeptides for protein phosphorylation Chem. 2005; PubMed Scopus Google Scholar) in an of monophosphorylated peptides but multiply phosphorylated peptides in the from an IMAC This suggested a binding of monophosphorylated peptides to the IMAC resin in an acidic compared with the multiply phosphorylated an to the two by of the was with of IMAC in of for the IMAC in the of a The IMAC was using the The phosphorylated peptides from the IMAC using and of and IMAC was and on reversed phase microcolumns and a MS using The MS mass of the acidic are in peptides from the IMAC multiply phosphorylated peptides detected of to monophosphorylated peptides and elution with of monophosphorylated peptides the in the elution buffer and in elution of multiply phosphorylated peptides and A elution step from the IMAC using in the elution of multiply phosphorylated peptides because the monophosphorylated peptides been on this, a phosphopeptide separation was and SIMAC A was with IMAC resin in the monophosphorylated peptides using and the multiply phosphorylated peptides from the IMAC resin using IMAC resin to acidic peptides are is to M.R. Jensen O.N. Roepstorff P. enrichment of phosphorylated peptides from mixtures using titanium dioxide Cell. Proteomics. 2005; 4: Full Text Full Text PDF PubMed Scopus Google Scholar). the from the IMAC separation was for phosphopeptides using chromatography or complex mixtures this enrichment step using chromatography is for the IMAC and the to the monophosphorylated are with acidic non-phosphorylated peptides. The SIMAC was using of the of the sample with the IMAC resin the was in a and peptides in the and and by and the MS mass the presence of monophosphorylated peptides The monophosphorylated peptides from the IMAC using acidic and the MS mass was by monophosphorylated peptides The multiply phosphorylated peptides using and the MS mass was by multiply phosphorylated peptides This the sequential elution from the IMAC resin with acid by in a complete coverage of the phosphorylated peptides. to the to a phosphoproteome we the of a step to to the of for of phosphorylated peptides in MS in of the can used to on the phosphorylated peptides. the ion from the loss of acid is for a of This has been applied to large scale phosphoproteomics studies (8Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, M. D. J. Gygi S.P. Large-scale characterization of Sci. S. A. 2004; PubMed Scopus Google Scholar). However, for phosphorylated peptides containing than in a loss of a acid and to the in many To increase the number of identified multiply phosphorylated peptides from the high from the SIMAC we the the loss of acid was and the was on the ion to the loss of phosphorylated peptides from 120 μg of using the SIMAC strategy, two and was using the on an MS and the other was using the optimized the loss of for phosphorylated peptides with than The of the the two are in using the we identified multiply phosphorylated peptides from the we identified multiply phosphorylated peptides using the optimized Despite the new doubly phosphorylated peptides we an increase in the identification of peptides with or using this compared with the this different are for the different SIMAC The SIMAC was applied to a protein from and was compared with a optimized S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 15: 21, 3635-3645Google Scholar, Jensen O.N. Larsen M.R. enrichment of phosphorylated peptides using titanium 1: PubMed Scopus Google Scholar). The cells with two mixtures for prior to This was to the on proteins from of of the in an increase in the phosphorylation is by this to an to cells are with external biological The total protein was with One of protein was to using and the peptides from a total of 120 μg of the protein to chromatography using an optimized S.S. Larsen M.R. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques.Rapid Commun. Mass Spectrom. 2007; 15: 21, 3635-3645Google Scholar, Jensen O.N. Larsen M.R. enrichment of phosphorylated peptides using titanium 1: PubMed Scopus Google and the sample of phosphorylated peptides was by MS using the A phosphorylated was as in a given sample it been in the with a of This was on and from other J.V. C. Mortensen P. Mann M. in and phosphorylation dynamics in signaling Scholar) phosphorylated peptides are by than peptides. A total of monophosphorylated multiply phosphorylated and non-phosphorylated peptides identified by the of the peptides identified A identified peptides with a of a of However, we this is an as many of the identified in the are amino or The have a than phosphorylated peptides. of the identified phosphorylated peptides are in A and The peptides. are by than protein of the protein is in the 120 μg of was to the SIMAC phosphopeptide enrichment method. The IMAC and to chromatography and by the MS using the the multiply phosphorylated peptides using the optimized for multiply phosphorylated peptides The number of monophosphorylated peptides identified in the IMAC and and for unique monophosphorylated peptides monophosphorylated peptides identified from the IMAC the of the of the IMAC. The an selectivity or binding of the IMAC resin for monophosphorylated peptides. in the amino acid was the monophosphorylated peptides identified in the different for The number of multiply phosphorylated peptides identified in the IMAC and and The of non-phosphorylated peptides identified in the and of the peptides identified A was for the SIMAC and in a of and the as we this is More than of the multiply phosphorylated peptides identified using SIMAC identified in the the of multiply phosphorylated peptides of monophosphorylated peptides from the unique phosphorylated peptides. of the phosphorylated peptides identified using the SIMAC are in A and The peptides. are by than protein of the protein is in the total from 120 μg of the optimized identified phosphorylated peptides of multiply the SIMAC identified phosphorylated peptides of multiply phosphorylated monophosphorylated peptides identified by identified by identified by and identified by SIMAC multiply phosphorylated peptides identified by identified by identified by and identified by SIMAC unique phosphorylation sites identified using the SIMAC compared with phosphorylation sites identified using the optimized We compared the of sites the two methods and the phosphorylated peptides identified using doubly and was the SIMAC identified doubly and phosphorylated peptides. Therefore the the two methods is the number of multiply phosphorylated peptides the SIMAC as many multiply phosphorylated peptides To the of the SIMAC on a we the phosphorylated peptides identified from the protein the 120 μg of total A study identified phosphorylation sites in this protein using of as M. D. J. Gygi S.P. Large-scale characterization of Sci. S. A. 2004; PubMed Scopus Google Scholar). We compared the of SIMAC with an optimized in The phosphorylated peptides identified from are in the phosphorylation sites identified in monophosphorylated and multiply phosphorylated peptides. SIMAC we identified phosphorylation sites from monophosphorylated and multiply phosphorylated peptides. the monophosphorylated peptides identified by SIMAC in the IMAC or the and the multiply phosphorylated identified in the for two peptides identified in of the identified phosphorylation sites in the and two been as the of the SIMAC in coverage of the phosphorylation sites from a protein from of SIMAC is a rapid and simple improves large scale The new is on sequential elution of monophosphorylated multiply phosphorylated peptides with acid or prior to MS analysis and the of IMAC and chromatography the use of biological or The separation of monophosphorylated peptides from the multiply phosphorylated peptides to the to analysis of the monophosphorylated or the multiply phosphorylated peptides in this new to a total of 120 μg of from than the number of phosphorylation sites identified compared with using the most optimized A total of monophosphorylated multiply phosphorylated and unique sites identified using the SIMAC in to monophosphorylated multiply phosphorylated and unique sites using the optimized method. Despite the SIMAC elution the of the it is a to using the optimized The the monophosphorylated peptides in the different SIMAC most of the monophosphorylated peptides in the However, many unique monophosphorylated peptides identified from the IMAC the of this with highly complex The the multiply phosphorylated peptides in the different SIMAC than of the multiply phosphorylated peptides identified using SIMAC identified in the the of the multiply phosphorylated peptides with optimized loss was as multiply phosphorylated peptides identified using optimized loss The optimized loss used in this study the identification of to than of the multiply phosphorylated peptides The use of optimized loss the two of acid peptides and to the of multiply phosphorylated peptides. with for this because this primarily in loss of the M.J. R. R. M.R. Protein kinase A phosphorylation by ion mass 2004; 4: PubMed Scopus Google Scholar, M.J. D.J. Shabanowitz J. D.F. for the detection of post-translational and proteins by mass 2005; 4: PubMed Scopus Google Scholar). The SIMAC is to other phosphoproteomics study combined with the protein or or using with different to increase the number of identified phosphorylation sites from complex samples. a of the phosphorylated ions detected by is identified using the To increase the number of identified phosphopeptides, the SIMAC combined with M.J. Shabanowitz J. Hunt D.F. A loss for phosphopeptide analysis by ion mass Chem. 2004; PubMed Scopus Google Scholar) or M.J. D.J. Shabanowitz J. D.F. for the detection of post-translational and proteins by mass 2005; 4: PubMed Scopus Google a coverage of the is for and for the human mesenchymal stem and A. are for S. and the for the of are for to the with