Analysis of Protein Phosphorylation in the Regions of Consecutive

Anal. Chem. 2007, 79, 3476-3486

Analysis of Protein Phosphorylation in the Regions of Consecutive Serine/Threonine Residues by Negative Ion Electrospray Collision-Induced Dissociation. Approach to Pinpointing of Phosphorylation Sites Marina Edelson-Averbukh,† Ru 1 diger Pipkorn,‡ and Wolf D. Lehmann*,†

Central Spectroscopy, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany, and Peptide Synthesis Facility, German Cancer Research Center, TP3 Im Neuenheimer Feld 580, 69120 Heidelberg, Germany

Pinpointing of phosphorylation sites by positive ion collision-induced dissociation (CID) in phosphopeptides containing consecutive Ser/Thr residues (Ser/Thr clusters) is frequently hampered by the lack of backbone cleavage between adjacent Ser/Thr or pSer/pThr sites. In this study, we demonstrate that in negative ion collisioninduced dissociation phosphorylated and unmodified residues of Ser/Thr clusters exhibit a very selective behavior toward cleavage of their N-Cr bonds. Ser/Thr clusters were defined as two and more consecutive serine or threonine residues in phosphopeptide sequences. Dissociation reactions at pSer are significantly more abundant than those of unmodified sites. Thr residues exhibit the same effect, but the cleavages occurring at pThr are generally less prominent than those at pSer. The correlation observed between the facility of the amine backbone bond dissociation of phosphopeptides and the presence of the phosphate group on the side chain residues of Ser and Thr is attributed to the different magnitudes of electron density on the Cr atoms of the amino acid in phosphorylated and unmodified forms. The results of this study indicate that the intensity ratio of the fragments generated by N-Cr bond cleavage within the phosphopeptide Ser/Thr clusters represents a reliable and general marker for pinpointing of phosphorylation sites. The presented data illustrate that negative ion electrospray CID is superior over the standard positive ion mode approach for the localization of protein phosphorylation inside Ser/Thr clusters. Reversible protein phosphorylation is the most important and ubiquitous posttranslational event known to occur in proteins. In eukaryotes, protein phosphorylation-dephosphorylation reactions play a central role in intracellular signal processing and in the communication between cells, resulting in countless functional * Corresponding author. Tel. ++ 49-6221-424563. Fax ++ 49-6221-424554. E-mail: [email protected]. † Central Spectroscopy. ‡ Peptide Synthesis Facility.

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consequences that depend on the particular target proteins.1-3 The phosphorylation of eukaryotic proteins primarily occurs on serine residues to give rise to O-phosphomonoesters. It has been estimated that the formation of phosphoserine accounts for ∼90% of all cellular protein phosphorylations, while the covalent modification of threonine and tyrosine residues counts for about 10 and 0.1%, respectively.4,5 Reversible protein phosphorylation in eukaryotes is often found in the regions of protein sequences exhibiting multiple Ser/Thr residues.1 In order to elucidate physiological and pathophysiological regulatory mechanisms of protein phosphorylation/dephosphorylation inside the cells, it is necessary to identify the exact O-phosphorylated amino acid residues. In recent years, mass spectrometry has become the most commonly used method for protein phosphorylation analysis.6-8 The majority of the MS-based approaches involve enzymatic digestion of phosphoproteins followed by CID analysis of the peptide mixtures. Typically, phosphorylated peptides are recognized on the basis of phosphatespecific fragments in the product ion mass spectra of their positively charged molecular ions. It is well-established that upon collision-induced dissociation phosphorylated residues of Ser and Thr release H3PO4 (98 Da), while the side chains of pTyr eliminate HPO3 (80 Da) to a smaller extent.9,10 In addition, CID mass spectra of pTyr-containing peptides exhibit a pTyr-specific marker ion at m/z 216.11,12 Pinpointing of protein phosphorylation sites by positive ion MS/MS within known amino acid sequences of (1) Marks, F. Protein Phosphorylation; VCH: Weinheim, 1996. (2) Hunter, T. Cell 2000, 100, 113. (3) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S. Science 2002, 298, 1912. (4) Hanks, S. K.; Quinn, A. M.; Hunter, T. Science 1988, 241, 42. (5) Hubbard, M. J.; Cohen, P. Trends Biochem. Sci. 1993, 18, 172. (6) McLachlin, D. T.; Chait, B. T. Curr. Opin. Chem. Biol. 2001, 5, 591. (7) Reinders, J.; Lewandrowski, U.; Moebius, J.; Wagner, Y.; Sickmann, A. Proteomics 2004, 4, 3686. (8) Carr, S. A.; Annan, R. S.; Huddleston, M. J. Methods Enzymol. 2005, 405, 82. (9) Hunter, A. P.; Games, D. E. Rapid. Commun. Mass Spectrom. 1994, 8, 559. (10) Tholey, A.; Reed, J.; Lehmann, W. D. J. Mass Spectrom. 1999, 34, 117. (11) Lehmann, W. D. Proceedings of the 32. Tagung der Deutschen Gesellschaft fu ¨ r Massenspektrometrie, Oldenburg, 1999, Poster P47, p 112. (12) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem. 2001, 73, 1440-1448. 10.1021/ac0623991 CCC: $37.00

© 2007 American Chemical Society Published on Web 03/28/2007

phosphopeptides is based on the detection of pairs of peaks with mass shift of 98 or 80 Da both for N- and C-terminal fragments (b- and y-type ions).13 Frequently, the tandem mass spectra of pSer/pThr-peptides do not show sequence-specific product ions carrying intact phosphoamino acid residues. In these cases, the location of the phosphate is deduced from the increased formation of dehydroalanine- or dehydroaminobutyric acid-containing fragment ions.14 Despite the power of positive ion CID for pinpointing protein phosphorylation sites, a successful application of this widespread approach is often challenging. This is mainly because of the high lability of the phosphate moiety upon CID, which leads to a rapid release of the phosphate group from pSer and pThr. These neutral loss fragmentations may suppress formation of sequence-specific backbone fragments15 and thus hinder pinpointing of the modified sites. This feature is especially critical for molecular ions that carry two or less protons. Since the presence of the highly acidic phosphate moieties reduces the charge state of phosphopeptides in the positive ion mode16-18 relative to their nonmodified analogues, weak sequence-specific peptide fragments are a common feature of phosphopeptide CID mass spectra. The outcome is that only limited information on amino acid sequences is generated for many phosphopeptides, especially for those with a long peptide chain, making it difficult to determine the location of the modified sites unambiguously. This situation is significantly aggravated when multiple phosphate groups are present. Pinpointing of protein phosphorylation by positive ion CID is even further complicated in the cases when the modification is located inside the regions of consecutive Ser/Thr residues of the peptide sequences. For such phosphopeptides, the phosphorylation analysis is very frequently hampered by the lack of backbone cleavages between adjacent Ser/Thr residues. Electron-capture dissociation (ECD)19 and electron-transfer dissociation (ETD)20 have been introduced as alternative techniques for phosphopeptide sequencing. The dissociation methods induce random N-CR bond cleavages of peptide backbones preserving labile phosphoserine and phosphothreonine side chains. ECD requires an FT-ICR mass spectrometer, the most expensive type of MS instrumentation. ETD is efficient only for highly charged precursor ions, which provide complex ETD spectra, the interpretation of which is frequently hampered by the limited resolving power of ion trap mass spectrometers.21 Although ECD and EDT are very powerful fragmentation methods for pinpointing of protein phosphorylation sites, the majority of protein phosphorylation analysis experiments are still performed using the standard collision-induced dissociation technique.

In this study, we demonstrate that negative ion collisioninduced dissociation of phosphopeptides provides an unambiguous pinpointing of phosphorylated sites within Ser/Thr clusters. Ser/ Thr clusters were defined as two and more consecutive serine or threonine residues in phosphopeptide sequences. Phosphorylation site pinpointing is possible due to the strikingly different propensity of phosphorylated and unmodified Ser/Thr residues to undergo heterolysis of their N-CR bonds. These amine backbone cleavages yield sequence-specific C-terminal fragments. Although negative ion MS/MS is extensively used for detection of protein phosphorylation,22-24 sequencing of phosphopeptides is generally performed by positive ion MS/MS. The reasons for the common use of the positive ion mode CID for localization of protein phosphorylation sites are the following: (i) there is generally lower sensitivity of negative ion MS compared to the positive ion mode, (ii) sequence information from negative ion MS/MS spectra is often more difficult to extract, because of a variety of side chain fragmentations of peptide anions,25 (iii) negative ion MS has limited compatibility with LC-MS configurations, and finally (iv) the body of experimental information on CID spectra of negatively charged peptides is still much smaller compared to positive ions. The results presented here show for the first time the decisive advantage of the negative ion MS/MS over the positive ion-based approach for pinpointing of phosphorylation sites located in Ser/ Thr clusters.

(13) Kinter, M.; Sherman, N. E. Protein Sequencing and Identification Using Tandem Mass Spectrometry; John Wiley: Chichester, 2000. (14) Pereg, Y.; Shkedy, D.; de Graaf, P.; Meulmeester, E.; Edelson-Averbukh, M.; Salek, M.; Biton, S.; Teunisse, A. F. A. S.; Lehmann, W. D.; Jochemsen, A. G.; Shiloh, Y. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 5056. (15) Resing, K. A.; Ahn, N. G. Methods Enzymol. 1997, 283, 29. (16) Janek, K.; Wenschuh, H.; Bienert, M.; Krause, E. Rapid Commun. Mass Spectrom. 2001, 15, 1593. (17) Stensballe, A.; Andersen, S.; Jensen, O. N. Proteomics 2001, 1, 207. (18) Thompson, A. J.; Hart, S. R.; Franz, C; Barnouin, K.; Ridley, A.; Cramer, R. Anal. Chem. 2003, 75, 3232. (19) Zubarev, R. A.; Kelleher, N. L.; McLafferty, F. W. J. Am. Chem. Soc. 1998, 120, 3265. (20) Syka, J. E. P.; Coon, J. J.; Schroeder, M. J.; Shabaniowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 9528. (21) Sweet, S. M. M.; Creese, A. J.; Cooper, H. J. Anal. Chem. 2006, 78, 7563.

RESULTS AND DISCUSSION The Phenomenon. Negative ion tandem mass spectra of singly charged molecular ions of the isomeric phosphopeptides FSIAP-pS-SLDPSNR (1) and FSIAPS-pS-LDPSNR (2) exhibit structure-specific features (Figure 1). The positional isomers 1 and 2 are characterized by different locations of phosphorylation

EXPERIMENTAL SECTION Mass Spectrometry. NanoESI-QTOF tandem mass spectrometry was performed on a Q-Tof2 instrument (Waters Micromass, Manchester, UK). Borosilicate capillaries manufactured in-house using a micropipet puller (type P-87, Sutter Instruments, Novato, CA) were coated with a semitransparent film of gold in a sputter unit type SCD 005 (BAL-TEC, Balzers, Liechtenstein). The software version used was MassLynx 3.5. Peptide analytes were dissolved in a 50/2/48% (v/v/v) mixture of acetonitrile/formic acid/water to give a concentration of ∼10-50 pmol/µL. Phosphopeptide Synthesis. For solid-phase synthesis of the phosphopeptides the Fmoc strategy26,27 was employed. A multiple automated synthesizer (Syro II, Multisyntech) was used for the peptide synthesis. Peptide chain assembly was performed by in situ activation of amino acid building blocks by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. The Fmoc-Thr(PO(OBzl)OH)-OH, Fmoc-Tyr(PO(OBzl)OH)-OH, and Fmoc-Ser(PO(OBzl)OH)-OH were purchased from Merck Biosciences GmbH.

(22) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180. (23) Townsend, R. R.; Lipniunas, P. H.; Tulk, B. M.; Verkman, A. S. Protein Sci. 1996, 9, 1865. (24) Neubauer, G.; Mann, M. Anal. Chem. 1999, 71, 235. (25) Bowie, J. H.; Brinkworth, C. S.; Dua, S. Mass Spectrom. Rev. 2002, 21, 87. (26) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (27) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404-3409.

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Figure 1. Negative ion mode product ion mass spectra of the singly charged molecules of (a) FSIAP-pS-SLDPSNR (1) and (b) FSIAPS-pSLDPSNR (2) (collision offset 61 V). The mass spectra of the isomeric phosphopeptides show a great difference: RA% of the m/z 838.3 z8fragment is significantly higher in the CID spectrum of peptide 1 while the extent of the formation of the m/z 751.3 z7- ion is much more prominent in the case of peptide 2.

within a cluster of two serines, and they are synthetic analogues of human Plk1 tryptic peptide 325-337 phosphorylated at Ser335.28 The most prominent difference between the CID spectra of the two phosphopeptides results from the signals at m/z 751.3 and 838.4. The m/z 751.3 fragment is the base peak in the mass spectrum of the peptide 2 while its relative abundance in the spectrum of the peptide 1 is only ∼25%. In addition, the deprotonated phosphopeptide 1 gives rise to a highly abundant fragment at m/z 838.4 whereas the CID decomposition of the second isomer (peptide 2) affords this fragment at a significantly lower abundance (Figure 1). The formation of the m/z 751.3 and 838.4 fragments is similarly efficient for the dianions of peptides 2 and 1, respectively (Figure 2). These product ions represent the base peaks in the corresponding CID spectra. Other efficient decomposition modes of the singly deprotonated peptides 1 and 2 are the elimination of phosphoric acid10 and the backbone cleavage of the Asp-Pro amide bond to give rise to m/z 1370.6 and b9- (m/z 996.4) ions, respectively (Figure 1). Similar to the molecular ion, the b9- fragment readily loses phosphoric acid, yielding the m/z 898.4 signal. The additional major fragments in the CID spectra of the [M - H]- ions are the ion obtained by the loss of C-terminal arginine (m/z 1214.5) from the [M - H - H3PO4]- ions and the product of water/ammonia elimination from Asp and Asn side chains of the m/z 1214.5 anion, respectively (Figure 1). The [M - H - H3PO4 - R]- fragments undergo weak elimination of C-terminal asparagine as well. The neutral loss of amino acid residues from the C-terminus of deprotonated peptides has been thoroughly discussed.29-32 In (28) Wind, M.; Kelm, O.; Nigg, E. A.; Lehmann, W. D. Proteomics 2002, 1, 1516. (29) Marzluff, E. M.; Campbel, S.; Rodgers, M. T.; Beauchamp, J. L. J. Am. Chem. Soc. 1994; 116, 7787. (30) Brinkworth, C. S.; Dua, S.; Bowie, J. H. Rapid Commun. Mass Spectrom. 2002, 16, 713.

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addition, the phosphopeptide MS2 spectra contain a relatively abundant y4- fragment (m/z 471.2) and the product of H2O loss from z5- ion (m/z 569.2), obtained by aspartic acid N-CR bond cleavage (Figure 1). Bowie and co-workers33,25 have proposed that the cleavage of the Asp amine bond is initiated by an enolate anion resulting from the abstraction of the amino acid side chain Cβproton. The CID spectra of the doubly charged anions of the phosphopeptides 1 and 2 exhibit the same backbone cleavage fragments as the singly charged analogues (Figure 2). In addition, the mass spectra show relatively abundant y1- (m/z 173.1) and [z2 - NH3]- (m/z 253) ions. The z2- fragment results from the cleavage of Asn amine bond, in analogy with the backbone cleavage at Asp.25 The tandem mass spectra of the doubly ionized phosphopeptide pair show abundant high-mass phosphorylation marker ions [M - 2H - PO3]- (m/z 1388.6)34 and relatively intensive signals of the dihydrogenphosphate anion H2PO4- (m/z 97.0). In the case of the doubly ionized phosphopeptides 1 and 2, the fragment m/z 1214.5 is a result of sequential neutral losses of C-terminal Arg and H2O from the [M - 2H - PO3]- ion. Positive Ion MS2 Mass Spectra of the Isomeric Phosphopeptides. In contrast to the negative ion CID spectra of 1 and 2, the mass spectra of the [M + 2H]2+ ions of this phosphopeptide pair are very alike (Figure 3). The MS/MS spectra show a variety of b- and y-type fragments the relative abundances of which are similar for both peptide systems. At the same time, the positive ion mass spectra of the isomeric phosphopeptides do not exhibit product ions derived from a dissociation of the amide backbone (31) Harrison, A. G. J. Mass Spectrom. 2004, 39, 136. (32) Li, Z.; Yalcin, T.; Cassady, C. J. J. Mass Spectrom. 2006, 41, 939. (33) Brinkworth, C. S.; Dua, S.; McAnoy, A. M.; Bowie, J. H. Rapid Commun. Mass Spectrom. 2001, 15, 1965. (34) Edelson-Averbukh, M.; Pipkorn, R.; Lehmann, W. D. Anal. Chem. 2006, 78, 1249.

Figure 2. Negative ion mode product ion mass spectra of [M - 2H]2- ions of (a) FSIAP-pS-SLDPSNR (1) and (b) FSIAPS-pS-LDPSNR (2) (collision offset 29 V). The MS/MS spectra of the [M - 2H]2- ions exhibit the same differences as observed for the [M - H]- ions.

Figure 3. Product ion mass spectra of the doubly protonated molecules of (a) FSIAP-pS-SLDPSNR (1) and (b) FSIAPS-pS-LDPSNR (2) (collision offset, 23 V). The lack of b- and y-type ions derived from the amide bond dissociation between the adjacent serine residues precludes spotting of the phosphorylation sites and thus differentiation between the two isomers.

bond between the two adjacent serines. Indeed, the signals of the corresponding b6+ and y7+ fragments (m/z 683.3 and 788.4 for peptide 1 and m/z 603.3 and 868.4 for peptide 2, respectively) are either completely absent in the acquired MS/MS spectra or their intensities are negligible (Figure 3). The b- and y-type fragments carrying two positive charges do not appear in the CID spectra as well. In addition, the signals of the secondary fragmentation products and of the ions derived from a pSer-Ser N-CR bond cleavage are also absent. As a result, there is no possibility to spot the phosphorylation sites in the sequences of the

phosphopeptides 1 and 2 and thus to distinguish between the two positional isomers using positive ion CID. It is reasonable to assume that the suppression of the backbone cleavages within the phosphopeptides serine clusters results from the stabilization of the cluster region by intramolecular hydrogen bonding involving the side chain OH groups of the phosphorylated and unmodified Ser residues. Origin of the m/z 751.3 and 838.4 Fragments. The difference of 87 between m/z 751.34 and 838.37 reveals that the corresponding fragments differ by one Ser residue. A correlation Analytical Chemistry, Vol. 79, No. 9, May 1, 2007

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Scheme 1. Origin of the m/z 751.3 and 838.4 Fragments in the CID Spectra of the Deprotonated Phosphopeptides 1 and 2a

a The product ions contain phosphopeptide C-termini and they are generated by N-CR bond cleavages at positions 7 and 8 of the peptides, followed by neutral loss of H3PO4 or H2O.

between the phosphopeptide sequences and the m/z values of the ions leads to the conclusion that the m/z 751.3 and 838.4 fragments are generated by Ser/pSer N-CR bond cleavages at positions 7 and 8, respectively (numbering from C-terminus). The fragments at m/z 751.3 and 838.4 contain the C-terminal parts of the peptide molecules. Hence, employing the fragment ion nomenclature introduced by Roepstorff and Fohlman,35 the product ions are z-type fragments. The reason for the equal m/z values obtained for the products of the N-CR bond dissociations at the position 7 of the peptides 1 and 2 (m/z 751.3) is in the fact that the amine bond backbone cleavages are accompanied by neutral losses of H2O and H3PO4, respectively (Scheme 1). The eliminations compensate the difference of 80 Da expected for the fragments arising from phosphorylated and unmodified Ser at positions 7. The m/z 851.4 fragments of the phosphopeptides 1 and 2 are generated by neutral loss of H3PO4 from the corresponding z8- ions (Scheme 1). It should be noted that no z-type fragments without neutral loss were observed in the CID spectra of the singly and doubly charged molecular ions of the isomeric phosphopeptides 1 and 2. In addition, no complementary c-type fragments, which incorporate phosphopeptides N-termini, appear in the tandem mass spectra (Figures 1 and 2). Besides, there are no z-type fragments of the other two serine residues (Ser-3 and Ser-12, from C-terminus). The major difference between the relative abundances of the m/z 751.3 and 838.4 fragments in the mass spectra of the singly and doubly deprotonated molecules of peptides 1 and 2 (Figures 1 and 2) implies that the side chain phosphorylation of serine has a dramatic effect on the efficiency of the amino acid N-CR bond cleavage. In order to verify this conclusion, the CID spectrum of the deprotonated nonmodified analogue FSIAPSSLDPSNR (3) has been acquired (Figure 4). The strong suppression of the m/z 751.3 [z7 - 18 Da]- and 838.4 [z8 - 18 Da]- ion signals in the mass spectrum presented in Figure 4 in comparison with the relative abundances of the m/z 751.3 and 838.4 fragments in the spectra of phosphopeptides 2 and 1, respectively, indicates clearly the influence of the phosphate group on the efficiency of the pSer N-CR bond heterolysis. The close similarity of the m/z 751.3 and (35) Roepstorff, P.; Fohlman, J. Biomed. Mass Spectrom. 1984, 11, 601.

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838.4 ion abundances in the CID spectrum of peptide 3 (FSIAPSSLDPSNR) indicates comparable probabilities for the dissociation of the backbone amine bonds at the peptide positions 7 and 8. The data of Figures 1 and 2 show that the phosphorylation of the Ser-7 or -8 in the sequence FSIAPSSLDPSNR increases the efficiency of the serine amine bond cleavages so significantly that the signals of the resulting pSer z fragments dominate all other backbone fragments of the phosphopeptides (b-, y-, and z-type ions). In addition, the CID spectra of the singly and doubly ionized phosphopeptides 1 and 2 (Figures 1 and 2) reveal that the efficiencies of the N-CR bond cleavages of phosphorylated serine residues are similar to amino acid side chain decompositions, namely, the elimination of H3PO4 (from [M - H]-) and the loss of PO3- ion from [M - 2H]2- ions. Efficiency of N-CR Bond Cleavage of pSer versus Ser. Further investigation of the phenomenon of the enhanced efficiency of the pSer amine bond dissociations, in comparison with those of the unmodified amino acid, was carried out by studying the CID behavior of phosphopeptide anions containing Thr and Tyr clusters. Two pairs of isomeric phosphopeptides were chosen for this study: FSIAPpTTLDPSNR (4) and FSIAPTpTLDPSNR (5), as well as FSIAPpYYLDPSNR (6) and FSIAPYpYLDPSNR (7). The peptides 4-7 are analogous to the modified peptides 1 and 2 differing only by the amino acids in positions 7 and 8 (the peptide cluster regions). Examination of the negative ion tandem mass spectra of the singly and doubly charged phosphopeptides 4-7 indicates that the substitution of pSer(Ser)-7 and Ser(pSer)-8 positions of the peptides 1 and 2 by Thr or Tyr residues (phosphorylated and unmodified) reduces significantly the relative abundances of the [z7 - NL]- and [z8 - NL]- fragments. Indeed, while the m/z 838.4 [z8 - H3PO4]- and 751.3 [z7 - H3PO4]- ions of peptides 1 and 2, respectively, exhibit highly abundant signals (Figures 1 and 2), the extent of the formation of the corresponding fragments from the Thr-containing peptides 4 and 5 (m/z 866.4 and 765.3) does not exceed 11% (Table 1). Furthermore, the replacement of the serine clusters of phosphopeptides 1 and 2 by the combinations of aromatic residues (peptides 6 and 7) leads to the complete suppression of the z-ion formation. It is noteworthy that, similarly to the behavior of Ser residues (Figures 1 and 2), the efficiencies of the N-CR bond cleavages at the unmodified Thr positions of peptides 4 and 5 are considerably lower than those of the phosphorylated sites (Table 1). The observed order in the efficiency of the N-CR bond dissociation (pSer > pT . pY) is well correlated with the magnitude of the electron density on the CR atom.36 The CID data reveal that the extent of the backbone cleavage decreases with an increase of the electron density on the central CH groups (Figures 1 and 2, Table 1). This correlation matches very well the observed enhancement of the amine bond dissociation for pSer and pThr residues, in comparison to their unmodified analogues (Figures 1 and 2, Table 1). Indeed, the conversion of the Ser/ Thr side chain hydroxyl groups to the phosphate ester form is expected to decrease the electron densities on the CR atoms, due to the more pronounced negative inductive effect (-I) of the side chain γ-oxygens of the modified residues. Thus, the experimental (36) March, J. Advanced Organic Chemistry,, 4th ed.; J. Wiley and Sons: New York, 1992; pp 12-19, 273-286.

Figure 4. Product ion mass spectrum of the [M - H]- ions of FSIAPSSLDPSNR (3) (collision offset 60 V). The similar intensities of the m/z 751.3 and 838.4 ions demonstrate comparable probabilities for the dissociation of the backbone amine bonds at positions 7 and 8 of the unmodified peptide. Table 1. Relative Abundances (RA%) of the Fragments Obtained by CID Dissociation of N-Cr Bonds at Positions 7 and 8 of Singly and Doubly Charged Molecular Anions of pThr-Peptides 4 and 5 [M - H]-

a

phosphopeptide

MW

FSIAPpTTLDPSNR (4)

1497.7

FSIAPTpTLDPSNR (5)

1497.7

[M - 2H]2-

position 7 ion (RA%)




[z7 - H2O]m/z 765.3 (2%)a [z7 - H3PO4]m/z 765.3 (11%)a

[z8 - H3PO4]m/z 866.4 (11%)a [z8 - H3PO4]m/z 866.4 (3%)a

[z7 - H2O]m/z 765.3 (1%)b [z7 - H3PO4]m/z 765.3 (8%)b

[z8 - H3PO4]m/z 866.4 (7%)b [z8 - H3PO4]m/z 866.4 (1%)b

64 V collision offset. b 31 V collision offset.

results imply that the differences in the efficiencies of the N-CR bond cleavages between the peptides 1 and 2 are the consequence of the different electrophilicities of the central CH groups of the phosphorylated and unmodified Ser/Thr residues. It should be noted that the observed order of the amino acid reactivity toward N-CR bond dissociation (pSer > pThr > Ser < Thr) is identical to that found in solution for the β-elimination reaction.37 The ratelimiting steps of the base-catalyzed H3PO4/H2O eliminations involve deprotonation of the central amino acid CH groups,38,39 the efficiency which is correlated with the magnitudes of the electron density on the CR atoms. Mechanism of z-Type Ion Formation. The correlation observed between the facility of the [z7 - NL]- and [z8 - NL]ion formation of the deprotonated phosphopeptides 1, 2, 4, and 5 and the magnitude of the electron density on the CR atoms of (37) Li, W.; Backlund, P. S.; Boykins, R. A.; Wang, G.; Chen, H.-C. Anal. Biochem. 2003, 323, 94. (38) Anderson, L.; Kelley, J. J. J. Am. Chem. Soc. 1959, 81, 2275. (39) Kalan, E. B.; Telka, M. Arch. Biochem. Biophys. 1959, 85, 273.

the cleavage sites allows one to assume that the decomposition reaction of the gaseous molecular anions is induced by a nucleophilic attack at the amino acid CH groups. It should be emphasized that, in principle, the increased bulkiness of the side chain pThr/Thr and pTyr/Tyr groups relative to pSer/Ser could cause the suppression of the z-ion formation of the phosphopeptides 4-7. However, the steric hindrance does not seem to be the reason for the different stabilities of the N-CR bonds since the z-ion formation is significantly more efficient for more bulky pSer compared to Ser (Figures 1 and 2, Table 1). It is apparent that upon CID conditions the interaction between the CR atoms of the decomposing phosphoamino acids and a peptide nucleophilic group is intramolecular. The absence of the analogous N-CR bond cleavage reaction of the phosphopeptides during positive ion MS/MS fragmentation leads to the conclusion that the attacking nucleophile carries a negative charge. In addition, it is clear that the moiety which attacks the amino acids R-carbon and creates a new covalent bonding with the atom is situated Analytical Chemistry, Vol. 79, No. 9, May 1, 2007

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Figure 5. Product ion mass spectra of the [M - H]- ions of (a) FSIAPS-pS-LDPSR (8) (collision offset 51 V), (b) FSIAPS-pS-LDPSR-NH2 (9) (collision offset 51 V), (c) FSIAPS-pS-LGPSR (10) (collision offset, 49 V), (d) FSIAPS-pS-LGPSR-NH2 (11) (collision offset 49 V), and (e) FSIAPSpS-LGPGR-NH2 (12) (collision offset 47 V). The comparison of the relative abundances of the pSer- z-type fragments in the CID spectra of the model phosphopeptides 8-12 reveals that carboxylic and amide functions of the peptide sequences are involved in the N-CR bond cleavages of the phosphorylated sites.

C-terminally to the cleavage site. The N-terminal part of the peptide sequence is eliminated during the fragmentation reaction as an amide, without change in its primary structure. The possibility of a nucleophilic attack of a deprotonated phosphate oxygen of pSer/ pThr at the amino acids CR atoms can be excluded because of the facial loss of 98 Da from the corresponding z-fragments. In order to investigate which functionalities of the negatively charged phosphopeptides initiate the efficient amine bond backbone cleavages of the phosphorylation sites within the peptide Ser/Thr clusters, a series of synthetic model phosphopeptides 8-12

FSIAPS-pS-LDPSR (8) FSIAPS-pS-LDPSR-NH2 (9) FSIAPS-pS-LGPSR (10) FSIAPS-pS-LGPSR-NH2 (11) FSIAPS-pS-LGPGR-NH2 (12) has been studied. The peptides chosen for the study are synthetic analogues of the peptide 2, differing by the number of functional groups in the C-terminal part of the peptide, which are prone to undergo deprotonation during electrospray ionization or CID activation of the molecular ions (carboxy, hydroxy, and amide).40-42 (40) Ewing, N. P.; Cassady, C. J. J. Am. Soc. Mass Spectrom. 2001, 12, 105. (41) Brinkworth, C. S.; Dua, S.; Bowie, J. H. Rapid Commun. Mass Spectrom. 2002, 16, 713. (42) Waugh, R. J.; Eckersley, M; Bowie, J. H.; Hayes, R. N. Int. J. Mass Spectrom. Ion Processes 1990, 98, 135.

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The abstraction of the proton from the peptide functional groups converts them to potential reacting centers of the z-ion formation. Thus, peptides 8-10 carry three sites that are able to attack the pSer CR atom after ionization (side chain residues of Asp-4 and Ser-2, C-terminal carboxyl/amide), while phosphopeptides 11 and 12 include only 2 and 1 of them, respectively. It is apparent that the singly and doubly charged molecular ions of phosphopeptides, which afford the z-fragments during MS/MS, do not carry a negative charge on the phosphate group because the ionization of the acidic phosphate ester would preclude the interaction of the residue with a nucleophile. It should be pointed out that C-terminal amidation and Gly substitution reactions have been performed for the sequence of peptide 8, instead of that of 2, in order to simplify the model peptides study (the possibility of the amide group attack has been examined in the CID behavior of the peptide 12). The MS/MS spectra of phosphopeptides 8-12 are presented in Figure 5. A comparison of the fragmentation patterns of peptides 8 and 9 (Figures 5a and b) reveals a small increase in the extent of the [z6 - H3PO4]- and [z7 - H3PO4]- ion formation for amidepeptide 9. Indeed, while the [z6 - 98]- fragment is the most abundant ion in the CID spectrum of the amidated peptide 9, the base peak in the spectrum of the peptide 8 belongs to the phosphopeptide molecular anion. In addition, the relative abundance of the [M - H - H3PO4]- fragment ion is higher in the spectrum of peptide 8 than that of the amidated analogue 9. Assuming that the derivatization of the C-terminal COOH group of peptide 8 increases the population of the peptide molecular anions carrying the charge on the Asp side chain carboxyl (pKa of the C-terminal amide is significantly higher than that of Asp

side chain carboxyl43,44) the enhancement of the z-ion formation for peptide 9 indicates involvement of the Asp residue in the N-CR bond cleavage fragmentation reactions. The relative insignificance of the C-end amidation effect on the facility of the z-ion formation of peptide 8 implies that the Asp residue is not the single functionality that initiates the peptide pSer-6/Ser-7 amine bond dissociation. A negative ion CID spectrum of model peptide 10 (Figure 5c), which contains a Gly residue at the position of Asp in 8, has been measured in order to assess the contribution of the Asp side chain to the process of the z-ion formation. The mass spectrum displays a decrease of more than 50% in the intensities of the [z6 - 98 Da]- and [z7 - 98 Da]- ion signals in comparison to these of peptide 8 (Figure 5a). To elucidate the identities of the functions that induce the pSer/Ser N-CR bond dissociations of the Gly-substituted peptide 10, CID behavior of the two additional model peptides 11 and 12 has been examined (Figure 5d and e). The designed sequence of peptide 11 has no carboxyl moieties while phosphopeptide 12 carries Gly substitution at position 2 from the C-terminus. The experimental results reveal a significant suppression of the [z6 - H3PO4]- ion signals in the spectra of peptides 11 and 12 relative to that observed for phosphopeptide 10 (the abundances of the [z7 - H3PO4]fragments in the spectra of 11 and 12 are negligible). At the same time, the comparison of the mass spectra of peptides 11 and 12 shows no difference in the extent of the pSer z-ion formation between the two peptides. Therefore, it is clear that (i) the hydroxyalkyl side chain group of Ser-2 of the peptides 1, 2, 4, 5, and 8-12 is not involved in the production of the z ions and (ii) the deprotonated C-terminal amides of phosphopeptides 11 and 12 are the functionalities which initiate their pSer N-CR bond cleavages. It should be noted that the very efficient release of 30 Da (formaldehyde38) from the [z6 - 98 Da]- fragments observed in the spectrum of peptide 11 (Figure 5e) provides an additional support to the claim that the phosphopeptide Ser-2 side chain group does not participate in the N-CR bond cleavage of pSer. If the serine residue had been involved in the fragmentation reaction, such elimination would have not been possible because of the formation of a covalent bond between the Ser side chain oxygen and CR atom of pSer. It is noteworthy that the facile expulsion of the formaldehyde molecules from the deprotonated serine residues of the intact phosphopeptides 11 and 12 gives rise to the splitting observed in the CID spectra of the peptides for their [M - H - H3PO4]- ion signals (Figure 5d and e). Besides, the exclusive initiation of the amine bond dissociations of peptides 11 and 12 by the ionized C-terminal amides has been confirmed by negative ion CID data of the peptide 10 methyl ester (FSIAPSpSLGPSR-COOme). As expected, the mass spectrum of the ester [M - H]- did not display the corresponding [z6 H3PO4]- and [z7 - H3PO4]- fragments. In addition, it should be noted that the inefficient neutral loss of 30 Da from the [M H - H3PO4]- fragments of peptides 8 and 9 indicates that most of the peptides molecular ions carry the ionized carboxyls and thus support the assumption that the amidation of the peptide 8 C-terminus concentrates the negative charge on its Asp residue. Finally, in similarity to the serine side chains (Figure 5d), a neutral

loss of 42 Da (HNdCdNH45) from the ionized arginine residue of the peptide 12 [z6 - H3PO4]- fragment leads to splitting of the z-ion signal (Figure 5e). A proposed mechanism for the pSer and pThr N-CR bond cleavage of phosphopeptide anions initiated by the peptide C-terminus is shown in Scheme 2. The mechanistic pathway of the fragmentation reaction induced by Asp and Asn deprotonated side chain functions is analogous to that outlined in Scheme 2. In accord with the experimental data described above, the z-ion formation is based on an intramolecular SN2 reaction between a deprotonated carboxyl/amide phosphopeptide functionality and the CR atom of a pSer/pThr residue. The approach between the two reacting centers is probably assisted by hydrogen bonding between the carbonyl oxygen of the attacking carboxylate/amide anion and the phosphate group. This assumption is made on the basis of the results obtained by the comparison between the CID spectrum of peptide 8 (FSIAPSpSLDPSR) with the spectrum of peptide 13 (FSIAPpSLDPSR), the sequence of which does not contain a Ser-Ser cluster. The spectrum of 13 displayed a decrease of ∼50% in the intensity of the [z6 - H3PO4]- fragment in comparison to the product ion signal of 8, whereas the relative abundances of other CID fragments were very similar. This finding correlates with the assumption that the probability for an intramolecular attack of a pSer/pThr/Ser/Thr cluster region is higher than that of the interaction with a lonely situated pSer/pThr site. The donor-acceptor interaction probably helps to direct the nucleophilic attack to the region of the Ser/Thr cluster of a peptide sequence. It should be noted that the occurrence of a similar H-bridging has been proposed by Bowie and co-workers for the fragmentation of unmodified Ser and Thr-containing peptides.46 According to the mechanism outlined in Scheme 2, the nucleophilic attack of the phosphoamino acid CR atom is followed by cleavage of the pSer/pThr N-CR bond and formation of an ion/ neutral complex A47,48 between the cleaved anionic N-terminal part of the phosphopeptide and the cyclic neutral fragment. The phosphorylated residue of the complex A uncharged component undergoes a CR-proton abstraction to produce the intermediate z-ion B. The next step is elimination of phosphoric acid from the cyclic anion B accompanied by heterolysis of its ester bond to give rise to the linear ketene-containing structure of the [z H3PO4]- fragments. The mechanism proposed for the elimination of phosphoric acid from the z fragments is analogous to that of the in-solution base-catalyzed dephosphorylation of pSer/pThrphosphopeptides.38,39 The neutral loss of 30 Da/42 Da from the [z6 - H3PO4]- fragments of peptides 11 and 12, respectively (Figure 5d and e) implies that the z ion is a mixture of tautomeric structures differing by the location of the negative charge (see Scheme 2). It should be noted that, in principle, there is another possibility for the mechanism of z-ion formation initiated by deprotonated side chains of Asp and Asn. In these cases, a succinimidecontaining structure of z-ions may be obtained following a nucleophilic attack of the Asp/Asn side chain carbonyls of the ion B by deprotonated backbone amine of adjacent amino acid. The formation of the 5-membered succinimide ring by Asp and

(43) Streitwieser, A; Heathcock, C. H.; Kosower, E. M. Introduction to Organic Chemistry; Prentice Hall, Inc.: Upper Saddle River, NJ, 1992. (44) Fersht, A. R. Enzyme Structure and Mechanism; Freeman: New York, 1984.

(45) Waugh, R. J.; Bowie, J. H.; Gross, M. L. Aust. J. Chem. 1993, 46, 693. (46) Brinkworth, C. S.; Dua, S.; Bowie, J. H. Eur. J. Mass Spectrom. 2002, 8, 53. (47) McAdoo, D. Acc. Chem. Res. 1993, 26, 295. (48) Longevialle, P. Mass Spectrom. Rev. 1992, 11, 157.


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Scheme 2. Proposed Mechanistic Pathway for the Formation of z-Type Fragments Derived from N-Cr Bond Cleavages of Phosphopeptide Modified Sites and Initiated by Ionized C-Terminusa

a The fragmentation reaction is an intramolecular S 2 process between a pSer/pThr C atom and a deprotonated carboxyl/amide N R group.

Asn has been observed both in the gas phase and in solution.49,50 To evaluate the probability of the mechanistic pathway involving the formation of the succinimide ring, the CID behavior of the negatively charged phosphopeptide 8 has been compared to that of a model peptide 14 (FSIAPS-pS-LEPSR). The sequence of peptide 14 contains glutamic acid (Glu) at the position of that of Asp in peptide 8. Since Glu residues cannot form the succinimide cyclic structures the high similarity observed for the intensities of the pSer z-fragments in the CID spectra of the phosphopeptides 8 and 14 ruled out the possibility of succinimide formation during N-CR bond cleavages of phosphopeptides. It is interesting to point out that, in contrast to the behavior of peptides 1 and 4, the N-CR bond cleavages of unmodified Ser/ Thr cluster positions of phosphopeptides 2, 5, and 8-14 are not accompanied by water elimination. Instead, the corresponding z8and z7- fragments of peptides 2, 5, and 8-14, respectively, lose phosphoric acid (Figures 1, 2, and 5, Table 1). The observed phenomenon indicates that, following amine bond cleavages of the unmodified Ser/Thr phosphopeptides cluster positions, the phosphate-containing cyclic neutrals of complex A undergo abstraction of a more acidic pSer/pThr CR-proton instead of the CH protons of the dissociating amino acids. As a result, a facile (49) Schlosser, A.; Lehmann, W. D. J. Mass Spectrom. 2000, 35, 1382. (50) Violand, B. N.; Schlittler, M. R.; Kolodziej, E. W.; Toren, P. C.; Cabonce, M. A.; Siegel, N. R.; Duffin, K. L.; Zobel, J. F.; Smith, C. E.; Tou, J. S. Protein Sci. 1992, 1, 1634.

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elimination of phosphoric acid from the z ions takes place, following the abstraction of the ester enolate proton, as shown in Scheme 2. The ionization of the more distant (relative to the cleavage site) modified position of the neutral C-terminal part supports the intermediacy of the ion/neutral complex A in the mechanism of the phosphopeptide z-ion formation reaction (Scheme 2). Practical Benefit of the Observed Phenomenon for Phosphorylation Site Pinpointing. We have tested the applicability for protein phosphorylation analysis of the prominent difference between the efficiencies of the N-CR bond cleavages of phosphorylated and unmodified Ser/Thr residues. To this end, the CID behavior both of synthetic peptides containing extended phosphorylated clusters and of synthetic sequences with naturally occurring phosphorylation sites have been investigated. The study of the usefulness of the new finding for identification of more intricate phosphorylation patterns than these of peptides 1-14 has been performed on peptides 15 (FSIAP-pSS-pS-SLDPSR) and 16 (FSIAPSS-pS-pSLDPSR). The model sequences of the doubly phosphorylated isomeric peptides 15 and 16 contain clusters of four serines with different locations of the two phosphate groups within these clusters. The negative ion CID mass spectra acquired for phosphopeptides 15 and 16 demonstrated that, as expected, the magnitudes of the fragments obtained by the pSer N-CR bond cleavages are significantly higher than these of the ions derived

Figure 6. Product ion mass spectrum (collision offset 70 V) of the [M - H]- ion of peptide 15 (FSIAP-pSS-pS-SLDPSR). The comparison of RA% of z ions obtained from N-CR bond cleavages at pSer sites (boldface) and those of the unmodified positions leads to the reliable identification of the two phosphorylated residues within the cluster of four serines. Table 2. Relative Abundances (RA%) of the [zn - NL]- Fragments in the CID Mass Spectra of [M - H]- Molecular Ions of Phosphopeptides 17-19 RA% of [zn - NL]phosphopeptidea 41VSEH-pS-SPEEEASPHQR56

369TS-pS-AMSGHSR378

191LLS-pS-PLRQEK200

(18)

(19)

MW (17)

1884.8 1099.4 1621.7

phosphorylated cluster site

unmodified cluster site

[z45 - 98]m/z 1316.6 (9%)b [z371 - 98]m/z 795.3 (100%)c [z194 - 98]m/z 907.4 (10%)d

[z46 - 18]m/z 1229.6 (-)b [z370 - 98]m/z 882.3 (-)c [z193 - 98]m/z 994.4 (-)d

a The subscripts on the peptide N- and C-temini refer to the numbers of amino acid in the sequences of the precursor proteins. b 82 V collision offset. c 50 V collision offset. d 55 V collision offset.

from the dissociation of the unmodified cluster positions. The CID spectrum of the [M - H]- precursor ion of peptide 15 is shown in Figure 6. One can see that the relative abundances of the [z8 - NL]- and [z10 - NL]- fragments are much more prominent that those of the [z7 - NL]- and [z9 - NL]- ions. Similarly, a CID spectrum of the [M - H]- ions of peptide 16 displayed total intensities of 115 and 60% for the [z7 - NL]- and [z8 - NL]fragments (respectively) while these of the [z9 - NL]- and [z10 - NL]- ions did not exceed 20%. The observed characteristic fragmentation schemes of the isomeric phosphopeptides 15 and 16 support the assumption that a comparison between the RA% of the [zn - NL]- fragments obtained from peptide Ser/Thr cluster N-CR bond cleavages is a reliable method for pinpointing of phosphorylation sites. It is noteworthy, that the doubly phosphorylated z fragments of peptides 15 and 16 release two molecules of phosphoric acid (see for example, Figure 6). The incomplete

character of the elimination reaction implies that the mechanism of the second H3PO4 neutral loss is not identical to that shown in Scheme 2 but rather involves the previously suggested 6-centered transition state.10 In addition, the power of the z ion-based method for localization of protein phosphorylation has been examined using a number of synthetic phosphopeptides with natural phosphorylation sites.51-53 A list of the tryptic peptides and relative abundances of the z fragments are given in Table 2. The experimental data indicate that the propensities of the pSer positions of peptides 17-19 to (51) Jin, M.; Bateup, H.; Padovan, J. C.; Greengard, P.; Nairn, A. C.; Chait, B. T. Anal. Chem. 2005, 77, 7845. (52) Neuscha¨fer-Rube, F.; Hermosilla, R.; Rehwald, M.; Ro ¨nnstrand, L.; Schu ¨ lein, R.; Wernstedt, C.; Pu ¨ schel, G. P. Biochem. J. 2004, 379, 573. (53) Gruhler, A.; Olsen, J. V.; Mohammed, S.; Mortensen, P.; Færgeman, N. J.; Mann, M.; Jensen, O. N. Mol. Cell. Proteomics 2005, 3, 310.


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undergo the amine bond cleavages are noticeably higher than these of the unmodified sites. Furthermore, the formation of the z fragments derived from N-CR bond cleavages of nonphosphorylated serine residues of peptides 17-19 was not observed at all (see Table 2). Thus, the negative ion CID mass spectra of the peptides 17-19 enable an unambiguous identification of the peptide phosphorylation sites within the serine clusters. The CID data of the peptide 369TS-pS-AMSGHSR378 (18) confirm the conclusion that the N-CR bond cleavages of phosphopeptides may be initiated by the peptide C-terminus. The decreased intensities of the pSer [zn - 98]- ion signals in the spectra of the peptides 41VSEH-pS-SPEEEASPHQR56 (17) and 191LLS-pS-PLRQEK200 (19) enable one to assume that the efficiency of the intramolecular SN2 reaction depends on the phosphopeptide conformation. If the conformation adopted by an ionized peptide chain is not favorable for the interaction between the phosphorylated Ser/Thr residues and the anionic carboxyl/amide functionality, the z-ion fragments are not expected to be very prominent. At the same time, the identities of the z-ion formation initiators (Figure 5 and Scheme 2) lead us to assume that the N-CR bond cleavages of phosphorylated positions inside serine (threonine) phosphopeptide clusters is a common feature of the negative ion collision-induced fragmentation of the modified peptides. CONCLUSION The results of this work show that negatively charged phosphopeptides undergo efficient N-CR bond cleavages at pSer/pThr

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residues to give rise to abundant [zn - H3PO4]- fragments. The extents of the pSer/pThr dissociation reactions are significantly higher than those of the nonphosphorylated Ser/Thr residues. In addition, peptide chain regions composed of multiple Ser/Thr residues provide more prominent z-type ions than isolated Ser or Thr residues. The present study reveals that negative ion CID fragmentation patterns of phosphopeptides enable direct determination of phosphorylated sites inside peptide Ser/Thr clusters. In contrast, pinpointing of phosphorylation sites by positive ion MS/MS in cluster-containing phosphopeptides is frequently hampered by a strong suppression of backbone cleavages between adjacent Ser/Thr residues. The strategy described in this work provides a solution for the numerous cases of positional ambiguity of the phosphate groups within Ser/Thr clusters of protein sequences following positive ion CID analysis. Finally, the selective CID behavior of phosphopeptides will help to establish negative ion tandem mass spectrometry as a powerful method not only for recognition of protein phosphorylation but also for phosphopeptide sequencing. ACKNOWLEDGMENT M.E.-A. gratefully acknowledges the financial support of the German Cancer Research Center by a guest scientist scholarship.

Received for review December 19, 2006. Accepted March 2, 2007. AC0623991

Analysis of Protein Phosphorylation in the Regions of Consecutive

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