New Approach for Analysis of the Phosphotyrosine Proteome and Its Application to the Chicken B Cell Line, DT40 Melissa D. Zolodz,† Karl V. Wood,‡ Fred E. Regnier,‡ and Robert L. Geahlen*,† Department of Medicinal Chemistry and Molecular Pharmacology and Department of Chemistry, Purdue University, West Lafayette, Indiana 47907 Received January 19, 2004
In this study, we have begun to analyze phosphotyrosyl and associated proteins present in a DT40 chicken B cell line overexpressing the nonreceptor protein-tyrosine kinase, Syk. An anti-phosphotyrosine antibody was used to select tyrosine-phosphorylated proteins. After tryptic digestion, peptides were subjected to a β-elimination reaction and phosphotyrosine-containing peptides were enriched via immobilized metal affinity chromatography. Several known substrates and candidate substrates for Syk and the location of 22 tyrosine phosphorylation sites were identified. Keywords: chicken B lymphocytes • mass spectrometry • proteomics • tyrosine-phosphorylated proteins • skimmer CID • phosphotyrosine immonium ion • DT40 cells • Syk
Introduction The reversible phosphorylation of proteins catalyzed by kinases and phosphatases is a common theme in the regulation of important cellular functions such as growth, metabolism, and differentiation.1 Approximately 5% of the vertebrate genome codes for kinases and phosphatases.2 Consequently, the phosphoproteome is an important target for study. In eukaryotic cells, the most widely examined phosphorylations occur on serine, threonine, and tyrosine residues. Phosphorylation on serine residues (pS) is the most frequent example of acidstable phosphorylation (∼90%), followed by threonine (pT) (∼10%). Although tyrosine phosphorylation (pY) occurs less frequently (∼0.05%), reversible phosphorylation on tyrosine plays an important role in both normal signal transduction cascades and aberrant signaling pathways encountered in malignant disease. Thus, there is considerable interest in identifying the substrates that mediate the effects of kinases and phosphatases on cellular responses. In this study, we have begun to analyze the phosphotyrosine proteome of the chicken B cell line, DT40. B cells are a critical component of the adaptive immune system and respond to multiple extracellular stimuli including antigens to undergo positive or negative selection and to proliferate and differentiate into antibody-producing cells. Changes in the phosphorylation of specific proteins on tyrosine are a hallmark of the earliest responses of B cells to antigenic stimulation. Although the proteome of B lymphocytes has been investigated recently in human and mouse cells,3,4 to our knowledge, the chicken B cell has not been investigated, most likely due to our incomplete knowledge of the chicken genome (the chicken genome project sponsored by the National Institutes of Health began in the spring of 2003 and is * To whom correspondence should be addressed. E-mail: geahlen@ purdue.edu. † Department of Medicinal Chemistry and Molecular Pharmacology. ‡ Department of Chemistry. 10.1021/pr049967i CCC: $27.50
2004 American Chemical Society
expected to be complete by 2004). However, chicken DT40 cells have proven to be a unique resource for the analysis of protein-tyrosine kinases and their roles in signal transduction due to the ability of these cells to undergo homologous recombination. This makes the preparation of somatic cell knockouts possible. Thus, contributions of individual kinases to signaling pathways can be more readily analyzed using genetic approaches. We have begun to explore new methodologies for the analysis of the phosphotyrosine proteome of DT40 cells. A DT40 cell line was used that overexpresses the nonreceptor proteintyrosine kinase, Syk, which is localized in both the cytoplasmic and nuclear compartments of these cells.5 Syk can be activated upon its recruitment to aggregated B cell antigen receptors at the plasma membrane or in response to oxidative stress, which inactivates cellular protein-tyrosine phosphatases.6 In this study, cells were treated with pervanadate, a potent proteintyrosine phosphatase inhibitor7, to nonselectively activate Syk to allow the detection and analysis of both potential nuclear and cytoplasmic substrates. Tyrosine-phosphorylated proteins were selected by adsorption to an immobilized anti-phosphotyrosine antibody. Bound proteins were eluted and subjected to digestion with trypsin and the resulting peptides were analyzed by reversed-phase nanoflow capillary-LC/MS/MS for the identification of the captured proteins. In addition, peptides containing phosphoserine or phosphothreonine were chemically dephosphorylated via a β-elimination reaction resulting in the formation of dehydroalanine and dehydrobutyric acid, respectively,8 in order to enrich for phosphotyrosine-containing peptides. Immobilized metal affinity chromatography (IMAC) was then used to isolate the remaining phosphorylated peptide(s) prior to LC/mass spectrometric analysis9 to determine, when possible, the location of the tyrosine phosphorylated residue in the peptide sequence. The tyrosine phosphorylated state of the peptide was verified with the detection of the phosphotyJournal of Proteome Research 2004, 3, 743-750
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Figure 1. Proteomic analysis method. Phosphotyrosyl and associated proteins were isolated via an anti-phosphotyrosine antibody column. After digestion, peptides were subjected to a β-elimination reaction. The tyrosine phosphorylated peptides were enriched from the collected mixture by IMAC. These peptides were analyzed by both reversed phase nanoflow capillary LC-MS/MS and skimmer CID LC-MS. Proteins were identified by database searching. *Analyzed by LC-MS/MS. *1 is the analysis of an aliquot of peptides right after digestion with trypsin. *2 indications analysis of peptides not captured by IMAC. *3 is the analysis of the IMAC eluate.
rosine immonium ion produced via skimmer CID.10 The experimental outline is shown in Figure 1.
Materials and Methods Cell Culture and Lysis. Syk-deficient DT40 cells overexpressing wild-type Syk have been described previously.11 To enhance the concentrations of tyrosine-phosphorylated proteins and to stimulate endogenous Syk, cells were first treated with pervanadate (100 µM) for 20 min at 37 °C. Cells (1 × 109) were then collected and resuspended in 12 mL lysis buffer containing 1% Triton-X100, 25 mM Hepes, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1.9 µg leupeptin, and 1.9 µg aprotinin. The mixture was incubated on ice for 15 min and then centrifuged at 1000 rpm for 5 min. The concentration of lysate was approximately 2 mg/mL as detected by Bradford analysis.12 Lysates were stored at -80 °C. Phosphotyrosine Protein Selection via Affinity Chromatography. Lysates were precleared with Protein A beads and the pH was adjusted to 7.4. The sample was then incubated on a column containing 1 mg of anti-phosphotyrosine monoclonal antibody covalently coupled to protein A agarose beads (Upstate Biotechnology, Lake Placid, NY) overnight at 4 °C, with rocking. The beads were then washed twice with binding buffer (20 mM Tris/HCl, pH 7.4, 10 mM EDTA, 100 mM NaCl, 1% NP-40, 0.2 mM Na3VO4, 0.01% sodium azide), followed by six washes with binding buffer lacking NP-40 to remove detergent and loosely associated proteins. Bound proteins were eluted with 2.5 column volumes of elution buffer containing 50 mM glycine, pH 2.3, 0.1% ethylene glycol and collected in 300 µL of 2 M ammonium acetate, pH 8.4. The column was then regenerated by extensive washing with binding buffer containing 2 M NaCl. Several incubations on the antibody beads and elutions were required to obtain protein levels detectable by Bradford. The eluates were pooled and lyophilized to a volume of 1 mL. Trypsin Digestion. The pH of the sample was adjusted to 8.5 with NH4OH. Proteins were denatured with urea, added to a final concentration of 6 M. Disulfide bonds were reduced with dithiothreitol (DTT) was added to a final concentration of 10 mM and the sample was incubated at 50 °C for 25 min. The 744
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sample was then alkylated with 40 mM iodoacetic acid in the dark for 1 h at room temperature. This was then followed by the addition of 40 mM DTT for 1 h at room temperature. Urea and small molecules were separated from the sample using a HiTrap desalting cartridge (10 000 molecular weight cutoff, Amersham Biosciences, Uppsala, Sweden). The samples were eluted with 1.5 mL of digestion buffer (25 mM ammonium acetate, pH 8.5), lyophilized to less than 1 mL, and pH adjusted to 8.5. Proteins were subsequently digested with trypsin (1:50 w:w enzyme:protein) at 37 °C overnight. An aliquot of the sample was pH adjusted and saved for LC/MS analysis (Figure 1, *1). β-Elimination Reaction. Considerable effort was taken to optimize the β-elimination reaction for the removal of the phosphate group from serine and threonine phosphorylated peptides. The purpose of this step is to decrease the complexity of the mixture captured by IMAC. The optimized reaction conditions were incubation in 0.85 N NaOH with 10% ethanol at 45 °C for 1 h. Samples were lyophilized (but not to dryness) to remove the ethanol and pH adjusted to less than 3 with formic acid. Immobilized Metal Affinity Chromatography (IMAC). NiNTA Agarose beads were purchased from Qiagen, Inc. (Valencia, CA). The Ni was removed with EDTA according to the method of Hart et al.13 The metal ion was replaced with Fe(III) by incubation with Fe(III)Cl3. Samples (500-750 µL) were adjusted to pH 2.5 and incubated on 50 µL Fe-NTA agarose beads for 30 min while tumbling. The supernatant was decanted and saved for LC-MS analysis (Figure 1, *2). The IMAC beads were washed twice with 2 column volumes of 100 mM acetic acid, followed by 2 column volumes each of 0.1 M NaCl/ 100 mM acetic acid; 100 mM acetic acid; 20% acetonitrile/100 mM acetic acid (twice); 100 mM acetic acid and finally with H2O. Peptides were eluted from the IMAC beads with 1 column volume of 5% NH4OH and rinsed again with an additional column volume of 5% NH4OH. The use of base instead of phosphate buffer to elute peptides from the IMAC beads alleviated the requirement for desalting the samples. Sample recoveries were similar to those eluted with phosphate buffer (data not shown). Samples were then lyophilized to less than 50 µL and the pH adjusted until acidic for LC/MS analysis (Figure 1, *3). Nanoflow HPLC/MS. Experiments were performed on a Bruker Esquire∼LC ESI-QIT mass spectrometer (Bruker Daltonics, Bremen, Germany). The standard Bruker ESI needle was replaced with a stainless steel (SS) tube (204 µm OD x 102 µm ID) (Small Parts, Inc Dallas, TX). The ends were bluntly cut at the Department of Physics Machine Shop (Purdue University, West Lafayette, IN). The needle was tapered according to the method by Alexander et al.14 Tapering the ESI needle lowers the detection limit of phosphopeptide analysis from 65 fmol/ µL to 800 amol/µL.15 The needle was glued to the ferrule inside the nebulizer needle assembly with epoxy to ensure the ESI needle position inside the source was fixed. Source conditions were as follows: the capillary was set to -4600 V, the end plate voltage was set to -3100 V, the nebulizer gas (N2) pressure was 17 psi and the drying gas (N2) flow rate was 5 L/min at 325 °C. A 75 µm I. D. × 280 µm O. D. × 15 cm (length) reversed phase PepMap C18 capillary column (3 µm particles, 100 Å pores) was purchased from LC Packings, Inc. (San Francisco, CA). The column was connected to a short piece of fused silica via a small Teflon tube. The fused silica connector capillary was coupled to the ESI needle through a SS reducing union (150 µm bore).
Phosphotyrosine Proteome Analysis
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Table 1. Parameters Used for Database Searches of NCBI Database Using MASCOT and MS-Tag Search Sites parameter species digest peptide mass accuracy cysteine modification masses are charge state number of missed cleavages
setting Gallus gallus, mouse, human, rat trypsin 1.2 Da carboxymethylation average +2, +1 2
An HP 1100 Binary HPLC pump (Hewlett-Packard) was coupled to the ESI-QIT for LC/MS experiments. The two mobile phases were A: 0.08% formic acid in H2O, and B: 0.068% formic acid in methanol. A linear gradient of 5 to 70% B was applied over 120 min followed by 70 to 85% B for 15 min. The flow rate from the HPLC outlet was split via a stainless steel T-junction using a piece of fused silica as a flow restrictor capillary in order to deliver a final flow rate of 250 nL/min (measured from the ESI needle). A Rheodyne injector valve with a 5 µL PEEK loop from Alltech (Deerfield, IL) was employed. For skimmer CID experiments, the capillary offset voltage was varied from 80 to 120 V to obtain the highest signal of the phosphotyrosine immonium ion (Im(pY)). The ion at m/z 216 was fragmented by in-trap CID for verification as the Im(pY) ion. For LC-MS/MS (in-trap CID of tryptic peptides) experiments, automatic MS/MS mode (MS, MS/MS every other scan) was compared to manual MS/MS. Manual MS/MS provided more comprehensive sequence data when compared to the automatic MS/MS mode (data not shown). As a result, manual MS/MS mode was employed for the experiments described in this paper. The data were acquired with Bruker Daltonics DataAnalysis software, version 3.0. Peptide sequences were derived by manual interpretation of the MS/MS data. Database Analysis. MASCOT and MS-Tag were used to match MS/MS spectra/peptide sequences against the National Center for Biotechnology Information nonredundant protein database. Parameters used are shown in Table 1. Assignments were confirmed by manual interpretation.
Figure 2. Western blot analysis of lysates from pervanadatetreated Syk-deficient DT40 cells (Syk-) and Syk-deficient DT40 cells expressing the exogenous kinase (Syk+). Phosphotyrosinecontaining proteins were separated by SDS-PAGE and detected by immunoblotting using an anti-phosphotyrosine antibody.
Results and Discussion We compared the pervanadate-induced phosphorylation of proteins on tyrosine in Syk-deficient DT40 B cells, lacking the gene for expression of endogenous Syk,16 to cells in which the expression of Syk was restored by transfection with a plasmid coding for the wild-type, murine kinase.11 As shown in Figure 2, resting cells contained few tyrosine-phosphorylated proteins as determined by Western blotting of whole cell lysates with an antiphosphotyrosine antibody. Treatment of Syk-deficient cells with pervanadate led to a modest increase in the phosphorylation of proteins on tyrosine while treatment of Sykexpressing cells resulted in the robust phosphorylation of numerous proteins. This indicated that the pervanadateinduced, tyrosine-phosphorylation of multiple proteins in DT40 cells was dependent on the expression of Syk. To begin to analyze the proteins phosphorylated in Sykexpressing cells, phosphotyrosyl-proteins present in lysates of pervanadate-treated cells were selected by adsorption to and elution from resin containing an immobilized, monoclonal antiphosphotyrosine antibody. Proteins captured by the antiphosphotyrosine antibody affinity matrix were isolated and subjected to digestion with trypsin. A sample of the digest was analyzed by LC/MS/MS. The LC/MS total ion chromatogram is seen in Figure 3A. Of these peptides, one was determined to
Figure 3. Total ion chromatograms of LC-MS analysis of A: aliquot after digestion, without further processing (*1), B: peptides not captured on IMAC beads (*2), and C: IMAC eluate (*3). Approximately 46 µg protein was digested.
be tyrosine-phosphorylated, two threonine-phosphorylated and two serine-phosphorylated. Five additional peptides showed either the loss of H3PO4 (-98 Da) or HPO3 (-80 Da), but the identity of the modified amino acid could not be deciphered from the tandem mass spectrum. A total of 87 peptides were subjected to in-trap CID in the LC/MS analysis and were searched via MASCOT and/or MS-Tag for possible protein identification. With the simultaneous analysis of multiple peptides, only those with the highest proton affinities are ionized and therefore detected in the mass spectrometer. Phosphorylated peptides typically have lower proton affinities due to the electronegativity of the phosphate group as compared to more electropositive (nonphosphorylated) peptides. Therefore, to detect phosphorylated peptides using mass spectrometry, it is Journal of Proteome Research • Vol. 3, No. 4, 2004 745
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Table 2. Proteins Consistent with MS/MS Data from Database Searches with Accession Numbers organism
chicken
chicken chicken chicken chicken mouse, rat chicken chicken mouse chicken mouse chicken chicken
chicken mouse (rat, human) rat, (human) chicken human, (mouse, rat) chicken chicken
chicken chicken chicken chicken chicken chicken human chicken chicken
746
protein (peptide)
histone H1 (GTLVQTR,LGLK, ALAAGGYDVEK, KPAGPSVTELITK, GTVQTK) histone H1 (methylated DNA binding protein) (KPAGPSVTELITK) histone H2A (AGLQFPVGR) macro histone H2 A1.1 (pSPSQKKTVSKK) histone H2B (LLLPGELAK, ESYSIYVYK) H3 histone, family 3B (pSSDPK) histone deacetylase 4 (LQETGLR) DNA-dependent kinase catalytic subunit (IIKHK, DQEK) DNA dependent ATPase SNF2h (ISpYGTNK) DNA(C5) methyl transferase 1 (LAGVTLGKR, FGGSGRSK) cold inducible RNA binding protein (YGQISEVVVK) myb-A protein (transcription activator) (VIYEAHK) nuclease sensitive element binding protein 1 (Y-box transcription factor) (NGYGFINR) c-myc (TSDSEENd) SET (VEVTEFEDIK) 40S ribosomal S19 (WVAKHK, DVNQQEFVR, AALQELLGK) ribosomal protein-L35 (VLTVINQTQK) ribosomal protein L31 (SAINEVVTR) G1 specific cyclin E (NMoINK) B cell phosphoinositide 3 kinase adaptor protein (GASRPVNdCmEGFpYPPP VPPR) protein tyrosine phosphatase (leukocyte) Gz-selective GTPaseactivating protein (EVSLDSR) tyrosine phosphatase CRYP-2 (NGLK) protein tyrosine kinase FAK (GNALEK) tyrosine kinase JAK1 (LSDPGIPITVLSR) tyrosine kinase CSK (c-SRC-kinase) (EKKFSTK) similar to chromosome 15 ORF 2 (HLQdFR) MAP kinase 9 (c-Jun Nterminal kinase 2) JNK 2 (APEVILGMGpYK) Vav2 (MTDDPMNNK)
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accession no.
modification
cell signaling
nuclear protein
P08288
ND
x
P08286
ND
x
P02263
ND
x
AAC28846
pS (S138)
x
CAA30590
ND
x
XP_229174
pS-p (S392)
x
P83038
ND
x
BAB91148
ND
x
AAK52454
pY (Y938)
x
Q92072
ND
x
NP_031731
ND
x
P52550
ND
x
Q06066
ND
x
P01109
Nd (N330)
x
NP_003002
ND
XP_218303
ND
AB046395; BAB21248
ND
CAA48925
ND
P49707
Mo (M116)
AAG48583
deamination (N618), pY (Y623), CMe (C619)
x
I50212/Q900815
ND
x
AAF00026
ND
x
AAB17968
ND
x
A45388
ND
x
AAD22628
ND
x
P41239
ND
x
gi 29732035
Qd (Q672)
P79996
pY (Y202)
x
AAL06250
ND
x
x
x
x
x
Phosphotyrosine Proteome Analysis
advantageous to separate them from other tryptic peptides with higher proton affinities. In addition, since the numbers of proteins or sites phosphorylated on tyrosine are low compared to those modified on serine or threonine, a method to enrich for these peptides would also be advantageous. To accomplish this, a sample of the tryptic phosphopeptides derived from the antiphosphotyrosine affinity-purified proteins was subjected to a β-elimination reaction to selectively decrease the content of phosphoserine and phosphothreonine. The treated peptides were further fractionated by adsorption to and elution from IMAC beads. Samples from both the IMAC flow-through and eluted fractions were further analyzed by LC/MS/MS. The LC/ MS total ion chromatograms are shown in Figures 3B and C. In the fraction not captured on the IMAC beads, six peptides were observed to be tyrosine-phosphorylated, one serinephosphorylated and one threonine-phosphorylated. Fourteen additional peptides were identified as being phosphorylated on an amino acid that could not be determined in this analysis. This observation indicated that the use of the β-elimination reaction successfully enriched for phosphotyrosine-containing peptides, but that not all of these were bound tightly by the IMAC resin. Ninety-seven peptides were subjected to MS/MS analysis from the IMAC flow-through fraction. Of the peptides captured by the IMAC resin, 22 were found to contain phosphotyrosine. In addition, five peptides contained phosphoserine and seven phosphothreonine, indicating that, while the β-elimination reaction was not complete, it did dramatically increase the ratio of tyrosine-phosphorylated to serine/threonine-phosphorylated peptides over what would be expected based on the relative endogenous levels of each phosphorylated amino acid. A total of 140 peptides were subjected to in-trap CID and database searching in the analysis of the IMAC eluate. Protein Identification. To identify candidate proteins, peptides were matched via MASCOT and/or MS-Tag to products from the chicken genome as well as the mouse, rat and/or human genomes since the chicken database is incomplete. To increase the stringency of the identification process, candidates were further compared against the bursal expressed sequence tag database17 to ensure that the corresponding genes are transcribed in bursal B cells. Proteins identified by database searching that meet these criteria are displayed in Table 2. The list includes several signaling molecules that are known to be phosphorylated on tyrosine in response to receptor engagement in lymphocytes. These include the B cell phosphoinositide 3-kinase adaptor protein (BCAP), which, when phosphorylated by Syk on tyrosine, interacts with the SH2 domains of the p85 subunit of phosphoinositide 3-kinase18 and Vav2, another Syk substrate, which serves as an exchange factor for Rac/Rhofamily GTPases.19 The cytoplasmic protein-tyrosine kinases, FAK (focal adhesion kinase)20 and JAK1 (Janus-family kinase 1)21 also are both phosphorylated and activated in B cells. The DNA-dependent protein kinase catalytic subunit is also known to be phosphorylated on tyrosine and to associate with both the c-Abl and Lyn protein-tyrosine kinases.21,22 The c-Src kinase, Csk, which is not generally thought to be phosphorylated on tyrosine, is known to bind via its SH2 domain to CBP/PAG, a membrane-raft-associated tyrosine-phosphorylated protein,23,24 suggesting that proteins can be recovered by this approach that bind indirectly to the anti-phosphotyrosine antibody-affinity resin. Nuclear Substrates. Syk is activated when recruited to the aggregated B cell antigen receptor (BCR) complex at the plasma
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Figure 4. LC/MS/MS spectra of DNA-dependent ATPase SNF2h (mouse) peptide. Inlay shows skimmer CID mass spectra.
membrane and is required for BCR-mediated signaling.25,26 The kinase, however, is localized to both the cytoplasmic and nuclear compartments,5 but no functional role for the kinase in the nucleus has been described. Syk is also activated by receptor-independent mechanisms in response to extracellular stress including exposure to high salt or H2O2.6,27 The later response is mediated by the inhibition of protein-tyrosine phosphatase activity. Thus, we were particularly interested in determining if any nuclear proteins could be identified among those binding to the immobilized anti-phosphotyrosine antibody in response to treatment of DT40 cells with pervanadate. The analysis did, in fact, identify several nuclear proteins as candidate substrates. Included among these was the DNA-dependent ATPase, SNF2h, a component of a chromatin remodeling complex.28 SNF2h has a tryptic peptide sequence of ISpYGTNK, which is completely conserved between mice and humans. While the chicken homologue has not yet been identified, an EST corresponding to chicken SNF2h is present in the bursal EST database. Figure 4 shows the tandem mass spectrum of this peptide. The difference between b2 and b3 as well as the difference between y4 and y5 is equal to 243 mass units which corresponds to the weight of a phosphotyrosine residue. In the skimmer CID analysis at this retention time, an ion at m/z 215.9 was observed consistent with the Im(pY) ion. Interestingly, three other nuclear proteins involved in chromatin remodeling, DNA-(cytosine-5)-methyltransferase (DNMT1), histone deacetylase and SET/TAF-Iβ, were also identified as candidate substrates. Figure 5A shows the tandem mass spectrum of a peptide with the sequence VEVTEFEDIK, detected in the fraction of peptides not captured on the IMAC beads. This sequence matches the human, rat, and mouse sequence for the SET/TAF-Iβ translocation (myeloid leukemiaassociated) protein.29 This protein was not present in the Gallus gallus genome database, but is present in the bursal EST database and contains the exact same sequence. The set gene, an oncogene that is associated with myeloid leukemogenesis, encodes a known inhibitor of protein phosphatase 2A.30 Additionally, Set/TAF-Iβ has been identified as a subunit in the cellular complex known as INHAT (inhibitor of acetyltransferases).31 The INHAT complex also contains the nuclear phosphoprotein pp32, another oncoprotein.32,33 The INHAT complex binds to histones, preventing histone acetyltransferases from acting on their substrates. It was discovered recently that Set/TAF-Iβ is also involved in gene silencing by inhibiting methylation of DNA.34 Both Set/TAF-Iβ and nuclear phosphoprotein pp32 were found in the analysis Journal of Proteome Research • Vol. 3, No. 4, 2004 747
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Figure 5. LC/MS/MS spectra of peptides from A: Set/TAF-1β translocation (myeloid leukemia-associated) protein, previously unidentified in chicken DT40 B cells. B: Peptide from the Histone H3, family 3B showing a dephosphorylated serine (dehydroalanine) residue. C: Peptide matching sequence from Histone H1.10 Methylated DNA-binding protein 2H1, Histone H1.03, and Histone H1.01.
of mouse B cell proteins from anti-phosphotyrosine antibody eluates as reported by Shu et al. from the Alliance for Cellular Signaling.35 In addition to proteins involved in chromatin remodeling, several transcriptional regulators were also identified as potential substrates. These included CNBP1, YB1, Myb-a and c-Myc. Of these, only chicken c-Myc has, to our knowledge, been described as a tyrosine-phosphorylated protein.36 Several histones were observed in our analysis. Histones are proteins involved not only in the packaging of DNA, but also the regulation of chromatin.37 Histone H3 and H1 variants were examined by LC/MS/MS of tryptic digests (Figures 5B-C). A number of histone H3 and H1 variants have been described in DT40 B cells.38,39 The peptide sequence for histone H3, family 3B, demonstrates that the first serine residue was phosphorylated but this phosphorylation was removed by the β-elimina748
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Figure 6. LC/MS/MS spectra of pY-containing peptides. Inlays are skimmer CID mass spectra. A-C: Phosphotyrosine-containing peptides of unknown protein origin.
tion as indicated by the loss of 69 mass units, which corresponds to a dehydroalanine residue (Figure 5B). In Figure 5C, the sequence ALAAGGYDVEK, derived from the tandem mass spectrum, matches to several variants of Histone 1, including variant 10 (methylated DNA binding protein 2-H1), variant 3, and variant 1. It is not yet clear whether histones were present due to direct, indirect or nonspecific interactions with the affinity resin. Histones were also identified in the Alliance for Cellular Signaling screen.35 Histones are known to be phosphorylated on serines, but generally not on tyrosines, although the phosphorylation of histone H3 on tyrosine in pharyngeal cells due to infection with group A streptococci has been reported.40 Additionally, histone H2B has been used in vitro as a substrate for Syk,41 indicating that it has the potential to be a candidate substrate. However, since histones are abundant proteins involved in numerous protein-protein interactions, verification of their actual phosphorylation on tyrosine must await further experimentation.
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Phosphotyrosine Proteome Analysis Table 3. Phosphotyrosine Containing-Peptide Partial Sequences (M+H)+
partial sequence
561.5 601.3 795.8 1037.4 787.4 1386.0 1644.2 1260.0 1226.4 1516.0 1201.6 1223.5 1690 1701 1093 994.6 453 1516 1756.6 906.4 846.6 769.2
WXpY 211-QpY 534-pY; 533(pSC)263; 389-pYK 716(pY)473; 717(X)604; 322(Y)159 571(X)458;530(CpY)179 F-pYAAGYXE-279 pY-1401;1498-R;1347(NVS)1047;436(HG)242 169-NXpYHXAA-229 1050(T)949;920(P)823;636(Q/KXD)281; pY-677 1313(X)1200;960(NVpYMX)260 P-1104;909(NYVS)446 or 795(pY)552; 407(N)293 1062(XXpYTM)361 N-1575; 814(pY)571 749-SpY-419 947-K, 838(SG)710; 604(NpY)247, 455(AG)327 734(pY)491 XPpY 401(CpYVDCH)1202 1198(C)1095; 743(pYT)399 646-pY 601-XFPSR; b1)pY WVpY-241
Several peptides could not be matched successfully to products from the available genome databases. Figure 6A-C shows a sampling of the tyrosine phosphorylated peptides that were not identified by database searching. The identity of the ions as being of the y or b type in Figure 6A was not possible. The ions observed provide the partial sequence ∼NXpYHXAA∼, where X represents I or L, which is indistinguishable by low energy in-trap CID. In Figure 6B the loss of the phosphotyrosine residue is observed between yn-2 and yn-3. At the same retention time, m/z 216 was observed. Only the last 2-3 C-terminal residues were not identified. A match for the sequence FYAAGYXE was not found by performing a BLAST search.42 As shown in Figure 6C, the y and b ion assignments could not be made but the partial sequence was found to be ∼NVpYMX∼. Skimmer CID analysis at retention time 88.4 min showed an ion at m/z 215.9. Unidentified peptides may belong to proteins that have not yet been identified in chicken, or they may represent new tyrosine phosphorylated sites. Even if the amino acid sequence of a protein from chicken is homologous to a previously identified protein in another species, a single amino acid substitution would change the m/z so that it will not match a peptide in the database. Many of the peptides analyzed by LC/MS/MS produced incomplete sequencing and were not able to be identified in the database analysis. The peptides that were partially sequenced and not identified by database searching are listed in Table 3. In this research, tyrosine phosphorylated and associated proteins isolated from extracts of Syk-deficient DT40 B cells expressing exogenous, wild-type Syk were analyzed via a double affinity selection method. The highest number of tyrosine phosphorylated sites were detected via LC/MS/MS when the anti-phosphotyrosine antibody selected proteins were digested with trypsin, subjected to a β-elimination reaction, and IMAC selection. Many proteins could not be identified, most likely due to absence from the protein databases or single amino acid substitutions. However, a number of proteins involved in signal transduction pathways and the regulation of gene expression were identified. These provide useful information for the design of further investigations into understanding putative Syk substrates and their roles in nuclear functions.
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