Comparative Assessment of Site Assignments in CID and Electron Transfer Dissociation Spectra of Phosphopeptides Discloses Limited Relocation of Phosphate Groups

In large scale mass spectrometry-based phosphoproteomics, a current bottleneck is the unambiguous assignment of the phosphorylation site within the peptide. An additional problem is that it has been reported that under conditions wherein peptide ions are collisionally activated the phosphate group may migrate to a nearby phosphate group acceptor, thus causing ambiguity in site assignment. Here, we generated and analyzed a statistically significant number of phosphopeptides. Starting with a human cell lysate, we obtained via strong cation exchange fractionation nearly pure phosphopeptide pools from trypsin and Lys-N digestions. These pools were subjected to nano-LC-MS using an Orbitrap mass spectrometer that is equipped with both CID and electron transfer dissociation with supplemental activation (ETcaD) functionality. We configured a method to obtain sequentially both ETcaD and CID spectra for each peptide ion. We exploited the resistant nature of ETcaD toward rearrangement of phosphate groups to evaluate whether there is potentially phosphate group relocation occurring during CID. We evaluated a number of peptide and spectral annotation properties and found that for ∼75% of the sequenced phosphopeptides the assigned phosphosite was unmistakably identical for both the ETcaD and CID spectra. For the remaining 25% of the sequenced phosphopeptides, we also did not observe evident signs of relocation, but these peptides exhibited signs of ambiguity in site localization, predominantly induced by factors such as poor fragmentation, sequences causing inefficient fragmentation, and generally poor spectrum quality. Our data let us derive the conclusion that both for trypsin- and Lys-N-generated peptides there is little relocation of phosphate groups occurring during CID. In large scale mass spectrometry-based phosphoproteomics, a current bottleneck is the unambiguous assignment of the phosphorylation site within the peptide. An additional problem is that it has been reported that under conditions wherein peptide ions are collisionally activated the phosphate group may migrate to a nearby phosphate group acceptor, thus causing ambiguity in site assignment. Here, we generated and analyzed a statistically significant number of phosphopeptides. Starting with a human cell lysate, we obtained via strong cation exchange fractionation nearly pure phosphopeptide pools from trypsin and Lys-N digestions. These pools were subjected to nano-LC-MS using an Orbitrap mass spectrometer that is equipped with both CID and electron transfer dissociation with supplemental activation (ETcaD) functionality. We configured a method to obtain sequentially both ETcaD and CID spectra for each peptide ion. We exploited the resistant nature of ETcaD toward rearrangement of phosphate groups to evaluate whether there is potentially phosphate group relocation occurring during CID. We evaluated a number of peptide and spectral annotation properties and found that for ∼75% of the sequenced phosphopeptides the assigned phosphosite was unmistakably identical for both the ETcaD and CID spectra. For the remaining 25% of the sequenced phosphopeptides, we also did not observe evident signs of relocation, but these peptides exhibited signs of ambiguity in site localization, predominantly induced by factors such as poor fragmentation, sequences causing inefficient fragmentation, and generally poor spectrum quality. Our data let us derive the conclusion that both for trypsin- and Lys-N-generated peptides there is little relocation of phosphate groups occurring during CID. Signaling events, many of which are regulated by dynamic protein phosphorylation, form the basis of inter- and intracellular communication. Cellular activation, for instance, by a specific activation of a membrane receptor often induces changes in phosphorylation in that receptor but also in hundreds of other proteins connected to that receptor in downstream pathways. Obtaining in-depth phosphopeptide profiles consisting of significant numbers of sites on thousands of proteins has now become accessible to the specialized proteomics community (1Reinders J. Sickmann A. State-of-the-art in phosphoproteomics.Proteomics. 2005; 5: 4052-4061Crossref PubMed Scopus (299) Google Scholar). A number of key technologies and strategies have led to this breakthrough. Peptide complexity reduction and/or phosphopeptide enrichment can be considered to be one of the most improved aspects of these phosphoproteomics profiling methodologies. A classic strategy for a phosphoproteome screen is to perform a fractionation of the sample followed by enrichment using a reagent with affinity for the phosphate group (2Gruhler 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 (693) Google Scholar). Strong cation exchange (SCX) 1The abbreviations used are:SCXstrong cation exchangeETcaDelectron transfer dissociation with supplemental activationETDelectron transfer dissociationPTMpost-translational modification. 1The abbreviations used are:SCXstrong cation exchangeETcaDelectron transfer dissociation with supplemental activationETDelectron transfer dissociationPTMpost-translational modification.- (3Motoyama A. Xu T. Ruse C.I. Wohlschlegel J.A. Yates 3rd, J.R. Anion and cation mixed-bed ion exchange for enhanced multidimensional separations of peptides and phosphopeptides.Anal. Chem. 2007; 79: 3623-3634Crossref PubMed Scopus (140) Google Scholar, 4Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villén J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12130-12135Crossref PubMed Scopus (1227) Google Scholar, 5Gauci S. Helbig A.O. Slijper M. Krijgsveld J. Heck A.J. Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.Anal. Chem. 2009; 81: 4493-4501Crossref PubMed Scopus (225) Google Scholar) and SDS-PAGE (“GeLCMS”) (6Everley P.A. Bakalarski C.E. Elias J.E. Waghorne C.G. Beausoleil S.A. Gerber S.A. Faherty B.K. Zetter B.R. Gygi S.P. Enhanced analysis of metastatic prostate cancer using stable isotopes and high mass accuracy instrumentation.J. Proteome Res. 2006; 5: 1224-1231Crossref PubMed Scopus (65) Google Scholar, 7Elortza F. Nühse T.S. Foster L.J. Stensballe A. Peck S.C. Jensen O.N. Proteomic analysis of glycosylphosphatidylinositol-anchored membrane proteins.Mol. Cell. Proteomics. 2003; 2: 1261-1270Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar)-based fractionation are the most common, although hydrophilic interaction chromatography (8Gilar M. Olivova P. Daly A.E. Gebler J.C. Orthogonality of separation in two-dimensional liquid chromatography.Anal. Chem. 2005; 77: 6426-6434Crossref PubMed Scopus (653) Google Scholar, 9Boersema P.J. Taouatas N. Altelaar A.F. Gouw J.W. Ross P.L. Pappin D.J. Heck A.J. Mohammed S. Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.Mol. Cell. Proteomics. 2009; 8: 650-660Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) and isoelectric focusing (IEF) (10Cargile B.J. Talley D.L. Stephenson Jr., J.L. Immobilized pH gradients as a first dimension in shotgun proteomics and analysis of the accuracy of pI predictability of peptides.Electrophoresis. 2004; 25: 936-945Crossref PubMed Scopus (135) Google Scholar, 11Krijgsveld J. Gauci S. Dormeyer W. Heck A.J. In-gel isoelectric focusing of peptides as a tool for improved protein identification.J. Proteome Res. 2006; 5: 1721-1730Crossref PubMed Scopus (93) Google Scholar) provide attractive alternatives. In the case of SCX and hydrophilic interaction chromatography, one can obtain nearly “uncontaminated” phosphopeptide enrichment when appropriate conditions and materials are chosen (5Gauci S. Helbig A.O. Slijper M. Krijgsveld J. Heck A.J. Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.Anal. Chem. 2009; 81: 4493-4501Crossref PubMed Scopus (225) Google Scholar, 12McNulty D.E. Annan R.S. Hydrophilic interaction chromatography reduces the complexity of the phosphoproteome and improves global phosphopeptide isolation and detection.Mol. Cell. Proteomics. 2008; 7: 971-980Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). The classic choice for the second step has been immobilized affinity chromatography (IMAC) (13Stensballe A. Andersen S. Jensen O.N. Characterization of phosphoproteins from electrophoretic gels by nanoscale Fe(III) affinity chromatography with off-line mass spectrometry analysis.Proteomics. 2001; 1: 207-222Crossref PubMed Scopus (361) Google Scholar), which can have its selectivity improved even further through the use of additives (14Stensballe A. Jensen O.N. Phosphoric acid the of Fe(III) affinity chromatography and mass spectrometry for and sequencing of 2004; 1721-1730Crossref PubMed Scopus Google Scholar) of the peptide D.J. Ross J. analysis by mass spectrometry and its to PubMed Scopus Google Scholar). In one of the most technologies used for enrichment of phosphopeptides has been Heck A.J. isolation the of phosphopeptides from using and Chem. 2004; PubMed Scopus Google Scholar) often with additives Jensen O.N. P. enrichment of peptides from peptide using Cell. Proteomics. 2005; 4: Full Text Full Text PDF PubMed Scopus Google Scholar, N. T. A. M. enrichment by chromatography for in proteomics Cell. Proteomics. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar), and it has been applied in an off-line form on as of the nano-LC-MS step Mohammed S. Gouw J.W. Heck A.J. and on phosphoproteomics applied to phosphorylation in Proteome Res. 2008; 7: PubMed Scopus Google Scholar, Yates 3rd, J.R. phosphopeptide enrichment for multidimensional liquid chromatography to mass Chem. 2007; 79: PubMed Scopus Google Scholar, S. S. Heck A.J. enrichment and analysis of phosphopeptides from Proteome Res. 2008; 7: PubMed Scopus Google Scholar). such as of enrichment with for improved mass characterization of protein Proteome Res. 2008; 7: PubMed Scopus Google Scholar) have to be In the specific case of the peptide have been to be A. Ross P.L. Pappin D.J. J. mass spectrometry of phosphorylation sites in the receptor signaling dynamic Cell. Proteomics. 2005; 4: Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. J. J. Li M. J. J. J. Bakalarski C.E. J. Li D. Gygi S.P. J. of signaling in 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, P.J. S. J. J. Heck A.J. and profiling of phosphorylation using a of phosphopeptide and stable Cell. Proteomics. Full Text Full Text PDF PubMed Scopus Google Scholar). The in of phosphoproteomics data has the community to on high analysis of the mass data to that has is the in the spectra of phosphopeptides as obtained by CID Annan R.S. S.A. of phosphopeptides in by 4: PubMed Scopus Google Scholar, J. of phosphopeptides in an ion mass PubMed Scopus Google Scholar). The is for ion dissociation of the used and the to use a ion P.J. Mohammed S. Heck A.J. and analysis by mass 2009; PubMed Scopus Google Scholar). The can often ion causing of the peptide to become even the use of ion the most of phosphoproteome there have been to this specific have been such as (2Gruhler 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 (693) Google Scholar, 4Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villén J. Li J. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12130-12135Crossref PubMed Scopus (1227) Google Scholar, Mohammed S. M. Heck A.J. Slijper M. Quantitative phosphoproteomics of signaling in Cell. Proteomics. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar) and J. Schwartz J.C. A activation method for improved phosphopeptide analysis by ion mass Chem. 2004; PubMed Scopus Google Scholar), which the of these the of the ion further to which form the for peptide there has also been a of in dissociation in the form of the activated dissociation cell J.V. Schwartz J.C. J. P. D. M. M. A. Mann M. S. A ion with high sequencing Cell. Proteomics. 2009; 8: Full Text Full Text PDF PubMed Scopus Google Scholar), which of the activation step and in P.J. Mohammed S. Heck A.J. and analysis by mass 2009; PubMed Scopus Google Scholar). transfer dissociation and electron dissociation have also the phosphate group during and activation N. S. A. profiling of phosphopeptides using electron transfer dissociation mass Natl. Acad. Sci. U.S.A. 2007; PubMed Scopus Google Scholar, A. J.E. D.L. J. D.J. of phosphorylation sites on proteins from by electron transfer dissociation mass Natl. Acad. Sci. U.S.A. 2007; PubMed Scopus Google Scholar, S. A. P. Heck A.J. proteomics of yeast and and phosphorylation Chem. 2008; PubMed Scopus Google Scholar, D.L. scale of protein phosphorylation by use of electron dissociation mass Cell. Proteomics. 2009; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). phosphopeptides the that there is one for the site of phosphorylation within the peptide. The in CID can the assignment of the in such a has been to in the of phosphopeptide there are a number of that site assignment J.V. F. Mortensen P. Mann M. in and phosphorylation in signaling 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, S.A. Villén J. Gerber S.A. J. Gygi S.P. A for protein phosphorylation analysis and site 2006; PubMed Scopus Google Scholar), that can T. M.A. an phosphorylation site assignment tool for Proteome Res. 2008; 7: PubMed Scopus Google Scholar) and electron data D.L. M. site of from mass Proteome Res. 2009; 8: PubMed Scopus Google Scholar). and the of and Proteome Res. 2008; 7: PubMed Scopus Google Scholar) phosphopeptide from a of that the phosphate group in CID for the through an and not through the as an step and group further using phosphopeptides to the phosphate the of rearrangement the phosphate group during CID The rearrangement the form of a relocation of the phosphate group to an group within the peptide. when a annotation of the one be to observe for the of the phosphate to as phosphate group of rearrangement and on protein phosphorylation site assignment using induced and Chem. 2008; PubMed Scopus Google Scholar). The that the is when there are and a for peptide PubMed Scopus Google Scholar) and that the be when are used for CID such as in an ion These have even the accuracy for phosphosite strong cation exchange electron transfer dissociation with supplemental activation electron transfer dissociation modification. strong cation exchange electron transfer dissociation with supplemental activation electron transfer dissociation modification. Here, we the of rearrangement the phosphate group during CID by a large scale phosphoproteomics data consisting of phosphopeptides sequentially and by both CID and electron transfer dissociation with supplemental activation (ETcaD) using an Orbitrap We the ETcaD spectrum to the phosphate group and it with the by the CID of human cell in was with and with For the trypsin of was with in followed by with trypsin to For the Lys-N of was with Lys-N in followed by with Lys-N to SCX was as (5Gauci S. Helbig A.O. Slijper M. Krijgsveld J. Heck A.J. Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.Anal. Chem. 2009; 81: 4493-4501Crossref PubMed Scopus (225) Google Scholar, N. Altelaar A.F. Helbig A.O. Mohammed S. Heck A.J. Strong cation fractionation of Lys-N-generated peptides the analysis of Cell. Proteomics. 2009; 8: Full Text Full Text PDF PubMed Scopus Google Scholar). In peptides from each were using an The applied was using pH as peptides were from the with pH a A for the of peptide was using a as A pH to pH to and to the of was The was for with high and with A for The applied during the SCX was were in for of the peptides were in SCX were analyzed on a Orbitrap An was equipped with a in and a in was acid in for and was with a from to acid in in in followed by a of in in a of in in and for The was from to was using a to The Orbitrap was in the to and spectra were from to in the Orbitrap with a of to a of in the ion The most ions a of were in the ion using CID a of and ETcaD a of The ETcaD reagent was to and the was to data by the mass a SCX CID ETcaD data were generated using Proteome with a of and the for the ETcaD with with and of from with within a of of the SCX for and peptides were trypsin trypsin Lys-N and Lys-N was used to these an from the human of the The were used for mass ion to as a and as a modification. The was as trypsin and the ion was as Peptide were a of and to an ion of first and peptides were considered for The for this peptide was as the of peptides that to a and is and for data was on the trypsin CID and Lys-N CID data using P. Gouw J.W. Olsen J.V. J. J. Foster L.J. Heck A.J. Andersen Mann M. an for mass spectrometry-based Proteome Res. PubMed Scopus Google Scholar). data was with and data of spectra are in the using the the of phosphate group rearrangement on the of a phosphorylation we in a sequentially both CID and D.L. M. Schwartz J.C. J.E. activation method for dissociation of peptide Chem. 2007; 79: PubMed Scopus Google Scholar) on The was using the proteomics The phosphopeptide was generated by of a of human with Lys-N N. Heck A.J. Mohammed S. Straightforward sequencing of peptides using a Lys-N 2008; 5: PubMed Scopus Google Scholar) trypsin followed by which was for the enrichment of peptides as (5Gauci S. Helbig A.O. Slijper M. Krijgsveld J. Heck A.J. Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.Anal. Chem. 2009; 81: 4493-4501Crossref PubMed Scopus (225) Google Scholar). SCX were further by and analyzed on an Orbitrap that was configured to peptide with both CID and The for the CID ETcaD a was with the of a which is the peptide for by ETcaD was considered identical to the for by CID. CID ETcaD were generated for both the Lys-N and trypsin and with the peptide obtained from CID and ETcaD were in a Lys-N data and a trypsin data In the trypsin data peptides were by and peptides were by these peptides were with CID and ETcaD ion spectra. For these for which from complementary is peptides been assigned identical phosphate group by further site of the The remaining peptides been assigned phosphate group obtain a of the of the phosphate group when from the CID the ETcaD spectrum the site we also evaluated the phosphate group by as in P. Gouw J.W. Olsen J.V. J. J. Foster L.J. Heck A.J. Andersen Mann M. an for mass spectrometry-based Proteome Res. PubMed Scopus Google Scholar), which is on an that the most ions in an the peptides with peptides been assigned identical phosphate group and peptides been assigned phosphate group The remaining peptides not be by the the of the phosphate group in the CID spectrum not be which was by phosphate group with nearly In this was phosphate group the CID spectra did not group of peptides with phosphate group potentially spectra with ions of phosphate group this is in The Lys-N data a to that of the trypsin data peptides were by and peptides were by The is by peptides which were by both CID and For of these peptides an identical of the phosphate group been from both the CID and ETcaD for peptides the site was for the trypsin data the CID spectra were with to the of the phosphate in the the of been assigned identical phosphate group and phosphate group The remaining peptides not be phosphate group in the CID these peptides as to the trypsin data the group of phosphopeptides phosphate group is potentially most In both data the of the phosphate group was identical for most peptides that been from CID and ETcaD spectra. this was for of peptides with complementary sequencing the phosphate group was from the and for the from the CID spectrum was by rearrangement of phosphate groups is to during CID in the ion but not during CID spectra can potentially ions of these have been both in of number of CID spectra and in of of the in the CID we have to many peptides with phosphate group In the the CID phosphate group was evaluated by we have a of peptides with phosphate group in CID. we that rearrangement it did not the of the phosphate group in the of identical phosphate group were CID and ETcaD spectra. of this rearrangement have during CID with the of the phosphate group by the by is that the group of peptides for which identical phosphate group were obtained CID spectra with of phosphate group but these have been of such that did not the the phosphate group within the groups of peptides for which phosphate group were the ions to phosphate relocation have been in the CID spectra such that the phosphate group was not reported by the In the case of phosphate group the not have been to the and the phosphate group to rearrangement in peptide groups and it have the CID spectra but not the ETcaD spectra has J.E. J. Peptide and protein analysis by electron transfer dissociation mass Natl. Acad. Sci. U.S.A. 2004; 101: PubMed Scopus Google Scholar). the of a peptide with identical acid but phosphate group have for a peptide when from the CID spectrum but not when from the ETcaD in is in the ion of a which is the of the ion of the most peptide and the ion of the peptide We used this ion as a to the of a phosphate group in CID and ETcaD P.J. Mohammed S. Heck A.J. and analysis by mass 2009; PubMed Scopus Google Scholar). For the ion we also used peptide with a the the acid was identical to the peptide In this as the peptides sequences for which the ion is are identical in acid and in the of the phosphate the ion is to the and of the ions that can the phosphorylation In the trypsin data for of the peptides for which complementary sequencing was and which be by a ion be for both CID and The remaining of the peptides did not have a peptide in In the Lys-N data for of a ion be for both CID and the remaining of the peptides ion in In both data many peptides a ion a and rearrangement In the trypsin data the of the ion for CID the ion for to the peptide groups that were generated by that peptides are the ion for which identical phosphate group been the for which phosphate group been a toward a ion in ETcaD with a ion in CID peptides with phosphate group a toward a ion in ETcaD The Lys-N data in groups of peptides that is the of the were for peptides for which phosphate group been found although in the trypsin data on the analysis of the ion of peptides with phosphate group we that these peptides were as such the CID and ETcaD spectra did not provide to phosphorylation The ion of the group of peptides with phosphate group a In both groups and the of phosphate group as by the ion was not to the spectral as by the ion the CID and ETcaD ion of peptides in these groups were the and most a was also when the CID and ETcaD ion were for the peptide a method that that peptide ion generally with peptide even spectral not S. A. P. Heck A.J. proteomics of yeast and and phosphorylation Chem. 2008; PubMed Scopus Google Scholar). when spectra from both peptide groups and we found that many peptides a CID a ETcaD ion CID ETcaD one of the of spectra was We also that many peptides phosphate group other properties that the of the phosphate group in the CID the ETcaD spectra. of the most properties is the number of and the number of the of the phosphate group in CID In both data we that peptides with phosphate group are toward numbers of that the assignment of the phosphate group in ETcaD of the is the of to the of the peptide as ions are to be in the ion M. of electron transfer dissociation mass Cell. Proteomics. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar, A. of induced dissociation and electron transfer Chem. 2008; PubMed Scopus Google Scholar, P.J. N. Heck A.J. Mohammed S. of on peptide electron transfer induced Chem. 2009; 81: PubMed Scopus Google Scholar). when CID and ETcaD spectra of peptides with phosphate group ions that have phosphate group rearrangement be In for peptide in these one of the factors the unambiguous even of the phosphate group in the CID ETcaD spectrum in a ion of phosphate group rearrangement have been it was not from for peptides a of this may not be the case for of peptide. We for phosphorylation site to peptides one phosphate to such peptides is by the SCX which the peptide pools (5Gauci S. Helbig A.O. Slijper M. Krijgsveld J. Heck A.J. Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.Anal. Chem. 2009; 81: 4493-4501Crossref PubMed Scopus (225) Google Scholar). peptides to the of the SCX of the which were subjected to the analysis In this trypsin data and supplemental peptides were by and were by that peptide spectra are in in the form of a these peptides were with CID and ETcaD from ion spectra. The spectra of peptides subjected to ion CID are by ETcaD the peptides are often as P.J. Taouatas N. Altelaar A.F. Gouw J.W. Ross P.L. Pappin D.J. Heck A.J. Mohammed S. Straightforward and de novo peptide sequencing by MALDI-MS/MS using a Lys-N metalloendopeptidase.Mol. Cell. Proteomics. 2009; 8: 650-660Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), the poor peptides of the been assigned identical phosphorylation sites by further site that most spectra and phosphorylation site were poor to the generally of was also in many peptides ion In with the for the for phosphate group rearrangement be found in this of conclusion Our SCX has been refined to phosphopeptides with a of and in which the of peptides (5Gauci S. Helbig A.O. Slijper M. Krijgsveld J. Heck A.J. Mohammed S. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach.Anal. Chem. 2009; 81: 4493-4501Crossref PubMed Scopus (225) Google Scholar). that peptides in the have a of for to the of the peptides within data and a for peptide PubMed Scopus Google Scholar). are in with and of rearrangement and on protein phosphorylation site assignment using induced and Chem. 2008; PubMed Scopus Google Scholar) that ions phosphate group rearrangement are for peptides in a for peptides in a even In we have the of phosphate group rearrangement under conditions used in a proteomics phosphate group from CID and ETcaD we in both data that for of phosphopeptides for which complementary sequencing of phosphate group were of the remaining also for significant phosphate group The spectrum of these was generally to factors such as poor and sequences causing inefficient although we that phosphate group rearrangement have it did not the of the in large data of trypsin- and Lys-N-generated with