Phosphate Group-Driven Fragmentation of Multiply Charged

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Anal. Chem. 2006, 78, 1249-1256

Phosphate Group-Driven Fragmentation of Multiply Charged Phosphopeptide Anions. Improved Recognition of Peptides Phosphorylated at Serine, Threonine, or Tyrosine by Negative Ion Electrospray Tandem Mass Spectrometry Marina Edelson-Averbukh,† Ru 1 diger Pipkorn,‡ and Wolf D. Lehmann*,†

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

The nanoelectrospray product ion spectra of multiply charged phosphopeptide anions reveal the occurrence of phosphate-specific high-mass fragment ions of the type [M - nH - 79](n-1)-. These so far unrecognized fragments, which are observed for phosphoserine-, phosphothreonine-, and phosphotyrosine-containing peptides, are the counterparts of the established inorganic phosphopeptide marker ion found at m/z 79 ) [PO3]-. The highmass marker ions are formed with high efficiency at moderate collision offset values and are particularly useful for sensitive recognition of pSer-, pThr-, and pTyr-peptides due to the low background level in MS/MS spectra at m/z values above those of the precursor ions. By virtue of this feature, the detection of the new phosphorylation-specific fragment ions appears to be more sensitive than the detection of the low-mass phosphate marker ion at m/z 79, where a higher interference by nonspecific background signals is generally observed. The number of phosphate groups within a phosphopeptide can also be estimated on the basis of the [M - nH - 79](n-1)- ions, since these exhibit an effective, sequential neutral loss of H3PO4 of the residing phosphate groups. A mechanistic explanation for the formation of the [M - nH - 79](n-1)ions from multiply charged phosphopeptides is given. The high-mass marker ions are proposed to originate from phosphopeptide anions, which carry two negative charges located at the phosphate group. A new search tool denominated “variable m/z gain analysis”, which utilizes these newly recognized high-mass fragments for spotting of phosphopeptides in a negative ion parent scan, is proposed. The findings strengthen the value of negative ion ESI-MS/MS for analysis of protein phosphorylation. The positive ion mode is the standard polarity for peptide analysis by mass spectrometry. This selection is a direct consequence of the highly informative sequence information that can * Corresponding author. Tel.: ++ 49-6221-424563. Fax: ++ 49-6221-424554. E-mail: [email protected]. † Central Spectroscopy. ‡ Central Peptide Synthesis Unit. 10.1021/ac051649v CCC: $33.50 Published on Web 01/14/2006

© 2006 American Chemical Society

be extracted from the fragment ions of protonated peptides through collision-induced dissociation (CID), which forms a stable basis for database-supported protein identification by tandem mass spectrometry.1 The widely accepted fragmentation mechanism model for the decomposition of protonated peptides is the socalled mobile proton model.2,3 In short, this model explains the CID of protonated peptides by the principle that in multiply protonated peptides the protons are located initially at the most basic sites, which are the peptide N-terminal amino group and the side-chain functional groups of K, R, and H. Upon collisioninduced activation, the extra protons become mobile and are capable of residing temporarily at all basic sites in the peptide, which include the peptide backbone amide nitrogens. Recently, the mobility and complete randomization of peptide acidic hydrogens including the amide hydrogens before collision-induced dissociation has been verified experimentally.4 CID of negative ions of peptides5 has been used only sporadically for structure analysis6 for a variety of reasons: (i) the ESI negative ion mode in general is less sensitive compared to the positive ion mode, (ii) sequence information from negative ion MS/MS spectra of peptides is more difficult to extract since an evenly spread backbone fragmentation is not generally observed, (iii) fragmentation of peptide anions is often governed by neutral loss reactions with involvement of side chains,5 and finally (iv) the body of experimental information on MS/MS spectra of peptide anions is still much smaller compared to positive ions. Currently, the majority of the few applications of negative ion MS in proteomics is connected to modified, strongly acidic peptides as, for instance, phospho- and sulfopeptides, which normally give (1) Mortz, E.; O’Connor, P. B.; Roepstorff, P.; Kelleher, N. L, Wood, T. D, McLafferty, F. W.; Mann, M. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 82648267. (2) Dongre, A. R.; Jones, J. L.; Somogyi, A.; Wysocki, V. H. J. Am. Chem. Soc. 1996, 118, 8365-8374. (3) Wysocki V. H.; Tsaprailis, G.; Smith, L. L.; Breci, L. A. J. Mass Spectrom. 2000, 35, 1399-1406 (4) Jorgensen, T. J. D.; Gardsvoll, H.; Ploug, M.; Roepstorff P. J. Am. Chem. Soc. 2005, 127, 2785-2793. (5) Bowie, J. H.; Brinkworth, C. S.; Dua, S. Mass Spectrom Rev. 2002, 21, 87107. (6) Boontheung, P.; Alewood, P. F.; Brinkworth, C. S.; Bowie, J. H.; Wabnitz, P. A.; Tyler, M. J. Rapid Commun. Mass Spectrom. 2002, 16, 281-286.

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abundant molecular ion signals especially when analyzed under close-to-neutral or slightly basic conditions.7 For peptides carrying tyrosine-O-sulfate (sTyr), the negative ion mode is attractive because the corresponding sTyr-peptide anions exhibit a much higher stability compared to their protonated counterparts.8,9 Peptides carrying a pSer, pThr, or pTyr residue in the negative mode give rise to specific anionic fragments at m/z 79 ) [PO3]-, m/z 97 ) [H2PO4]-.10-12 Besides, the deprotonated molecules of p-Ser- and pThr-containing peptides show a characteristic neutral loss of phosphoric acid (98 Da) in analogy with the reaction observed in the positive ion mode, which is commonly used for the detection of the protonated phosphopeptides. Protonated molecules of phosphopeptides modified at tyrosine exhibit loss of HPO3 (80 Da) and produce a pTyr-specific marker ion at m/z 216.13,14 Unfortunately, the efficiency of the formation of the phosphate-specific fragments from the positively charged phosphopeptides is sequence-dependent,15 which may hamper phosphoprotein analysis in the positive ion mode. In addition, the detection of phosphopeptides containing multiple aspartic and glutamic acid residues by positive ion MS often fails because of the highly acidic nature of the peptides.16 Several negative ion LC-ESI-MS/MS strategies based on the detection of the phosphate marker anions have been established, where the phosphate anions are used for detection of peptide or protein phosphorylation either by LC-CID-ESI-MS11,17,18 or by precursor ion scanning.19,20 However, under normal operating conditions, the transmission for low-mass species in quadrupole time-of flight (Q-TOF) instruments is low (in the order of 5%)21 and the transmission of selected mass regions can be increased only at the expense of the other parts of the spectra.22 The Q-TOF mass spectrometers are powerful for detection of posttranslational modifications of proteins due to their high accuracy, resolution, and sensitivity. In addition to the poor transmission of the phosphate anions, the background level in the low-mass region of MS/MS spectra is generally increased compared to the high-mass region. Furthermore, the approach based on the monitoring of the phosphate reporter ions might provide false-positive results because of the possible in(7) Carr, S. A.; Huddleston, M. J.; Annan, R. S. Anal. Biochem. 1996, 239, 180192. (8) Rappsilber, J.; Steen, H.; Mann, M. J. Mass Spectrom. 2001, 36, 832-833. (9) Salek, M.; Costagliola, S.; Lehmann, W. D. Anal. Chem. 2004, 76, 51365142. (10) Huddleston, M. J.; Annan, R. S.; Bean, M. F.; Carr, S. A. J. Am. Soc. Mass Spectrom. 1993, 4, 710-717. (11) Ding, J.; Burkhart, W.; Kassel, D. B. Rapid Commun. Mass Spectrom. 1994, 8, 94-98. (12) Tholey, A.; Reed, J.; Lehmann, W. D. J. Mass Spectrom. 1999, 34, 117123. (13) Lehmann, W. D. Proceedings of the 32. Tagung der Deutschen Gesellschaft fu ¨ r Massenspektrometrie, Oldenburg, 1999; Poster P47, p 112. (14) Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Anal. Chem. 2001, 73, 1440-1448. (15) Salek, M.; Alonso, A.; Pipkorn, R.; Lehmann, W. D. Anal. Chem. 2003, 75, 2724-2729. (16) Kocher, T.; Allmaier, G.; Wilm, M. J. Mass Spectrom. 2003, 38, 131-137 (17) Jedrzejewski, P. T.; Lehmann, W. D. Anal. Chem. 1997, 69, 294-301. (18) Beck, A.; Deeg, M.; Moeschel, K.; Schmidt, E. K.; Schleicher, E. D.; Voelter, W.; Haring, H. U.; Lehmann, R. Rapid Commun. Mass Spectrom. 2001, 15, 2324-2333. (19) Neubauer, G.; Mann, M. J. Mass Spectrom. 1997, 32, 94-98. (20) Annan, R. S.; Huddleston, M. J.; Verma, R.; Deshaies, R. J.; Carr, S. A. Anal. Chem. 2001, 73, 393-404. (21) Chernushevich, I. V.; Ens, W.; Standing, K. G. Anal. Chem. 1999, 71, 452A461A. (22) Chernushevich, I. V. Eur. J. Mass Spectrom. 2000, 6, 471-480.

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solution formation of noncovalently bound adducts of the phosphates present in the solution and nonphosphorylated peptides of the analyte.23,24 In the present work, we have observed that upon CID conditions multiply charged phosphopeptide anions produce highmass phosphate-specific marker fragments as a general phenomenon. Their detection at high sensitivity is compatible with highsensitivity detection of the complete mass range of organic fragment ions. In this study, the occurrence of these fragment ions is documented, a mechanism for their formation is proposed, the utility of the marker ions for phosphopeptide detection is demonstrated, and a new MS/MS data analysis mode for phosphopeptide detection is developed. EXPERIMENTAL SECTION Mass Spectrometry. NanoESI-QTOF tandem mass spectrometry was performed on a Q-Tof2 instrument (Waters Micromass, Manchester, U.K.). 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). Negative ion parent ion scanning was performed at 31 V offset with a step width of 2 Da. Neutral loss and m/z gain () neutral gain option of the software) data analyses were also performed with a peak width of 2 Da. The software version used was MassLynx 3.5, and 3D collision offset plots were prepared using Origin version 6.1. Protein Digestion. Ovalbumin (Sigma-Aldrich, Taufkirchen, Germany) was purified by 1D gel electrophoresis, reduced by dithiothreitol, and alkylated by iodoacetamide as described.25,26 Digestion was performed in-gel at pH 8.0 using a mixture of trypsin and AspN (Roche Diagnostics, Mannheim, Germany). Phosphopeptide Synthesis. For solid-phase synthesis of the phosphopeptides, we employed the Fmoc strategy27,28 in a multiple automated synthesizer (Syro II, Multisyntech). Peptide chain assembly was performed using in situ activation of amino acid building blocks by 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate. Fmoc-Thr(PO(OBzl)OH)-OH, FmocTyr(PO(OBzl)OH)-OH, and Fmoc-Ser(PO(OBzl)OH)-OH were purchased from Merck Biosciences GmbH. RESULTS AND DISCUSSION Phosphate Group-Driven Fragmentation of Phosphopeptide Anions. Following CID, singly charged pS- or pT-phosphopeptide anions show neutral loss of H3PO4 () 97.977) and the marker ions [PO3]- at m/z 79 () 78.959 Da) and [H2PO4]- at m/z 97 () 96.970 Da).12 In this study, we demonstrate that, in addition to the above fragments, multiply charged phosphopeptide anions show high-mass phosphorylation-specific fragment ions, which are complementary to the established inorganic phosphate (23) Henry, K. D.; Williams, E. R.; Wang, B. H.; McLafferty, F. W.; Shabanowitz, J.; Hunt. D. F. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 9075-9078. (24) Chowdhury, S. K.; Katta, V.; Beavis, R. C.; Chait, B. T. J. Am. Soc. Mass Spectrom. 1990, 1, 382-388. (25) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Anal. Chem. 1996, 68, 850-858. (26) Kinter, M.; Sherman, N. E. An in-gel-digestion protocol. In Protein Sequencing and Identification Using Tandem Mass Spectrometry; John Wiley: Chichester, 2000; Chapter 6.4, pp 152-160. (27) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149-2154. (28) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37, 3404-3409.

Figure 1. Negative ion tandem mass spectra of phosphopeptide dianions. (a) The [SA-pT-PEALAFVR]2- ion mainly decomposes into two complementary pairs of fragment ions at m/z 79/m/z 1159.7 and at m/z 97/m/z 1141.7 (collision offset, 18-22 V), a weak neutral loss of H3PO4 is observed in addition; (b) the [HYQP-pY-APPR]2- ion mainly decomposes into the complementary pair of fragment ions at m/z 79 and 1126.6. (collision offset, 18-22 V). The ion at m/z 137 represents an ion generated from the background.

ion at m/z 79. Figure 1a shows the MS/MS spectrum of the [M - 2H]2- ion of the phosphopeptide SA-pT-PEALAFVR as an example. This phosphorylation site was recently spotted in choline acetyltransferase by positive ion ESI-MS/MS.29 The MS/MS spectrum recorded at moderate collision offset (18-22 V) shows two complementary fragment ion pairs at the m/z values 79/1159.7 and 97/1141.7 as the most abundant fragment ions. These ion pairs clearly identify the investigated peptide as phosphopeptide. It should be noted that the fragment at m/z 1141.7 can also be formed by loss of water from the [M - 2H - 79]- ion. Upon CID, phosphotyrosine-containing peptide anions preferentially form the phosphate marker ion at m/z 79 whereas the fragment at m/z 97 is of minor abundance.12 This is due to the fact that phosphotyrosine represents an ester of phosphoric acid with an aromatic hydroxyl function, so that the C-O bond in pY is stronger compared to the corresponding C-O bond in pS and pT, where phosphoric acid is esterified with an aliphatic hydroxyl (29) Dobransky, T.; Brewer, D.; Lajoie, G.; Rylett, R. J. J. Biol. Chem. 2003, 278, 5883-5893.

function. A typical MS/MS spectrum of the [M - 2H]2- ion of a pY-peptide is shown in Figure 1b. The sequence of this peptide occurs in a T-cell-specific surface glycoprotein. This high-mass fragment of the type [M - 2H - 79]- is particularly useful for the identification of phosphotyrosine phosphopeptides, since the pY typical neutral loss of HPO3 is generally of low abundance. In positive ion mode, a pY-specific marker ion at m/z 216 exists;13,14 however, the abundance of this ion is also sequence-dependent.15 Formation of the [M - 2H - 79]- Fragments. The fragmentation behavior of the [M - 2H]2- ions of 25 synthetic phosphopeptides (12 with pSer, 9 with pThr, 4 with pTyr, Table 1) has been investigated in this study. All peptides studied showed a fragmentation behavior similar to that of the peptides selected for Figure 1, with some variation in relative ion abundances. On this basis, we conclude that the described fragmentation behavior is a generally occurring property of doubly charged anions of pS-, pT-, and pY-phosphopeptides. Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

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Table 1. Sequences and Molecular Weight Data of the Synthetic Phosphopeptides Analyzed in This Studya pS-phosphopeptides IRV-pS-INEK FRG-pS-GDTSNF SA-pS-PEALAFVR FSIAPS-pS-LDPSNR FSIAP-pS-SLDPSNR F-pS-IAPSSLDPSNR FSIAPSSLDP-pS-NR FSIAP-pS-pS-LDPSNR F-pS-IAPS-pSL-DP-pS-NR F-pS-IAP-pS-pSL-DP-pS-NR F-pS-IAP-pS-pSL-DP-pS-NR GSEEESVKGSEEE-pS-VK pT-phosphopeptides pT-PEALAFVR FRGSGD-pT-SNF TW-pT-LSGTPEY SA-pT-PEALAFVR DCRR-pT-ISAPVVRPK ITI-pT-PNRITITPNR GSEEESVKGSEEE-pT-VK TW-pT-LCGTPEYLAPEY-LAPEIILSK IADPEHDHTGFL-pT-EYVATR pY-phosphopeptides TPD-pY-FL ISL-pY-DNPDQ HYQP-pY-APPR DQQD-pY-FFPK

mol wt (mono) 1037.5 1166.4 1226.6 1469.7 1469.7 1469.7 1469.7 1549.7 1629.7 1709.7 1709.7 1788.7 1082.5 1166.4 1233.5 1240.6 1249.5 1688.9 1802.8 2214.1 2251.0 834.32 1143.5 1207.5 1266.5

a Upon negative ion CID, the dianions of these peptides all exhibit phosphate-specific high-mass fragments (see Figure 1).

MS/MS spectra of the peptides were recorded in the range from 10 to 60 V. A three-dimensional display of the collision offset plots allows a comprehensive view on the peptide fragmentation behavior. In Figure 2, the MS/MS spectra of a nonphosphorylated peptide (FSIAPSSLDPSNR) and of its phosphorylated analogue (FSIAPSSLDP-pS-NR) are shown in this way. The latter one contains a phosphorylation site found in a human polokinase.30 The quantitatively most important difference between the MS/ MS spectra of the nonphosphorylated (Figure 2a) and the phosphorylated peptide (Figure 2b) is the occurrence of an intense

Table 2. Sequences and Molecular Weight Data of the Synthetic Multiply Phosphorylated Peptides Analyzed in This Study peptide sequence

mol wt (mono)

F-pS-IAP-S-S-LDP-pS-NR F-S-IAP-pS-pS-LDP-S-NR F-S-IAP-S-pS-LDP-pS-NR F-pS-IAP-S-pS-LDP-pS-NR F-pS-IAP-pS-pS-LDP-pS-NR

1549.62 1549.62 1549.62 1629.59 1709.55

double band of fragment ions around m/z 1300. The band with the higher m/z value is the [M - 2H - 79]- ion; the lower band is caused by a neutral loss of 30 Da () CH2O, formaldehyde) from the [M - 2H - 79]- ion.31 In Figure 2b, the intensities of the marker ions at m/z 79 and 97 are of low abundance, which is probably due to the reduced ion transmission of the Q-TOF analyzer in the low-mass region.21 Recently, pSer and pThr marker ions at m/z 166 and 180, respectively, have been reported to occur in negative ion ESI tandem mass spectra of phosphopeptides.32 These findings could not be confirmed in our study. MS/MS of Multiply Phosphorylated Peptide Anions. Examination of CID mass spectra of phosphopeptides containing multiple phosphorylation sites reveals a characteristic fragmentation pattern of such phosphopeptide anions. The observed behavior enables us to determine the exact number of phosphorylated sites on the basis of the number of subsequent neutral losses of H3PO4 from the [M - 2H - 79]- fragments. A series of multiply phosphorylated synthetic variants of the tryptic peptide FSIAPSSLDPSNR (Table 2) has been examined in this study. As expected, abundant high-mass fragments of the type [M - nH 79](n-1)- were observed in the CID spectra of each species. The phosphate-specific fragment ions showed neutral loss of the residual phosphate groups as H3PO4. As an example, the product ion spectrum of the peptide that carries four phosphoserine groups is presented in Figure 3. The spectrum displays the loss of three molecules of H3PO4 from the high-mass marker ion [M - 2H 79]-, indicating correctly the presence of four pSer residues in the peptide sequence. It should be noted that neutral loss of

Figure 2. Three-dimensional display of the collision offset plots of a doubly charged peptide anion and of its phosphorylated analogue; (a) FSIAPSSLDPSNR; (b) FSIAPSSLDP-pS-NR. The most striking effect induced by the presence of pSer is the occurrence of an abundant singly charged fragment, which represents the [M - 2H - 79]- ion. 1252 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

Figure 3. Product ion spectrum of the [M - 2H]2- ion of the quadruply phosphorylated peptide F-pS-IAP-pS-pS-LDP-pS-NR. The peptide dianion shows an abundant [M - 2H - 79]- fragment, which shows neutral loss of up to three units of H3PO4 originating from the three pSer residues present in this fragment.

Scheme 1. Proposed Fragmentation Mechanism for [M - nH - 79](n-1)- Anions of Phosphopeptides (pSer as an Example)a

a By collisional activation, a small population of ions is formed with both negative charges located at the phosphate group. In a free gaseous phosphopeptide ion, this structure effectively decomposes by shift of an electron pair into the complementary fragments [PO3]and [M - nH - 79](n-1)-.

H3PO4 is also observed from the molecular [M - 2H]2- ion; however, the ion at [M - 2H - 4H3PO4]2- is of extremely low abundance. Mechanism for Formation of the [M - nH - 79](n-1)Ions. The charge distribution within multiply charged phosphopeptide anions seems to be the key to understanding of the origin of the phosphorylation reporter ions in their MS/MS spectra. A plausible mechanistic pathway for the decomposition of doubly deprotonated phosphopeptides leading to the formation of the [M - nH - 79](n-1)- ions is displayed in Scheme 1. Upon formation by electrospray ionization, the two negative charges of the [M 2H]2- ions of phosphopeptides are probably located at the two most acidic groups, which are the phosphate group (pKa, first proton 2.1) and the carboxy terminus (pKa ∼3). Upon collisional activation, a mixture of structurally different anions is created by internal redistribution of mobile protons. At moderate collision offset, many of these charge isomers will not undergo characteristic fragmentation reactions. For instance, a negative charge on a C-terminal carboxy group will not induce backbone fragmentation at moderate activation energies (data not shown). A negative charge at a deprotonated backbone will not generally induce backbone fragmentation with high efficiency. However, if the charge is located at the amide nitrogen that is C-terminal to an Asp or Asn residue, it may effectively induce succinimide formation and backbone cleavage.33 Through the presence of the phosphate group with two acidic protons, phosphopeptides have a unique property, namely, to create a structure with two adjacent

negative charges at their phosphate group (structure A in Scheme 1). This type of structure cannot be formed in other unmodified or modified peptides. It is reasonable to assume that the structure A is one of the many charge isomers of phosphopeptide dianions that are generated upon collisional activation conditions. Because of the low stability, which is expected for such gaseous phosphoester dianions, it is highly probably that once this structure is formed it undergoes efficient heterolysis of its phosphate ester bond to give rise to two singly charged anions as displayed in Scheme 1. To verify the hypothesis that the unstable phosphopeptide dianion carrying the two negative charges at the phosphate group is not initially formed upon negative ion ESI, we compared the negative ion ESI spectra of the peptide VDNIRSApT and of its methyl-esterified form V-D(me)-NIRSA-pT-(me) (me, methyl). Examination of the spectra of the two peptides revealed that the protection of the two carboxylic groups of the peptide VDNIRSApT suppresses completely the formation of a doubly deprotonated molecular ion upon negative ion ESI conditions. The nanoESI spectrum of the phosphopeptide VDNIRSApT showed an abundant signal of the doubly charged molecular ion [M - 2H]2-. In contrast, the spectrum of the methyl-esterified form indicated that (30) Wind, M.; Kelm, O.; Nigg, E.; Lehmann, W. D. Proteomics 2002, 2, 15161523. (31) Edelson-Averbukh, M.; Pipkorn, R.; Lehmann, W. D. To be submitted. (32) Carlberg, I.; Hansson, M.; Kieselbach, T.; Schroder, W. P.; Andersson, B.; Vener, A. V. Proc. Natl. Acad. Sci U. S. A. 2003, 100, 757-762. (33) Schlosser, A.; Lehmann, W. D. J. Mass Spectrom. 2000, 35, 1382-1390.

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Figure 4. Collision offset plots showing the formation of the [PO3]ion at m/z 79 and of the [H2PO4]- ion at m/z 97 produced from singly and doubly charged phosphopeptide anions of SA-pT-PEALAFVR as indicated. At low collision offset, the favored formation of the [PO3]fragment from the doubly charged precursor ion is evident. For the mechanism of its formation, see Scheme 1.

multiple deprotonation of this peptide does not occur. The absence of the dianion in the ESI spectrum of the peptide V-D(me)-NIRSApT-(me) confirms that the labile structure A of the doubly ionized peptide, which decomposes with formation of the [M - 2H 79]- fragment, is not generated by electrospray ionization but mostly is the result of an additional activation in the collision cell. Further evidence for the fragmentation mechanism given in Scheme 1 comes from collision offset plots, which show the intensity of the inorganic marker ions m/z 79 [PO3]- and m/z 97 [H2PO4]- as a function of the collision offset, during their formation from singly and doubly charged phosphopeptide anions. These results are summarized in Figure 4. As demonstrated in Figure 4, with increasing collision offset first the m/z 79 fragment is formed from doubly charged anions. There is no precursor-product relationship between [H2PO4]-

and [PO3]-, at least not at collision offset values below 30 V. This finding supports the model that [PO3]- is formed from an activated state, which is postulated to be the OPO3]2- group. In our earlier LC-ESI-skimmerCID-MS studies,17 we performed CID collision offset plots and observed sigmoidal curves for both the fragments m/z 79 and 97, with optimal fragment ion abundances above 6070 V. The precursor ions under these conditions represented mainly a mixture of singly and doubly charged anions, so that the specific contribution of selected charge states could not be recognized. In view of the results of Figure 4, we would generally recommend lower collision offset values for such studies, since (i) the ion transmission of MS/MS systems is more favorable for low offset values and since (ii) the background is generally lower in MS/MS spectra produced with moderate offset values. Analysis of an Ovalbumin Digest. To evaluate the usefulness of the newly described high-mass phosphopeptide fragment for identification of protein phosphorylation sites, we analyzed chicken ovalbumin as a reference phosphoprotein. For this system, two major phosphorylation sites at Ser-68 and Ser-344 are known. Ovalbumin was digested in-gel with a mixture of trypsin and AspN and investigated by negative ion parent ion scanning. As shown in Figure 5, this data set was analyzed in different ways (extracted ion trace, neutral loss, m/z gain) for the presence of phosphopeptides. The extracted ion trace for m/z 79 did not give an indication for the presence of phosphopeptides (Figure 5a). The neutral loss analysis for m/z 49 (Figure 5b) indicated three phosphopeptides, which contain the two established phosphorylation sites in ovalbumin (see Figure 6). The high-mass satellites in the peak groups in Figure 5b correspond to metal adducts of the phosphopeptides, which also exhibit neutral loss of H3PO4. An example of an m/z gain analysis is shown in Figure 5c. This is a rarely used MS/MS mode, which can be employed for detection of intermolecular reaction products with increased mass34,35 orsas in this casesfor detection of fragmentation processes that create ions with increased m/z value due to their reduced charge state caused by loss of a charged fragment. In contrast to the extracted ion trace for m/z 79, the m/z gain analysis

Figure 5. Negative ion nanoESI parent scan of a trypsin + AspN digest of ovalbumin (offset value, 31 V): (a) extracted ion trace of m/z 79; (b) neutral loss analysis for m/z 49, tailored for detection of loss of H3PO4 from doubly charged anions; (c) m/z gain analysis for m/z 625, tailored for detection of 336EAGREVVG-pS-AEAGV;349 (d) total ion current. Both neutral loss and m/z gain analysis provide a positive identification of the ovalbumin phosphorylation sites. In contrast, the marker ion formation of m/z 79 in this case is of too low abundance to allow spotting of phosphopeptides. 1254 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

Figure 6. MS/MS spectra of the parent ion scan file shown in Figure 5, recorded at m/z 703, 883, and 1019. Three phosphopeptides containing the two established phosphorylation sites of ovalbumin are detected. The “high-mass” fragments are visible in each spectrum. (cam, carbamidomethyl).

for 625, which identifies the phosphopeptide 336EAGREVVG-pSAEAGV349 on the basis of the formation of the [M - 2H - 79]ion, clearly indicates the phosphorylation of Ser-344 of ovalbumin (see Figures 5c and 6c). Similarly, the m/z gain analysis for m/z 803 (m/z difference between the [M - 2H]2- and the [M - 2H - 79]- ion) and 922 (m/z difference between [M - 2H]2- and the [M - 2H - 79 - H2O]- ion) reveals the presence of the two other phosphorylated species in the trypsin + AspN digest mixture of ovalbumin: 333EINEAGREVVG-pS-AEAGV349 and 67D-pS-IAEQcamC-GTSVNVHSSLR,84 respectively (data not shown). It should be noted that for the peptides 333-349 and 67-84 the abundances of the corresponding high-mass marker ions are much lower than that for the shorter peptide 336-349. This effect may be attributed to software restrictions that do not allow use of a mass-dependent collision offset for the parent ion scan. The phosphate-driven fragmentation behavior described above for dianions of phosphopeptides is also observed for ions with higher charged states. Thus, product ion spectrum of triply charged molecular ion [M - 3H]3- of the peptide 67-84 from ovalbumin reveals that the trianion loses [PO3]- to give rise to an abundant [M - 3H - 79]2- ion accompanied by a [M - 3H 79 - 18]2- ion. The high-mass phosphate-specific species are the most abundant fragments of the triply deprotonated phosphopeptide. The ions are important reporters of the presence of a phosphate group in the peptide 67-84, since no elimination of H3PO4 from the molecular ion [M - 3H]3- is observed. Variable m/z Gain Analysis for Multiply Charged Phosphopeptide Anions. The high-mass phosphate-specific fragments of [M - 2H]2- ions of phosphopeptides exhibit a characteristic m/z difference relative to their precursor ions, which is defined by the equation

m/z(fragment ion) - m/z(molecular ion) ) [ m/z(molecular ion) × 2 - 79 ] - m/z(molecular ion) Thus, this mass difference can be calculated for each molecular ion, and this mathematical correlation can be used to design a variable m/z gain analysis. With the software available for this study, only a constant m/z gain analysis could be performed. To

Figure 7. Evaluation of the parent ion scan file shown in Figure 5 with a variable m/z gain analysis, which was set up to detect the [M - 2H - 79]- fragments formed from [M - 2H]2- ions of phosphopeptides. In the selected m/z range of the parent ion scan file, the variable m/z gain analysis detects the [M - 2H]2- ion of the peptide 336EAGREVVG-pS-AEAGV349 at m/z 702.8.

demonstrate the utility of a currently unavailable software function for a variable m/z gain analysis, we performed a manual simulation using as data file the parent ion scan shown in Figure 5. The m/z range from 650 to 750 containing the signal of the [M - 2H]2ion of the peptide EAGREVVG-pS-AEAGV at m/z 702.8 was selected. For each m/z value in this range, we calculated the specific m/z gain, performed a m/z gain analysis with the resulting value, and documented the ion intensity of the m/z gain trace at the selected m/z value. Then, these data points were plotted as a function of the m/z value. Figure 7 displays the result of this simulation. As expected, this variable m/z gain analysis showed a peak at m/z 703, corresponding to the [M - 2H]2- ion of the aforementioned phosphopeptide 336EAGREVVG-pS-AEAGV.349 A variable m/z gain scan is useful for detection of any class of multiply charged ions, where one building block with constant m/z value is lost, whereas the other part consequently exhibits a (34) Schwartz, J. C.; Wade, A. P.; Enke, C. G.; Cooks, R. G. Anal. Chem. 1990, 62, 1809-1818. (35) Chen, H.; Zheng, X.; Cooks, R. G. J. Am. Soc. Mass Spectrom. 2003, 14, 182-188.

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variable mass value. The addition of this search tool to a standard software of tandem mass spectrometers would increase the versatility of MS/MS analyses, allowing combination of the results of extracted ion traces and neutral loss traces with the complementary results of a variable m/z gain analysis. Thus, it can be summarized that negative ion MS/MS spectra provide a triplet of features for specific detection of phosphopeptides, (i) marker ions (79 and 97), (ii) constant neutral loss (H3PO4 for pSer and pThr; HPO3 for pY), and (iii) variable m/z gain based on the [M - nH - 79](n-1)- marker ions for pSer, pThr, and pY. Precursor ion scanning for the fragments [M - nH 79](n-1)- has advantages over the conventional detection of the marker ions PO3- at m/z 79 and H2PO4- at m/z 97. The transmission of the high-mass fragments in quadrupole time-oflight instruments is much better than that of the low-mass phosphate anions, and the background in the high-mass region is lower than in the low-mass region. In addition, negative ion product spectra of peptides contain less sequence-specific fragment ions than the corresponding positive ion mode spectra. The

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sum of these features effects that MS/MS scan modes for detection of phosphopeptides in the negative ion mode exhibit very specific results, in particular when a neutral loss scan or an m/z gain scan is employed. A prerequisite for the latter is multiple deprotonation, and our study demonstrates that phosphopeptides of diverse sequences and lengths are forming doubly negatively charged ions. It is our belief that the finding of the phosphatespecific marker ions reported here will help to establish negative ion ESI-MS/MS as one of the most versatile and specific tandem MS techniques for the spotting of phosphopeptides. ACKNOWLEDGMENT M.E.-A. gratefully acknowledges the financial support of the Minerva Foundation of the Max-Planck-Society.

Received for review September 15, 2005. Accepted December 6, 2005. AC051649V