Abstract

The mass spectrometric analysis of protein phosphorylation is still far from being routine, and the outcomes thereof are often unsatisfying. Apart from the inherent problem of substoichiometric phosphorylation, three arguments as to why phosphorylation analysis is so problematic are often quoted, including (a) increased hydrophilicity of the phosphopeptide with a concomitant loss during the loading onto reversed-phase columns, (b) selective suppression of the ionization of phosphopeptides in the presence of unmodified peptides, and (c) lower ionization/detection efficiencies of phosphopeptides as compared with their unmodified cognates. Here we present the results of a study investigating the validity of these three arguments when using electrospray ionization mass spectrometry. We utilized a set of synthetic peptide/phosphopeptide pairs that were quantitated by amino acid analysis. Under the applied conditions none of the experiments performed supports the notions of (a) generally increased risks of losing phosphopeptides during the loading onto the reversed-phase column because of decreased retention and (b) the selective ionization suppression of phosphopeptides. The issue of ionization/detection efficiencies of phosphopeptides versus their unphosphorylated cognates proved to be less straightforward when using electrospray ionization because no evidence for decreased ionization/detection efficiencies for phosphopeptides could be found. The mass spectrometric analysis of protein phosphorylation is still far from being routine, and the outcomes thereof are often unsatisfying. Apart from the inherent problem of substoichiometric phosphorylation, three arguments as to why phosphorylation analysis is so problematic are often quoted, including (a) increased hydrophilicity of the phosphopeptide with a concomitant loss during the loading onto reversed-phase columns, (b) selective suppression of the ionization of phosphopeptides in the presence of unmodified peptides, and (c) lower ionization/detection efficiencies of phosphopeptides as compared with their unmodified cognates. Here we present the results of a study investigating the validity of these three arguments when using electrospray ionization mass spectrometry. We utilized a set of synthetic peptide/phosphopeptide pairs that were quantitated by amino acid analysis. Under the applied conditions none of the experiments performed supports the notions of (a) generally increased risks of losing phosphopeptides during the loading onto the reversed-phase column because of decreased retention and (b) the selective ionization suppression of phosphopeptides. The issue of ionization/detection efficiencies of phosphopeptides versus their unphosphorylated cognates proved to be less straightforward when using electrospray ionization because no evidence for decreased ionization/detection efficiencies for phosphopeptides could be found. Protein phosphorylation is one of the most prevalent intracellular protein modifications that is of pivotal importance in numerous cellular processes including cell differentiation, proliferation, and migration. It is estimated that ∼30% of all proteins in a cell are phosphorylated at any given time. However, this number is in stark contrast to the actual number of phosphorylation sites found so far. For instance, the Phospho.ELM database (phospho.elm.eu.org) currently lists 1703 experimentally verified phosphorylation sites for 556 different proteins derived from eukaryotes, the human protein reference database (www.hprd.org) lists 3652 reported phosphorylation sites on 1240 human proteins, and PhosphoSite (www.phosphosite.org) lists 6084 non-redundant phosphorylation sites on 2430 human and mouse proteins. However, a recent study by Beausoleil et al. (1Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villen 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: 12130-12135Google Scholar) gave some idea about the expected number of phosphorylation in the cell when the authors performed a large scale phosphoproteomics study on HeLa cell nuclei. Although their method was biased against basic phosphopeptides where His, Lys-Pro, and/or Arg-Pro residues are in close proximity to the phosphoamino acid residue, more than 2000 phosphorylation sites on 967 nuclear proteins were found. Considering the fact that protein phosphorylation analysis is of major interest in numerous laboratories around the world it is surprising that more information about protein phosphorylation sites has not been gathered since the discovery of protein phosphorylation. This raises the question as to why it is still such a challenge to perform unbiased protein phosphorylation analysis. One inherent reason is the generally low phosphorylation stoichiometry of most of the proteins such that phosphopeptides are grossly underrepresented in the generated complex peptide mixtures (see below). In addition, numerous reasons can be listed for not being able to identify and localize a phosphorylation site in a given protein, which affects all stages of phosphorylation analysis, i.e. sample preparation, analysis, and data interpretation. Examples for problems during the sample preparation include e.g. omission of phosphatase inhibitor, overestimation of the degree of phosphorylation because of the lack of information about the phosphorylation stoichiometry when using phosphospecific antibodies, and/or underestimation of the heterogeneity of the phosphorylation due to nonspecific 32P-labeling. However, even if it was ensured that a particular protein sample is phosphorylated by using other methods such as radiolabeling it is often the case that the mass spectrometric analysis does not provide any information about the sites of phosphorylation. Three main reasons are commonly used to explain as to why the phosphopeptides were missed during the mass spectrometric analysis: (i) increased hydrophilicity and hence reduced retention of phosphopeptides on reversed-phase materials, (ii) selective suppression of their ionization/detection efficiencies in the presence of large amounts of unphosphorylated peptides, and (iii) lower detection efficiencies of phosphopeptides as compared with their unphosphorylated cognates. In this study we tried to scrutinize these different arguments often brought forward with the aim to draw the attention to the most prevalent problem(s) of mass spectrometric phosphorylation analysis and to improve the analysis where it is most fruitful/promising. All solvents used were of HPLC-grade quality from Burdick and Jackson (VWR International). All chemicals were purchased from Sigma unless otherwise noted. The synthetic peptides were HPLC-purified and quantitated in duplicate by amino acid analysis. Stock solutions were prepared using freshly calibrated pipettes. The phosphopeptide and its unphosphorylated counterpart were mixed in at least two different defined ratios (final concentrations 30–150 nm). To avoid carry-over problems during the LC/MS analyses, the solutions of two different peptide pairs were analyzed in an alternating fashion. The standard protein digest was prepared by digesting 50 μm protein solution overnight at 37 °C using sequencing grade trypsin (w/w 1:10, Roche Biochemicals). All electrospray ionization experiments were performed using a QSTAR XL hybrid mass spectrometer (AB/MDS Sciex) hyphenated with microscale capillary reversed-phase HPLC (Famos autosampler (LC Packings), Agilent 1100 HPLC pump (Agilent)). The columns were packed in-house using Magic C18 (5 μm, 200 Å, Michrom BioResources) beads. The buffer compositions are as follows: buffer A: 2.5% acetonitrile, 0.2% formic acid; buffer B: 2.5% H2O, 0.2% formic acid. For the quantitation experiments a 5-min gradient was used with mass spectra being acquired every 0.15 s. Data analysis and quantitation was done using the Analyst software package provided by Applied Biosystems/MDS Sciex. “Phosphopeptides often are lost during the loading onto the reversed-phase columns because the addition of anionic/acidic phosphate groups increases hydrophilicity resulting in reduced retention.” This is one reason often used to explain why a phosphorylation analysis using LC/MS failed. To test this we used a set of peptide/phosphopeptide pairs that varied in length from 7 to 17 amino acids, resembling the size of peptides commonly observed in tryptic digests. Cysteine and methionine-containing peptides were not included in this study to avoid problems associated with partial oxidation. All peptide pairs were analyzed using water/acetonitrile/0.2% formic acid buffers as mobile phases and Magic C18 as stationary phase; Magic C18 is a reversed-phase material commonly used in proteomics applications. Interestingly, despite the common belief that phosphopeptides are more hydrophilic than their unphosphorylated cognate, all singly phosphorylated peptides tested eluted off the reversed-phase column after the unmodified complement irrespective of the number of basic amino acid residues (His, Lys, Arg; Table I). One example is presented in Fig. 1A; it shows the selected ion chromatograms (XICs) 1The abbreviation used is: XIC, selected ion chromatogram. of RNYSVGS and RNYpSVGS Table I, Peptide species 2), the shortest peptide/phosphopeptide pair investigated. Although there is a considerable overlap in the elution profiles, the phosphopeptide clearly elutes after the unphosphorylated cognate. Of the three doubly phosphorylated peptides in this test set, two phosphopeptides eluted significantly later than the singly phosphorylated or unphosphorylated peptide from the reversed-phase column; one example is shown in Fig. 1B. The third doubly phosphorylated peptide with the sequence DQAVpTEpYVATR Table I, Peptide species 27) was the only peptide whose modified form eluted before its unmodified complement (Fig. 1C). It was noted that this particular peptide was the only phosphopeptide in our sample set in which the number of phospho moieties exceeded the number of basic amino acid residues. This led us to hypothesize that phosphorylation does increase the hydrophilicity of peptides; however, if the peptide contains basic amino residues that are positively charged under standard LC(MS) conditions, the increase of hydrophilicity can be overcompensated by charge neutralization, i.e. reduction of the net charge, thereby reducing the overall hydrophilicity.Table ITest peptidesPeptide speciesPeptide sequenceZΔRTIon./detec. efficiency ratiomin1LLLRLpSENSG2++ 0.11.4 (0.13)2RNYpSVGS2++ 1 indicates that the unphosphorylated peptide has a better ionization/detection efficiency, whereas a ratio 2 upon phosphorylation. This reflects the lack of general understanding of what determines the absolute ionization/detection efficiencies of peptides. The ionization/detection efficiency ratio unphosphorylated peptide versus phosphopeptide increases with increasing the charge state. The extent of increase, however, varies significantly. The observed increases varied from 11% (SRARIG(pS)DPLAYEPK: 2+ → 3+ Table I, Peptide species 21 and 22)) to about 800% (KTQA(pS)QGTLQTR: 2+ → 3+ Table I, Peptide species 8 and 9)). One example is presented in Fig. 4, which shows charge states of 2+ to 4+ of the peptide GLGTRTG(pS)NLDRDKL Table I, Peptide species 14–16; please note that the peptide/phosphopeptide were combined in a 2:1 mixture). A clear decrease of the phosphopeptide ion signal relative to the unphosphorylated form is apparent with increasing charge state. Although the number of doubly phosphorylated peptides investigated is too small to draw any general conclusions about their ionization/detection efficiencies as compared with their unphosphorylated cognates, it is still interesting to note that all three test peptides show relative ionization/detection efficiencies similar to those of the singly phosphorylated species. Even in the one case where the number of phosphorylation sites is larger than the number of basic amino acid residues (e.g. DQAV(pT)E(pY)VATR Table I, Peptide species 27)), the ionization/detection efficiency ratio of peptide versus phosphopeptides is only 2.0. It was observed that for the three highly basic peptides, the most highly charged species of the unmodified cognate had ionization/detection efficiencies 2 to 3 orders of magnitude better than the phosphorylated complement (peptide species 16, 20, and 23). One example is given in Fig. 4. The triply charged phosphopeptide GLGTRTGpSNLDRDKL Table I, Peptide species 16) is more efficiently ionized and detected than the unmodified cognate GLGTRTGSNLDRDKL. However, the quadruply charged unmodified species shows a vastly better ionization/detection efficiency as compared with its phosphorylated form, which is almost completely absent. A significant fraction of the molecules of these “exceptional” species show this very high charge state because of the overall proton affinity of the peptides. As soon as the proton affinity is reduced by e.g. phosphorylation, harboring four protons becomes energetically unfavorable, and the ion signal intensity of this charge state becomes negligible. The rather large standard deviations in these exceptional cases result from the limited dynamic range of the instrument used for the study. The findings in this part of the study clearly show a general statement, stating that phosphopeptides show lower ionization/detection efficiencies than their unphosphorylated cognates, does not apply to electrospray ionization mass spectrometry as the majority of the phosphopeptides tested actually show better ionization/detection efficiencies than their unphosphorylated cognates. Similar ionization/detection efficiencies were also found for tryptic-like peptides, i.e. those with only one basic amino acid residue. This finding is supported by other studies from our own and other groups, which determined similar ionization/detection efficiencies for truly tryptic peptide/phosphopeptide pairs, i.e. peptides that were generated by tryptic digestion of proteins (4Steen H. Jebanathirajah J.A. Springer M. Kirschner M.W. Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3948-3953Google Scholar, 5Tsay Y.G. Wang Y.H. Chiu C.M. Shen B.J. Lee S.C. A strategy for identification and quantitation of phosphopeptides by liquid chromatography/tandem mass spectrometry.Anal. Biochem. 2000; 287: 55-64Google Scholar, 6Hegeman A.D. Harms A.C. Sussman M.R. Bunner A.E. Harper J.F. An isotope labeling strategy for quantifying the degree of phosphorylation at multiple sites in proteins.J. Am. Soc. Mass Spectrom. 2004; 15: 647-653Google Scholar); all these studies report similar ionization/detection efficiencies for tryptic peptides and their unphosphorylated cognates. This raises the questions (or highlights the lack of knowledge about) what determines the ionization/detection efficiencies of peptides. Some preliminary studies have been published investigating the effect of different physicochemical properties of peptides on the ionization/detection efficiencies during electrospray ionization. These studies highlighted a positive correlation between the hydrophobicities and the ionization/detection efficiencies of peptides as well as between pI values and ionization/detection efficiencies (e.g. Refs. 7Cech N.B. Krone J.R. Enke C.G. Predicting electrospray response from chromatographic retention time.Anal. Chem. 2001; 73: 208-213Google Scholar, 8Nielsen M.L. Savitski M.M. Kjeldsen F. Zubarev R.A. Physicochemical properties determining the detectionprobability of tryptic peptides in Fourier transform mass spectrometry. A correlation study.Anal. Chem. 2004; 76: 5872-5877Google Scholar, 9Pan P. Gunawardena H.P. Xia Y. McLuckey S.A. Nanoelectrospray ionization of protein mixtures: solution pH and protein pI.Anal. Chem. 2004; 76: 1165-1174Google Scholar). However, when following their line of argument that the phosphorylated form of a particular peptide should have lower ionization/detection efficiencies than their unmodified cognate because both the hydrophobicity and pI are thought to decrease with the covalent attachment of a phosphate group. Although the pI of a peptide is certainly reduced upon phosphorylation, the effect of phosphorylation on the hydrophobicity is not as clear. As described above, some kind of charge compensation/internal salt bridges/zwitterion formation might actually decrease the hydrophilicity of basic amino acid-containing peptides in diluted formic acid/acetonitrile/water solutions upon phosphorylation. However, a more detailed understanding of the determinants of the ionization/detection efficiencies of peptides still has to be established. Such a study must include the composition of the peptides as well as instrumental parameters and the composition of the spray solution. The importance of the latter was underscored by a phosphorylation analysis on the yeast transcription factor Pho4 performed in our laboratory. The same sample was analyzed twice. The following parameters were varied in the two LC/MS experiments: (i) a different electrospray emitter was used, and (ii) the LC buffer system was changed from water/acetonitrile/0.4% acetic acid/0.005% heptafluorobutyric acid to water/acetonitrile/0.2% formic acid. When observing the signal intensities of the ions corresponding to the phosphopeptide TSSSAEGVVVASE(pS)PVIAPHGSTHAR and its unmodified cognate it was noted that the triply charged species show almost identical intensity ratios for both sets of experimental conditions, whereas the signal intensity ratio of the quadruply charged peptide/phosphopeptide is significantly different, changing from 1.2 to 0.33 (Fig. 5). It should be noted that measuring relative ionization/detection efficiencies of peptides and their phosphorylated cognates is not only of educational importance in the context of this study but can be used for stable-isotope-free quantitation of protein phosphorylation stoichiometries as we have recently shown (4Steen H. Jebanathirajah J.A. Springer M. Kirschner M.W. Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 3948-3953Google Scholar). Once such an ionization/detection efficiency ratio has been determined the degree of phosphorylation can easily be calculated based on the ion signal intensity ratio corrected by the relative ionization/detection efficiencies. Because there is a lack of information regarding the factors influencing ionization/detection efficiencies, the ratios have to be determined empirically. However, it is foreseeable that this property can be theoretically estimated in the future once a better understanding of ionization/detection efficiencies has been provided. This would greatly simplify the task of quantitation of protein phosphorylation stoichiometries. The data presented in this study shed some light onto some of the often used reasons explaining why mass spectrometric phosphorylation analysis is so difficult. The reasons investigated included the issue of increased hydrophilicity because of phosphorylation, the concern of selective suppression of phosphopeptides in the presence of unphosphorylated peptides, and the anecdotal lower ionization/detection efficiencies of phosphopeptides as compared with their unphosphorylated cognates. A set of synthetic peptide/phosphopeptide pairs was purified to homogeneity and quantitated by amino acid analysis. These peptide/phosphopeptide pairs were then used for numerous mass spectrometric experiments, which allowed one to address these issues separately. Several conclusions could be drawn from the results of these experiments.