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Originally published In Press as doi:10.1074/mcp.T400019-MCP200 on March 7, 2005.
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Molecular & Cellular Proteomics 4:809-818, 2005.
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

Technology

Identification of Phosphopeptides by MALDI Q-TOF MS in Positive and Negative Ion Modes after Methyl Esterification*,S

Chong-Feng Xu{ddagger}, Yun Lu{ddagger}, Jinghong Ma§, Moosa Mohammadi§ and Thomas A. Neubert{ddagger},§,

From the {ddagger} Skirball Institute of Biomolecular Medicine, § Department of Pharmacology, New York University School of Medicine, New York, New York 10016


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of ß-casein and {alpha}-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.

Reversible phosphorylation of serine, threonine, and tyrosine residues is one of the most common and important regulatory modifications of proteins and often is a key event in cellular signal transduction (1, 2). In recent years, mass spectrometry has become a key technology for characterization of protein phosphorylation and phosphoproteome analysis. Two complementary ionization techniques, MALDI and ESI, in combination with a variety of mass analyzers, have been used to identify phosphopeptides and determine the phosphorylated amino acids on the peptides (3–16). In most cases, characterization of phosphopeptides by MS requires selection of phosphorylated peptides from complex peptide mixtures resulting from proteolysis of phosphorylated proteins followed by MS/MS to confirm the phosphorylation and to identify the phosphorylated amino acid residues on phosphopeptides containing more than a single serine, threonine, or tyrosine residue. Despite many recent advances in methodology, identification of phosphorylation sites on proteins remains a difficult challenge (17).

High sensitivity, resolution, and mass accuracy make Q-TOF MS a powerful tool for the characterization of phosphopeptides. The MALDI Q-TOF allows for increased efficiency and sample throughput because identification of phosphopeptides by MS and characterization by CID MS/MS can be performed on a single sample spot (18). However, a limitation of this type of experiment is the low ionization efficiency of phosphopeptides in positive ion mode, resulting in low sensitivity of phosphopeptide detection and consumption of a large portion of the sample during the search for phosphorylated precursor peptides before MS/MS can be performed (18). Phosphorylated precursor ions can be detected by comparing MALDI spectra of a single sample taken in positive and negative ion modes, with phosphopeptides demonstrating greater relative ion intensities in negative ion mode (19, 20). However, this approach suffers from poor specificity for phosphopeptides because of the high background of nonphosphorylated acidic peptides in negative ion mode caused by the ability of carboxylate groups on glutamate or aspartate residues to develop negative charges in a manner similar to that of phosphate groups. In this article, we show that removal of these acidic groups by methyl esterification (21, 22) can greatly diminish the ion intensity of these acidic nonphosphorylated peptides in negative ion mode and therefore greatly increase the selectivity of the method for phosphopeptides in peptide mixtures. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of a model phosphoprotein, ß-casein, and its minor contaminant {alpha}-s-casein.

The fibroblast growth factor receptors (FGFRs)1 are a family of tyrosine kinase receptors that play critical roles in human skeletal development. Gain of function mutations in the tyrosine kinase domain of FGFRs are responsible for a number of human skeletal disorders. The degree of clinical severity associated with the mutations correlates with the level of constitutive kinase activity in these mutants (23–25). A method for rapidly comparing phosphorylation sites on various mutants of FGFRs and then correlating phosphorylation status with receptor activity would be very useful for understanding the molecular basis for receptor gain of function and potentially facilitate development of therapeutic interventions. We used our method to characterize phosphopeptides on the in vitro phosphorylated kinase domain of the N549H mutant of the FGFR2, which is responsible for the severe craniosynostosis disorder known as Crouzon syndrome (26). After in vitro phosphorylation by incubation with ATP, isolation by SDS-PAGE, and in-gel tryptic digestion, we identified 10 phosphopeptides containing five phosphorylation sites from 100 fmol of the mutant kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. These identified tyrosine phosphorylation sites correspond to those previously found to be phosphorylated on the kinase domain of FGFR1 (27), demonstrating that our method can be used to rapidly characterize phosphorylation sites on low levels of receptors or other proteins such as can be obtained in biological experiments.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—
2,6-Dihydroxyacetophenone (DHAP), {alpha}-cyano-4-hydroxycinnamic acid (CHCA), dihydroxybenzoic acid (DHB), diammonium hydrogen citrate, and acetyl chloride were purchased from Sigma-Aldrich (St. Louis, MO). Sequencing grade modified trypsin was purchased from Promega Co. (Madison, WI). Ammonium bicarbonate, TFA, [Glu1]-Fibrinopeptide B, BSA, ß-casein (from bovine milk, purity >90% by electrophoresis,) and the monophosphopeptide and tetraphosphopeptide from ß-casein were from Sigma Chemical Co. HPLC grade water, acetonitrile, and methanol were purchased from Fisher Scientific (Hanover Park, IL). Coomassie Blue R-250 and precast 12% SDS-PAGE gels were purchased from Bio-Rad.

Preparation of in Vitro Phosphorylated FGFR Kinase Domain—
Affinity purified kinase domain of FGFR2 harboring the N549H mutation was concentrated using a Centricon filtration device to a concentration of 10 mg/ml as measured by A280. To generate the phosphorylated form, the kinase domain was incubated with 5 mM ATP and 10 mM MgCl2. The course of phosphorylation was monitored by native gel analysis until the phosphorylation was complete (~10 min). The phosphorylation reaction was stopped by adding 20 mM EDTA. For preparation of in-gel digests for mass spectrometry analysis, the sample buffer was prepared by mixing Bio-Rad Laemmli sample buffer with mercaptoethanol at a ratio of 20:1 (v/v). Two picomoles of the phosphorylated kinase domain was diluted in this sample buffer at a ratio of 1:1 (v/v), denatured at 95 °C for 5 min. The sample was separated on a 12% SDS-PAGE gel at 100 V. Proteins were visualized by staining with Coomassie Blue R-250.

In-solution Digestion and in-Gel Digestion of Proteins—
For in-solution digestion, proteins were dissolved in 25 mM ammonium bicarbonate and denatured by heating in 95 °C water for 5 min and then digested in 25 mM ammonium bicarbonate by sequence-grade trypsin at a ratio of 1:50 (enzyme/protein) for 5 h at 37 °C. The in-gel digestion of the phosphorylated FGFR2 kinase domain was carried out according to the protocol of Shevchenko et al. (28), except that the reduction and alkylation step was omitted.

Formation of Methyl Esters—
The methanolic HCl solution was prepared by the dropwise addition of 160 µl of acetyl chloride to 1 ml of dry methanol (22). The protein tryptic digests up to 250 pmol in amount were lyophilized and redissolved in 50 µl of 2 M methanolic HCl regent. When more than 250 pmol of peptides were methylated, the volume was 200 µl of methanolic HCl. Methyl esterification was allowed to proceed for 2–3 h at room temperature. Solvent was removed by lyophilization, and the resulting samples were redissolved in 30% acetonitrile in 0.2% TFA.

Preparation of Matrix for MALDI Q-TOF Mass Spectrometry—
A 50 mM solution of the DHAP matrix was prepared by dissolving 15.2 mg of 2,6-dihydroxyacetophenone in 1 ml water/methanol (10:90, v/v) followed by the addition of 100 mM diammonium hydrogen citrate in water at a ratio of 1:1 (v/v).2 CHCA matrix was prepared by dissolving 2 mg of {alpha}-cyano-4-hydroxycinnamic acid in 1 ml of water/acetonitrile (50:50, v/v) containing 0.1% TFA.

MALDI Q-TOF Mass Spectrometry—
Sample and matrix were mixed at a ratio of 1:1 (v/v), and 1.0 µl of this mixture was spotted onto the MALDI sample stage. Positive and negative ion MALDI Q-TOF mass spectra were acquired with a Micromass Q-TOF Ultima MALDI mass spectrometer (Waters, MA). The instrument was operated in V mode with a mass resolution of ~10,000, which enabled the discrimination of carboxyl methylation (+14 atomic mass units) and asparagine/glutamine methylation (+15 atomic mass units). Laser pulses were generated by a nitrogen laser (337 nm) with laser energy of 350 µJ per pulse. Mass spectra were acquired and processed by Masslynx 4.0 software (Micromass Ltd., Manchester, United Kingdom). A total of 200–800 laser shots were averaged per mass spectrum, the background was subtracted, and the spectrum was smoothed using a mass window appropriate for the significant peak widths. Known peptide masses were used as internal mass standards.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Phosphorylation Sites on ß-Casein—
ß-Casein is a model phosphoprotein used to develop and test methods to characterize phosphopeptides. Fig. 1 shows positive and negative mode MALDI Q-TOF mass spectra of 140 fmol of a tryptic digest of ß-casein before and after methylation. As shown in Fig. 1, a and b, and previously by us and others (19, 20), the phosphopeptides (indicated by asterisks) have higher relative abundance in the negative ion MALDI spectrum compared with the positive ion spectrum. However, the relative difference between the positive and negative ion intensities of the phosphopeptides is subtle and difficult to observe in the case of the monophosphorylated peptides of m/z 1949.94 from ({alpha}-casein) and 2059.81 (FQpSEEQQQTEDELQDK, calculated monoisotopic MW = 2060.82) from ß-casein (Fig 1b). In addition to the tetraphosphopeptide (RELEELNVPGEIVEpSLpSpSpSEESITR, calculated MW = 3121.26) at m/z 3120.25 and several resulting neutral loss fragments resulting from the phosphopeptides, numerous nonphosphorylated peptides also can be observed in the negative ion mode spectrum. Fig. 1, c and d, show positive and negative ion spectra of 140 fmol of the same tryptic digest of ß-casein, but after methylation. In this case, the phosphorylated peptides (designated by asterisks and listed in Table I) produce the predominant peaks in the negative ion spectrum and demonstrate a much greater relative increase in negative ion mode compared with positive ion mode. Indeed, 17 phosphorylated peptides from the minor contaminants {alpha}-s1-casein and {alpha}-s2-casein (present at less than 14 fmol based on the manufacturer’s analysis that the ß-casein is more than 90% pure) can be observed in Fig 1d. These peptides contain all 5 previously described phosphorylation sites from ß-casein, all 8 phosphorylation sites from the contaminant {alpha}-s1-casein, and 8 of the 10 previously described phosphorylation sites on {alpha}-s2-casein. We also identified one phosphorylation site on {alpha}-s2-casein (Ser-143) not reported in the ExPaSy database. The sensitivity of the method is further illustrated in Fig. 1, e and f, in which both of the phosphopeptides, containing all five phosphorylation sites from the ß-casein digest, can be seen at the 7 fmol level in negative ion mode. Although the positive ion mode signal intensity of many nonphosphorylated peptides increased after methylation (e.g. peptide LLYQEPVLGPVR of m/z 1383.79 in Fig. 1a compared with m/z 1411.75 in Fig. 1c), the intensity of the major methylated peptide species decreased in some cases because of partial methylation of glutamine and asparagine residues (see below), which leads to additional peak complexity for these peptides (e.g. peptide SPAQILQWQVLSNTVPAK of m/z 1980.10 in Fig. 1a compared with m/z 1994.00, 2008.98, 2023.95, etc. in Fig. 1c).



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  		FIG. 1. MALDI Q-TOF MS of methylated and nonmethylated ß-casein tryptic digest in positive and negative ion modes. a, spectrum of 140 fmol of ß-casein digest acquired in positive ion mode; b, spectrum of 140 fmol of ß-casein digest acquired in negative ion mode; c, spectrum of 140 fmol of methylated ß-casein digest acquired in positive ion mode; d, spectrum of 140 fmol of methylated ß-casein digest acquired in negative ion mode; e, spectrum of 7 fmol of methylated ß-casein digest acquired in positive ion mode; f, spectrum of 7 fmol of methylated ß-casein digest acquired in negative ion mode. Predominant phosphopeptides species are labeled (*), undermethylated (one or more carboxylate groups not methylated) are labeled (–), and methyl ester side products of deamidated Gln or Asn residues are labeled (+). Each spectrum is the sum of 740 laser shots. Base peak (maximum) ion counts are shown in the upper right corner of each spectrum. The 12 phosphopeptides identified in d, containing five phosphorylation sites from ß-casein and 17 phosphorylation sites from the minor contaminants {alpha}-s1 and {alpha}-s2-casein, are listed in Table I.

 

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  		TABLE I Phosphopeptides detected in a methylated tryptic digest of 140 fmol of ß-casein

MW (without methylation) is the calculated molecular weight of the phosphopeptides before methylation; MW-H (after methylation) is the observed m/z of the singly charged methylated phosphopeptides measured in the negative ion spectra shown in Figs. 1 and 2.

 
Identification of Phosphorylation Sites in a Mixture of BSA and ß-Casein Tryptic Digests—
To demonstrate that the method can be used to study more complex peptide mixtures, a similar analysis was carried out on a tryptic digest of a mixture of 140 fmol of BSA and 140 fmol of ß-casein (Fig. 2). The presence of the BSA peptides increased the complexity of the spectra of the unmethylated peptides in both positive and negative modes (Fig. 2, a and b) and the methylated peptides in positive ion mode (Fig 2c). However, only phosphopeptides produced peaks of significant ion intensity in the negative ion spectra of the methylated peptide digest (Fig. 2d). In Fig. 2f, three phosphopeptides (including one missed cleavage peptide) containing all five phosphorylation sites from ß-casein and four of the {alpha}-casein phosphopeptides can be observed in negative ion mode spectra of 14 fmol of the methylated peptide mixture (containing less than 2 fmol of the {alpha}-casein contaminants). These results indicate that our method is capable of identifying small amounts of phosphopeptides directly from simple protein mixtures without the need for further purification or enrichment.



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  		FIG. 2. MALDI Q-TOF MS of methylated and nonmethylated mixtures of ß-casein and BSA tryptic digests in positive and negative ion modes. a, spectrum of 140 fmol of nonmethylated digest mixture acquired in positive ion mode; b, spectrum of 140 fmol of nonmethylated digest mixture acquired in negative ion mode; c, spectrum of 140 fmol of methylated digest mixture acquired in positive ion mode; d, spectrum of 140 fmol of methylated digest mixture acquired in negative ion mode; e, spectrum of 14 fmol of methylated digest mixture acquired in positive ion mode; and f, spectrum of 14 fmol of methylated digest mixture acquired in negative ion mode. c–f, signal intensity of m/z above 2500 is increased 10-fold. Predominant phosphopeptides species are labeled (*), undermethylated (one or more carboxyl groups not methylated) are labeled (–), and methyl ester side products of deamidated Gln or Asn residues are labeled (+). The labeled phosphopeptides are listed in Table I.

 
In each negative ion spectrum of the methylated peptides, the major phosphopeptide peaks, designated by asterisks in the figures, correspond to the peptides with complete methylation of carboxylate groups. For example, in Figs. 1, d and f, and 2, d and f, the peak at m/z 2157 corresponds to FQpSmEmEQQQTmEmDmELQmDmK, where m indicates the methyl esterified acidic or C-terminal amino acid residue, whereas 3232 corresponds to the tetraphosphorylated peptide with complete methylation of carboxylate groups. Minor peaks, designated by (–), correspond to phosphopeptides with incomplete methyl esterification of acidic groups. Some peptides, indicated by (+) in Figs. 1–3, undergo deamidation followed by methyl esterification on side chains of asparagine and/or glutamine, resulting in a mass increase of 15 Da because of substitution of methyl-oxide (-OCH3) for the amine group (-NH2) (29). Consistent with the results of He et al. (29), we have found that this side reaction is unavoidable even when using dehydrated methanol and extensively drying the peptides, given that the reaction must proceed for a minimum of 2–3 h to ensure nearly complete methyl esterification of acidic groups (21). We evaluated the methylation of Glu1-Fibrinopeptide B peptide (sequence EGVNDNEEGFFSAR) and a tryptic digest of {alpha}-s-casein tryptic digest in a time course experiment. Methyl esterification of acidic groups of most peptides was more than 95% complete after 2 h without side product (amine) methylation in 60 and 80% of peptides containing 2 to 4 and mono Gln/Asn, respectively. However, those peptides with multiple acidic amino acids and Gln/Asn, such as the tetraphosphopeptide in ß-casein and the pentaphosphopeptide in {alpha}-s1-casein, required 3 h to achieve complete methylation of acidic groups on 95% of the peptides.



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  		FIG. 3. MALDI Q-TOF MS of 10% of the methylated tryptic peptides from the in-gel digestion of 1 pmol of autophosphorylated FGFR2 kinase domain operated in positive (a) and negative (b) modes. The peaks with mass difference of 80 in b indicate different levels of phosphorylation (e.g. peaks of m/z 1717.74, 1797.71, and 1877.71 are the mono-, di-, and triphosphorylated forms of the peptide 580–592). a, inset, shows the MS/MS spectrum of the diphosphorylated species of peptide 650–659 mDINNImDpYpYKmK, M+H+ = 1487.63. c, MS/MS spectrum of the nonmethylated monophosphorylated peptide of sequence RPPGMEYpSYDINR from the activation loop of the FGFR2 kinase insert region. Labeled phosphopeptides are listed in Table II.

 
Identification of FGFR2 Autophosphorylation Sites—
We used the method to identify phosphorylation sites on the N549H mutant FGF receptor kinase domain after incubation with ATP to induce autophosphorylation. The minimum amount of autophosphorylated kinase domain (2 pmol, 76 ng) required to visualize by Coomassie Blue staining was subjected to SDS-PAGE. After electrophoresis, the corresponding gel band (37 kDa) was excised for in-gel digestion with trypsin, and half of the resulting tryptic peptides were methyl esterified as described under "Experimental Procedures." The results of the analysis of 10% of the methylated peptides (100 fmol before electrophoresis and digestion) by MALDI Q-TOF in positive and negative modes are shown in Fig. 3 and Table II.


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  		TABLE II Phosphorylation sites identified in autophosphorylated kinase domain of the N549H mutant of the FGF receptor 2

MW (without methylation) is the calculated molecular weight of the phosphopeptides before methylation, and MW-H (after methylation) is the observed m/z of the singly charged methylated phosphopeptides measured in the negative ion spectrum of 100 fmol of a tryptic digest of FGFR2 kinase domain shown in Fig 3. The peptide his tag + 459–473 (sequence GSSHHHHHHSQDPMLAGVSEYELPEDPK) was only observed by analysis of the tryptic digest of 500 fmol of the FGFR2 kinase domain. For comparison with results published previously, amino acid numbers refer to the sequence of full-length FGFR2, not the kinase domain construct used in our experiments.

 
Fig. 3b shows 10 phosphopeptides containing four tyrosine and one serine phosphorylation sites (Table II). All of the major peaks in the spectrum acquired in negative ion mode (Fig. 3b) are caused by FGFR2 phosphopeptides with the exception of the peak of m/z 1342.58. Tandem mass spectra in positive ion mode of the phosphopeptides were acquired from the same spot on the MALDI sample plate to confirm the identity of the phosphopeptides, and in most cases to determine the phosphorylated amino acids. The sequences of these phosphopeptides are shown in Table II. Fig. 3a (inset) shows the MS/MS spectrum for the precursor ion of m/z 1487.02 of sequence mDINNImDpYpYKmK. This peptide is derived from the activation loop of the kinase domain. Autophosphorylation of both corresponding tyrosine residues in FGFR1 is required for up-regulation of kinase activity (27). The MS/MS spectrum supports phosphorylation on the two tyrosine residues. The immonium ion of phosphotyrosine is observed at m/z 216.04, peaks at m/z 1407.65 and 1327.72 indicate the loss of one and two HPO3 from the molecular ion (MH+ = 1487.62), respectively, and the y4 to y7 fragment ions are clearly seen.

Interpretation of the previous spectrum was straightforward because both potential sites on the peptide were phosphorylated. In many cases MALDI Q-TOF MS/MS can also be used to identify phosphorylated amino acids on peptides with several potential phosphorylation sites (18). Fig. 3c shows the positive ion MS/MS spectrum of the monophosphorylated peptide RPPGMEYpSYDINR (MH+ = 1677.71). Lack of immonium ion at 216.04, the abundant b7 (831.38) and c7 (848.41) ions, as well as a minor b5 ion, and lack of b7 + 80, c7 + 80, and b5 + 80 ions indicated that the tyrosine residues were not phosphorylated or were phosphorylated at very low stoichiometry. The loss of H3PO4 from the b8 ion indicated that the serine was phosphorylated, which is also consistent with the loss of H3PO4 from c9, b10, c11, b12, and the molecular ion. We concluded that most or all of the monophosphorylated peptides are phosphorylated on serine, which has not been previously reported. For comparison, we have shown the MS/MS spectrum of both methylated and nonmethylated forms of this peptide in supplemental Fig. S1. We find no systematic advantage or disadvantage from fragmenting either form of the phosphopeptides but often perform MS/MS analyses of both forms to provide more complete information. The doubly and triply phosphorylated forms of this peptide were also observed (Table II). The peptide is derived from the kinase insert region, and the equivalent tyrosines on the highly similar FGFR1 have been shown to be phosphorylated in cells using 32PO4 labeling and Edman degradation (27). An additional N-terminal phosphopeptide was detected by analysis of 500 fmol of the kinase domain digest (Table II). This peptide is derived from the juxtamembrane region preceding the core kinase domain and contains a tyrosine residue previously shown to be phosphorylated upon receptor activation (27). Positive and negative ion mode spectra of methylated and nonmethylated tryptic digests of 500 fmol of the FGFR2 kinase domain are shown in supplemental Fig. S2, and sequence coverage of the FGFR2 with and without methyl esterification is shown in supplemental Fig. S3.

In summary, we demonstrated that the FGFR2 kinase domain contains a total of six phosphorylation sites, including four identified tyrosine residues and an identified serine residue as well as an additional phosphopeptide containing an undetermined phosphorylation site. This result is consistent with a native gel showing the time course of phosphorylation of the FGFR2 kinase domain, which shows nearly complete phosphorylation of five or six sites after 10 min of incubation with ATP (supplemental Fig. S4). The result is also consistent with a mass of the intact phosphoprotein as determined by MALDI-TOF MS of 37,458 Da (data not shown). This mass is 505 Da higher than the predicted average mass of the nonphosphorylated protein, 36,953 Da, suggesting that six phosphate groups modified the FGFR kinase domain at high stoichiometry. However, the mass of the intact protein alone is not definitive because of variable and uncertain stoichiometry of phosphorylation as well as the possibility of additional posttranslational modifications. Although we detected some potentially multiphosphorylated peptides that contained partially phosphorylated sites (Table II and supplemental table), we did not detect any nonphosphorylated versions of the phosphopeptides in any of our experiments.

Comparison of DHAP, DHB, and CHCA Matrices—
We used DHAP as the matrix because it is a "cooler" matrix than CHCA (30) and resulted in less PSD at the relatively high fixed laser energy (350 µJ/pulse) of our MALDI Q-TOF instrument. Fig. 4 shows positive ion MALDI Q-TOF mass spectra of 500 fmol of two synthetic standard phosphopeptides from ß-casein using CHCA (Fig. 4a) and DHAP (Fig. 4b) as matrix. The loss of H3PO4 is significantly greater in Fig. 4a than in 4b for both the mono- and tetraphosphorylated peptides, which decreases the ion intensity generated by the intact phosphopeptides as well as increases spectrum complexity. The increased sensitivity is also demonstrated by the clear presence of an additional peak at m/z 2967.23 in Fig. 4b but not Fig. 4a, which corresponds to a contaminant in the peptide mixture because of a missing N-terminal arginine residue. A comparison of positive and negative ion mode spectra of methyl esterified ß-casein digest using CHCA and DHAP matrices is shown in supplemental Fig. S5, also demonstrating less ß elimination of phosphate in the DHAP spectra. Whereas we found that use of DHB as matrix improved the ion intensity for nonmethylated phosphopeptides in both positive and negative ion modes, and inclusion of phosphoric acid improved sensitivity in negative ion mode (31), lack of improvement of the signal for methylated phosphopeptides makes DHB suboptimal for our method (data not shown).



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  		FIG. 4. Positive ion mode MALDI Q-TOF MS of 500 fmol of two ß-casein-derived standard phosphopeptides (mono- and tetraphosphorylated) using 2 mg/ml CHCA as matrix (a) and 7.6 mg/ml DHAP as matrix (b). Peptides are listed in Table I, and the peak at m/z 2967.23 corresponds to the tetraphosphorylated peptide missing a single Arg residue.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have shown that comparing MALDI-TOF spectra of mixtures of phosphorylated and nonphosphorylated peptides acquired in positive and negative modes can be useful for identifying the phosphopeptides based on the relatively higher ion intensities generated by the phosphorylated peptides in negative ion mode. However, a limitation of this method is its poor specificity based on the tendency of acidic peptides to exhibit behavior similar to that of phosphopeptides. We have greatly improved the selectivity of the method by methylation of carboxylate groups on the peptides with methanolic HCl (22) to suppress the contribution of these groups to formation of negative charges on the peptides in negative ion mode. We then analyze the phosphopeptides on the same sample spot by CID in the MALDI Q-TOF instrument to confirm phosphopeptide identification and locate the phosphorylated amino acid(s).

We have demonstrated the utility of the method by identifying all of the known phosphorylation sites on low femtomolar amounts of a simple protein mixture, including ß-casein and its contaminant {alpha}-s-casein. We have also shown that the method can be used to rapidly monitor all previously identified phosphorylation sites on subpicomolar amounts of the medically important N549H mutant FGFR2 tyrosine kinase domain. This study is the first characterization of the autophosphorylation sites on the FGFR2. Based on a previous study of the highly homologous (more than 90% identical) FGFR1 kinase domain, we expected to find six tyrosine phosphorylation sites on the kinase domain of FGFR2. Indeed, four of our identified tyrosine phosphorylation sites correspond to those on FGFR1, and we found a fifth site on a tryptic peptide that contains tyrosine 466, which corresponds to phosphorylated tyrosine 463 on FGFR1. We did not find phosphorylation on tyrosine 733 of FGFR2, which corresponds to tyrosine 730 on FGFR1, which was shown by Mohammadi et al. (27) to be phosphorylated. However, the crystal structure of FGFR1 kinase domain shows that tyrosine 730 is poorly solvent-exposed and therefore would be expected to be a poor substrate for phosphorylation (32). We found an additional phosphorylation on serine 587, which we believe was phosphorylated by a heterologous serine kinase during protein expression (data not shown).

Although each of the phosphorylation sites on these two proteins is phosphorylated to very high stoichiometry, we also believe the method is capable of detecting phosphorylation events at low stoichiometry. We detected 16 of 18 previously reported phosphorylation sites as well as an additional site not previously reported on {alpha}-casein that was present at less than 10% of the ß-casein in the same sample. After methylation, the ratio of intensity (ion counts) of phosphorylated peptides in negative ion mode compared with positive ion mode was more than 25 greater than that of nonphosphorylated peptides (see supplemental table), further suggesting the method should efficiently detect phosphopeptides of low stoichiometry. However, it is possible for the method to fail to detect phosphorylated peptides when enzymatic digestion of a phosphoprotein fails to yield a sufficient quantity of phosphopeptides for MALDI-TOF MS analysis, when the resulting phosphopeptides are too small or too large to be efficiently detected by MALDI-TOF MS, or when a highly abundant nonphosphorylated peptide has nearly the exact same molecular weight as the phosphorylated peptide.

The identification of multiphosphorylated peptides by mass spectrometry has been especially challenging because of low ionization efficiency in positive ion mode and a high propensity for nonspecific adsorption to metallic and hydrophilic surfaces (33). For example, without methyl esterification, the tetraphosphopeptide of ß-casein (containing eight acidic amino acids and four phosphates) and the pentaphosphopeptide from {alpha}-s1-casein (amino acids 59–79, containing six acidic amino acids and five phosphates) were difficult to detect by MALDI-TOF or nanoflow LC-MS (Q-TOF) in positive ion mode at the 50 pmol level, or several pmol in negative ion mode for the {alpha}-s1-casein pentaphosphorylated peptide (data not shown and Kim et al. (33)). However, after methyl esterification, the limit of detection by MALDI Q-TOF in negative ion mode for these peptides decreased dramatically to the low femtomolar level. The most likely explanation is that methyl esterification achieves this effect by neutralizing the carboxylate groups on the aspartate and glutamate side chains as well as the carboxyl terminus of each peptide. The phosphate groups of phosphopeptides are then the only remaining acidic groups, and only phosphopeptides ionize efficiently in negative ion mode. In this case, the ionization of phosphopeptides is less likely to be suppressed by non-phosphopeptides, so that the negative ion mode spectrum of a mixture of methyl-esterified phosphorylated and nonphosphorylated peptides is similar in ion detection efficiency to that of purified phosphopeptides. By comparing negative and positive ion spectra of methylated peptides, low abundance phosphorylated peptides can be discriminated from highly abundant nonphosphorylated peptides that produce detectable signals in negative ion mode because the nonphosphorylated peptides produce much stronger relative signals in positive ion mode.

The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides. An advantage of the method is that it can be used to characterize peptides phosphorylated on serine, threonine, or tyrosine residues. Another advantage is that the method also can be used with other MALDI instrumentation that can be operated in positive and negative ion modes. Because the method relies on methylating carboxyl groups on peptide mixtures before mass spectrometry, it could be used for relative quantification experiments using stable isotopic labeling of the methyl groups (29), though partial methylation of glutamine and asparagine residues would complicate analyses of such experiments. In addition, our method is an ideal complement to enrichment of phosphopeptides from complex mixtures using immobilized metal affinity chromatography IMAC (22). Such pre-enrichment would enable the method to be used in large scale analyses of the phosphoproteome.


   ACKNOWLEDGMENTS
 
We thank Dr. Vivek Shetty and Dr. Ignatius Kass (Waters Corp.) for helpful advice and Dr. Scot R. Weinberger (Ciphergen, Inc.) for helpful advice about the DHAP/diammonium hydrogen citrate MALDI matrix.


   FOOTNOTES
 
Received, November 24, 2004, and in revised form, February 4, 2005.

Published, MCP Papers in Press, March 7, 2005, DOI 10.1074/mcp.T400019-MCP200

1 The abbreviations used are: FGFR, fibroblast growth factor receptor; DHAP, 2,6-dihydroxyacetophenone; DHB, dihydroxybenzoic acid; CHCA, {alpha}-cyano-4-hydroxycinnamic acid. Back

2 S. R. Weinberger, personal communication. Back

* This work was supported by National Institutes of Health Shared Instrumentation Grants 1-S10-RR14662 and 1-S10-RR017990 (to T. A. N.) and National Institutes of Health grants 1-R21-NS44184 (to T. A. N.) and R01-DE013686 (to M. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked ‘advertisement’ in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this article (available at www.mcponline.org) contains supplemental material. Back

To whom correspondence should be addressed: Skirball Institute, Lab 5-18, New York University School of Medicine, 540 First Ave., New York, NY 10016. Tel.: 212-263-7265; Fax: 212-263-8214; E-mail: neubert{at}saturn.med.nyu.edu


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