Martin R. Larsen

Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented. Reversible phosphorylation of proteins regulates the majority of all cellular processes, e.g. proliferation, differentiation, and apoptosis. A fundamental understanding of these biological processes at the molecular level requires characterization of the phosphorylated proteins. Phosphorylation is often substoichiometric, and an enrichment procedure of phosphorylated peptides derived from phosphorylated proteins is a necessary prerequisite for the characterization of such peptides by modern mass spectrometric methods. We report a highly selective enrichment procedure for phosphorylated peptides based on TiO2microcolumns and peptide loading in 2,5-dihydroxybenzoic acid (DHB). The effect of DHB was a very efficient reduction in the binding of nonphosphorylated peptides to TiO2 while retaining its high binding affinity for phosphorylated peptides. Thus, inclusion of DHB dramatically increased the selectivity of the enrichment of phosphorylated peptides by TiO2. We demonstrated that this new procedure was more selective for binding phosphorylated peptides than IMAC using MALDI mass spectrometry. In addition, we showed that LC-ESI-MSMS was biased toward monophosphorylated peptides, whereas MALDI MS was not. Other substituted aromatic carboxylic acids were also capable of specifically reducing binding of nonphosphorylated peptides, whereas phosphoric acid reduced binding of both phosphorylated and nonphosphorylated peptides. A putative mechanism for this intriguing effect is presented. Phosphorylation is among the most widespread post-translational modifications in nature, and it has been estimated that more than 30% of the proteins in a given mammalian cell at some point during their expression are phosphorylated (1Hubbard M.J. Cohen P. On target with a new mechanism for the regulation of protein phosphorylation.Trends Biochem. Sci. 1993; 18: 172-177Google Scholar). Phosphorylation and dephosphorylation of proteins regulates a large number of biological processes such as signal transduction (2Graves J.D. Krebs E.G. Protein phosphorylation and signal transduction.Pharmacol. Ther. 1999; 82: 111-121Google Scholar), molecular recognition and interaction, and other cellular events. A fundamental understanding of these biological processes at the molecular level thus requires a characterization of the phosphorylated sites in the proteins. It is therefore essential to develop sensitive and selective methods for this task. A wide variety of methods are known for characterization of phosphorylated proteins. The most widely used have been peptide sequencing using Edman degradation combined with 32P labeling. This method is well established and very robust but has several limitations. For example, in Edman degradation the peptides have to be separated before the analysis using liquid chromatography. This decreases the overall sensitivity and increases analysis time, and it is therefore not well suited for analysis of complex samples. Recently a number of MS-based strategies have been developed that are relatively sensitive and in many cases easier to perform than Edman degradation with respect to handling complex mixtures (e.g. Ref. 3McLachlin D.T. Chait B.T. Analysis of phosphorylated proteins and peptides by mass spectrometry.Curr. Opin. Chem. Biol. 2001; 5: 591-602Google Scholar). The increased sensitivity is especially needed for low stoichiometric phosphorylation. However, presently none of these MS-based methods can individually provide a complete characterization of a phosphorylated protein. For the MS-based strategies, it is common that the phosphorylated protein is enzymatically degraded to peptides, which are subsequently analyzed by MS to detect a mass increment of 80 Da per phosphate group. Because sulfonation gives the same mass shift, this strategy is often combined with phosphatase treatment to specifically cleave off the phosphate group from the peptide. This mass shift can be monitored by MS as a loss of 80 Da. This differential peptide mass mapping can be combined with purification of peptides using microcolumns packed with material of increasing hydrophobicity (4Larsen 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 I and dynamin III.Mol. Cell. Proteomics. 2004; 3: 456-465Google Scholar). In MALDI-TOF MS operating in reflector ion mode, the loss of phosphoric acid in the gas phase is often detected from phosphorylated peptides as a poorly resolved peak originating from metastable fragmentation (5Annan R.S. Carr S.A. Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry.Anal. Chem. 1996; 68: 3413-3421Google Scholar). The exact site of phosphorylation can often be localized using tandem MS; however, the loss of phosphoric acid upon CID is frequently observed as the major fragmentation pathway, and this may interfere with the interpretation due to inadequate fragmentation of the peptide backbone. The phosphate group is believed to have an effect on the ionization of phosphorylated peptides in MS, resulting in decreased signal intensity for phosphorylated peptides in the presence of non-phosphorylated peptides (i.e. an ion suppression phenomenon). Matrix additives like ammonium citrate (6Asara J.M. Allison J. Enhanced detection of phosphopeptides in matrix-assisted laser desorption/ionization mass spectrometry using ammonium salts.J. Am. Soc. Mass Spectrom. 1999; 10: 35-44Google Scholar) or phosphoric acid (7Kjellstrom S. Jensen O.N. Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins.Anal. Chem. 2004; 76: 5109-5117Google Scholar) have been shown to enhance the relative abundance of phosphorylated peptides in the presence of non-phosphorylated peptides in MALDI MS. To reduce the suppression of phosphorylated peptides caused by the presence of non-phosphorylated peptides, it is advantageous to prepurify the phosphorylated peptides, especially from complex peptide mixtures. Enrichment of phosphorylated peptides from peptide mixtures using IMAC is widely used (8Neville D.C. Rozanas C.R. Price E.M. Gruis D.B. Verkman A.S. Townsend R.R. Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.Protein Sci. 1997; 6: 2436-2445Google Scholar, 9Posewitz M.C. Tempst P. Immobilized gallium(III) affinity chromatography of phosphopeptides.Anal. Chem. 1999; 71: 2883-2892Google Scholar, 10Figeys 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: 1811-1818Google Scholar, 11Li S.H. Dass C. Iron(III)-immobilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides.Anal. Biochem. 1999; 270: 9-14Google Scholar, 12Ficarro S. Chertihin O. Westbrook V.A. White F. F. P. J. analysis of Evidence of phosphorylation of a protein and during Biol. Chem. Scholar, Jensen O.N. analysis of in vivo phosphorylated proteins by metal ion affinity chromatography and mass Cell. Proteomics. Scholar, S. P. M. Jensen O.N. to the Cell. Proteomics. Scholar). this the phosphorylated peptides are by their affinity to metal like or However, frequently non-phosphorylated peptides, are also by this method J. White analysis by mass spectrometry and its to Scholar). the by has been shown to enhance the of the binding J. White analysis by mass spectrometry and its to Scholar). in the of this is which the sensitivity of this procedure and increases the of the In addition, often a and of and and these the MS analysis and interpretation J. on Mass and for Mass Scholar). this method requires of the from the peptide to the of the and this is known to resulting in decreased sensitivity D. a for Mass Spectrom. 2001; Scholar, O. S. analysis of peptide from for protein in Scholar). by and has also been widely used for affinity purification and of phosphorylated peptides (e.g. Ref. Y. Chait B.T. Enrichment analysis of phosphorylated proteins as a for the 2001; 19: Scholar). However, this strategy from several of and sensitivity and of D.T. Chait B.T. Improved affinity purification strategy for enrichment of phosphopeptides.Anal. Chem. Scholar). Recently a strategy was by M.J. at the level of phosphopeptides from using and Chem. 2004; 76: Scholar) was used as an to IMAC for the selective enrichment of phosphorylated peptides to liquid chromatography tandem MS. used an TiO2 to a and with this analysis of phosphorylated peptides was However, the selectivity of this method was by the detection of several non-phosphorylated peptides that were also by their TiO2 we a new and procedure for using TiO2 microcolumns that the binding selectivity of TiO2 toward phosphorylated peptides, phosphorylated peptide characterization from low level phosphorylated proteins. peptides from proteins were used to and the In addition, the method was with the IMAC was from and material were from were from acid used 2,5-dihydroxybenzoic phosphoric was from The was from for loading were from The was from a were from a TiO2 number from other and were of the and were from protein was in ammonium and with at for peptides originating from a of of peptides originating from of and and to a of peptides originating from a of of the phosphorylated proteins and and and of the non-phosphorylated peptides and TiO2 microcolumns with a of were packed in A of material was of a using an and at the of the The as a to the the that the used for or loading the the TiO2 which of peptides to the The TiO2 were in and an of this on the of the was the by a was used to the as M.R. Roepstorff P. powder as an or to material for and of peptide mixtures to matrix-assisted laser Scholar, J. R. Roepstorff P. purification and based on for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass Mass Spectrom. 1999; Scholar). The of for selective binding of phosphorylated peptides was The procedure was from the method M.J. at the level of phosphopeptides from using and Chem. 2004; 76: Scholar). In the peptide was In procedure peptides were TiO2 in The were with of and the peptides were with of ammonium of the was with of and of matrix in phosphoric on the MALDI was the same as procedure A with the that the peptides were with In procedure the peptides were the TiO2 in and the were with of the DHB in and with of before the peptides were using of In procedure the peptides were the TiO2 in DHB of in The were with of the DHB and of The peptides were using of To the selective of phosphorylated peptides relative to non-phosphorylated peptides a of acid were as TiO2 microcolumns were with peptide The peptides were the TiO2 microcolumns in a of of the acids in with phosphoric and 2,5-dihydroxybenzoic In the of was from the loading the peptides the TiO2 the were with of was with of was on a to MALDI MS For LC-ESI-MSMS analysis the peptides were by material and by and to in microcolumns used for and of peptides were using as in M.R. Roepstorff P. powder as an or to material for and of peptide mixtures to matrix-assisted laser Scholar, J. R. Roepstorff P. purification and based on for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass Mass Spectrom. 1999; Scholar). The from the TiO2 microcolumns were in acid to a of and microcolumns using The were with of The peptides were using of matrix the MALDI IMAC purification of phosphorylated peptides was to C. phosphorylation of the proteins regulates in Biol. Chem. 2004; Scholar) with of metal were in of 30% and in of of this was with the peptide in a of of for with the was a and a was used to the the were with the The peptides were using of and using microcolumns to MALDI MS analysis MALDI MS was using a mass with or a MALDI mass were in reflector Mass spectrometric analysis was using the or the analysis and peptide were using the For analysis of phosphorylated peptides, DHB in phosphoric acid was used as the that inclusion of phosphoric acid in the MALDI matrix increases the relative abundance of peptides (7Kjellstrom S. Jensen O.N. Phosphoric acid as a matrix additive for MALDI MS analysis of phosphopeptides and phosphoproteins.Anal. Chem. 2004; 76: 5109-5117Google Scholar). Because some of the proteins used in this and several peptides the were all using phosphoric acid in the matrix LC-ESI-MSMS analysis was using a mass A The was used for of the peptide to MS detection The peptides were and on a and at by an increasing of an A was for The most in the were and by per The were to a using the and the resulting was the protein using an of the procedure was using peptides originating from (i.e. peptide of and and the are with of and A of the phosphorylated peptides derived from and and their molecular is shown in of the phosphorylated peptide binding selectivity of the TiO2 microcolumns and of the was by the relative of the non-phosphorylated peptides with of the phosphorylated of observed phosphorylated peptides derived by of and and of phosphate was by MALDI tandem peptide signal at a new of the in the to the is in to the This from an The was by MALDI tandem was by MALDI tandem by MALDI tandem MS. of in the was by MALDI tandem was by MALDI tandem The peptide signal at a new of the in the to the is in to the This from an The was by MALDI tandem was by MALDI tandem by MALDI tandem MS. of in the in a new A analysis of a of of by MALDI MS using a in which the peptide is with and DHB matrix phosphoric in detection of a of the phosphorylated peptides with The MALDI MS from the TiO2 enrichment of phosphorylated peptides from peptide using the purification as by M.J. at the level of phosphopeptides from using and Chem. 2004; 76: Scholar) with ammonium is shown in A number of non-phosphorylated peptides were observed with phosphorylated peptides with were observed from the peptides. with ammonium the same was subsequently with of and the MALDI MS analysis of of this very phosphorylated peptides that with a of the phosphorylated peptides, whereas most of the phosphorylated peptides. using not in in the of phosphorylated peptides from the TiO2 In purification for enrichment of phosphorylated peptides using both TiO2 and IMAC acid has been used as the loading The for this in the loading is to that the in the peptides are whereas the of phosphoric acid is and therefore the phosphate group have a at However, a of non-phosphorylated peptides to IMAC or TiO2 J. White analysis by mass spectrometry and its to Scholar, M.J. at the level of phosphopeptides from using and Chem. 2004; 76: Scholar). The of an group an phosphate increases the e.g. the of phosphoric acid decreases to upon (i.e. J. F. of metal ion with phosphate and Biol. Chem. 1996; Scholar). Thus, we that the of the phosphate group also decreases it is to a peptide. which has a of was used in the for the purification of phosphorylated peptides using TiO2. A TiO2 was with peptide in with the phosphorylated peptides were from the TiO2 with of and the MALDI MS analysis of of this in the MALDI shown in the intensity of the phosphorylated peptides increased relative to the non-phosphorylated peptides, a more selective enrichment of phosphorylated peptides was used as loading However, a number of non-phosphorylated peptides were observed using this of phosphorylated peptides from IMAC material using the DHB matrix has been shown to the of some phosphorylated peptides from this material R. the of phosphopeptides on IMAC and their analysis by MALDI Am. Soc. Mass Spectrom. Scholar). Because the binding of phosphorylated peptides to TiO2 is to its ion M.J. at the level of phosphopeptides from using and Chem. 2004; 76: Scholar) and is to the binding observed in IMAC we to the phosphorylated peptides with the DHB matrix The enrichment of phosphorylated peptides from peptide using TiO2 in by in and of the phosphorylated peptides with is shown in from peptide were a new TiO2 as however, with the peptides were the MALDI target using DHB matrix in Phosphoric acid was the and the was subsequently analyzed by MALDI MS non-phosphorylated peptides were detected as by the in the the of some of the phosphorylated peptides. Thus, all phosphorylated peptides were by the TiO2 resin with the DHB matrix from the same with a number of phosphorylated peptides with with the procedure the was with In addition, a low abundance and number of non-phosphorylated peptides were observed in In DHB is to the phosphorylated peptides, whereas the TiO2 and the phosphate group to be and be by However, non-phosphorylated peptides can be from TiO2 by binding of DHB increasing the selective binding of phosphorylated peptides. Because DHB is capable of peptides from the TiO2 was in which peptide was a TiO2 in of a DHB matrix in The was subsequently with of the DHB and of The peptides were with of and of this was with of and of matrix on the MALDI The resulting MALDI peptide mass is shown in a of were detected of which all phosphorylated peptides, and were detected from non-phosphorylated peptides. the of the signal at The from the loading was on the MALDI target and analyzed for the presence of phosphorylated peptides. non-phosphorylated peptides be detected not The very low at and and phosphate from the protein as by MALDI tandem MS not The of DHB in the loading was to have a large effect on the of non-phosphorylated peptides TiO2. A of were using peptide and of DHB in the loading from peptide were TiO2 microcolumns in and DHB The MALDI mass the mass and Da from the with are shown in This that the number of non-phosphorylated peptides decreases with the increasing of In the the peptides were in from non-phosphorylated peptides were observed of as as DHB decreased the abundance of these peptides. A in the of DHB to the of these non-phosphorylated peptides to TiO2 as is by their in the mass the peptides in the are observed at DHB This a suppression of the ionization of peptides in the presence of non-phosphorylated peptides. The using TiO2 microcolumns were with peptides derived from a protein low of a peptide was analyzed by TiO2 microcolumns using the In this a of a phosphorylated peptides in are The number of peptides derived from the proteins by is for and a mass of Da. from peptide were analyzed by MALDI MS using the method phosphorylated peptides be detected with due to the ion suppression effect caused by the non-phosphorylated peptides. A of peptides from peptide was a TiO2 using the procedure by M.J. at the level of phosphopeptides from using and Chem. 2004; 76: Scholar). The peptides were off the using of and the peptides were subsequently using a from which the peptides were the MALDI target using the matrix The resulting MALDI MS peptide mass is shown in the same phosphorylated peptides be however, a of non-phosphorylated peptides was observed in the The was using of acid in the loading The resulting MALDI MS peptide mass is shown in a of non-phosphorylated peptides was observed in the but the relative signal intensity of the phosphorylated peptides was increased with loading in In addition, phosphorylated peptides be peptide in DHB in in the selective purification of phosphorylated peptides with with non-phosphorylated peptides a of phosphorylated peptides was The purification of the using in the loss of at phosphorylated peptides and not to this However, these phosphorylated peptides were detected by MALDI MS purification of the from the by using a graphite (4Larsen 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 I and dynamin III.Mol. Cell. Proteomics. 2004; 3: 456-465Google Scholar) not The abundance of the phosphorylated peptides was increased using the DHB as loading with acid or the same of The same was in all the other in this This a more efficient ionization for phosphorylated peptides in the of non-phosphorylated peptides. The effect of the inclusion of DHB in the loading and procedure for complex mixtures was using peptide from peptide were TiO2 microcolumns of the same in and DHB The resulting MALDI peptide mass from of of the with of is shown in The of DHB caused a high number of non-phosphorylated peptides to to the The number of non-phosphorylated peptides decreased with the increasing of DHB to non-phosphorylated peptides were In addition, the peptides were more detected a of DHB was due to decreased ion suppression This that the is more complex a of DHB is needed to the binding of non-phosphorylated peptides. For very complex a DHB of to a is highly The selective enrichment of phosphorylated peptides using TiO2 microcolumns was with from peptide and were using TiO2 microcolumns and IMAC The were in both cases with and the peptides were using microcolumns and from this the MALDI MS target using of the matrix with DHB from IMAC has been shown to the of phosphorylated peptides. However, in this was used to the binding selectivity of phosphorylated TiO2 and In addition, with DHB matrix the for like liquid chromatography to MS. The resulting MALDI peptide mass from the TiO2 are shown in and the MALDI peptide mass from the IMAC are shown in The to the detected phosphorylated peptides are by in A and D. peptide the purification methods well with respect to number of detected phosphorylated peptides. However, a number of non-phosphorylated peptides were observed in the IMAC increasing and the of the TiO2 method the IMAC method with respect to number of detected phosphorylated peptides and reduction of the number of non-phosphorylated peptides in the (e.g. and This a more selective binding of the phosphorylated peptides on the TiO2 than on the IMAC of the IMAC e.g. by loading in a more the selectivity of the IMAC of peptide was a TiO2 in DHB in and the phosphorylated peptides were by This peptide was with acid and analyzed by The resulting ion were by the and a of phosphorylated peptides were phosphorylated peptides to not In to the phosphorylated peptides, non-phosphorylated peptides were with the with MALDI MS e.g. more than phosphorylated peptides were the LC-ESI-MSMS showed a toward monophosphorylated peptides as several peptides were not detected by the LC-ESI-MSMS in the LC-ESI-MSMS analysis was by S. P. M. Jensen O.N. to the Cell. Proteomics. Scholar) this as observed a of peptides with phosphorylated peptides using A effect has been observed in a number of by group using both MALDI tandem MS and R. O. P. and J. D. The for this is presently not It is from the that the presence of DHB in the loading dramatically the selective of phosphorylated peptides on TiO2. We this effect to a for binding sites on TiO2 non-phosphorylated peptides and DHB The large of DHB thus with non-phosphorylated peptides for to the of whereas phosphorylated peptide binding is To the molecular of DHB that are for this intriguing effect we a number of acid as well as other acids and their effect on the selective of phosphorylated peptides from complex peptide mixtures. from peptide were TiO2 microcolumns in of the phosphoric and The peptides were using and and on microcolumns to MALDI MS The phosphorylated peptide binding selectivity was by the relative of these peptides with of non-phosphorylated peptides. MALDI mass from TiO2 enrichment of phosphorylated peptides using acids in the loading phosphoric and The that DHB was the most efficient acid to of nonphosphorylated peptides while retaining the of TiO2 to phosphorylated peptides. In phosphoric acid was not as as DHB to reduce binding of nonphosphorylated peptides, and it to the of some of the phosphorylated peptides. For example, the relative of the phosphorylated peptides at and were dramatically reduced the loading phosphoric whereas the relative of these peptides were in the of the other acid or acid in the loading very to from whereas carboxylic acid very to that of acid not The in of nonphosphorylated peptides the DHB acid acid acid carboxylic acid phosphoric acid Thus, the substituted aromatic carboxylic acids (i.e. and are than the carboxylic acid (i.e. and for binding of non-phosphorylated peptides to TiO2. In with this have shown that substituted aromatic carboxylic acids acid and to the of whereas carboxylic acids acid and very with TiO2 In analysis of the of aromatic carboxylic acids to and from A Scholar). phosphate to TiO2 with affinity to substituted aromatic carboxylic acids TiO2 from an in 1999; Scholar), but it to be in binding of nonphosphorylated peptides. a high TiO2 binding affinity of the loading is not the for the of phosphorylated peptide binding In this it is to that the binding of phosphate to TiO2 from that of substituted aromatic carboxylic For example, the binding of acid to TiO2 is a In analysis of the of aromatic carboxylic acids to and from A Scholar, S. M. of on the of acid and using TiO2 Chem. Scholar), whereas the of phosphate to the of TiO2 is a complex TiO2 from an in 1999; Scholar) The for an phosphate binding site on TiO2 is thus to from that of an binding site for a substituted carboxylic In this phosphate with phosphorylated peptides for binding sites on whereas DHB other binding sites that to be to by non-phosphorylated peptides. binding sites on TiO2 may from a of the TiO2 but the is also to binding sites on TiO2 by the of TiO2 to a of C. and characterization of among on at 5: Scholar). also to a in binding of non-phosphorylated peptides carboxylic acids (e.g. are more than acids (e.g. In we the selective of phosphorylated peptide binding by DHB to an with non-phosphorylated peptides for binding sites on TiO2. This effect is by the of a of sites on of phosphate and to TiO2 from In analysis of the of aromatic carboxylic acids to and from A and TiO2 from an in 1999; We used DHB to enhance the selective enrichment of phosphorylated peptides by TiO2 This novel in a in the selectivity of purification of phosphorylated peptides from complex mixtures of non-phosphorylated and phosphorylated peptides. In with procedure in of selectivity and sensitivity of phosphorylated peptide In addition, the TiO2 purification was than per and can be used in with high liquid chromatography to or However, the toward monophosphorylated peptides in LC-ESI-MSMS observed in this that both mass spectrometric methods be in the analysis of phosphorylated peptides.

Protein phosphorylation is a key regulator of cellular signaling pathways. It is involved in most cellular events in which the complex interplay between protein kinases and protein phosphatases strictly controls biological processes such as proliferation, differentiation, and apoptosis. Defective or altered signaling pathways often result in abnormalities leading to various diseases, emphasizing the importance of understanding protein phosphorylation. Phosphorylation is a transient modification, and phosphoproteins are often very low abundant. Consequently, phosphoproteome analysis requires highly sensitive and specific strategies. Today, most phosphoproteomic studies are conducted by mass spectrometric strategies in combination with phospho-specific enrichment methods. This review presents an overview of different analytical strategies for the characterization of phosphoproteins. Emphasis will be on the affinity methods utilized specifically for phosphoprotein and phosphopeptide enrichment prior to MS analysis, and on recent applications of these methods in cell biological applications.

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. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar, 9Li S.H. Dass C. Iron(III)-immobilized metal ion affinity chromatography and mass spectrometry for the purification and characterization of synthetic phosphopeptides.Anal. Biochem. 1999; 270: 9-14Crossref PubMed Scopus (112) Google Scholar, 10Neville D.C.A. Rozanas C.R. Price E.M. Gruis D.B. Verkman A.S. Townsend R.R. Evidence for phosphorylation of serine 753 in CFTR using a novel metal-ion affinity resin and matrix-assisted laser desorption mass spectrometry.Protein Sci. 1997; 6: 24362445Google 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, 12Posewitz M.C. Tempst P. 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