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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.
Larsen et al. (Wed,) studied this question.