From Discovery Technologies, Novartis Institute for Biomedical Research, Inc., Cambridge, MA 02139
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ABSTRACT |
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It is estimated that only 0.050.1% of protein phosphorylation occurs on tyrosine residues (2). Thus, it has been challenging to extensively document tyrosine phosphorylation events in a signaling pathway. Monoclonal antibodies that specifically recognize phosphotyrosine (pTyr)1 have been widely used in research related to protein tyrosine phosphorylation. Tyrosine-phosphorylated proteins can be effectively immunoprecipitated from cell lysate by these antibodies. Conventionally, after immunoprecipitation, proteins are separated and quantified by SDS-PAGE or two-dimensional PAGE. The protein spots bearing differences between the control and the treated samples are identified using in-gel proteolysis and MS. Phosphopeptides can be selectively enriched from in-gel digested samples by IMAC if phosphorylation sites are to be identified (3). Alternatively, parent ion scanning of phosphate- or pTyr-specific marker fragment ions may be used (4) to detect phosphopeptides in the in-gel-digested samples, and phosphorylation sites can be identified by MS/MS. However, these gel-based approaches are not very effective (5) and are not amenable to high throughput studies. In contrast, solution-based phospho-proteomics are preferred.
Rapid advances have been made in the area of technology development regarding phosphopeptide enrichment in recent years. In particular, methyl esterification of acidic side chains of peptides has been demonstrated to dramatically reduce the nonspecific background in IMAC-based approaches. This strategy has allowed for the identification of over 200 phosphopeptides from a yeast lysate (6). However, due to the presence of the overwhelming serine/threonine phosphorylation, it is still difficult to assess the global dynamics of tyrosine phosphorylation using this method alone. Enrichment of pTyr-containing proteins prior to IMAC enrichment has been shown to significantly improve the detection of tyrosine phosphorylation (7).
Interferons (IFNs) are a group of secreted proteins that belong to the cytokine family. They are classified as type I IFNs (including IFN, IFNß, and IFN
) and type II IFN (IFN
). Type I interferons, such as IFN
and IFNß, are produced by numerous types of cells, including leukocytes and fibroblasts, that elicit different effects depending on the type of cell that receives the signal (8). The anti-viral, anti-proliferative, and immuno-modulatory activities of IFN
have been utilized in treatment for chronic myelogenous leukemia and hepatitis B and C for many years (911). It is known that IFN
elicits its effect by binding to specific membrane receptors (IFNAR1 and IFNAR2c) on the cell surface and activates the receptor-associated tyrosine kinases, which in turn activate a series of transcription factors known as signal transducers and activators of transcription or STAT (12). However, the mechanistic details of IFN
function are still poorly understood. Evaluation at a global scale of tyrosine phosphorylation events involved in the signaling of IFN
will help us gain insight of this important pathway.
This article describes an improved double enrichment method that allows quantitative study of signaling events in the aspect of tyrosine phosphorylation. Using this method, we studied IFN signaling pathway in Jurkat cells. Tyrosine-phosphorylated proteins were immunoprecipitated from control and treated Jurkat cell lysates using a mixture of two pTyr-specific monoclonal antibodies. Furthermore, tryptic digest of the immunoprecipitated proteins was methyl-esterified, and phosphopeptides were enriched by IMAC and analyzed by reverse phase (RP) LC-MS/MS. Protein and phosphorylation site identification was achieved by data base search using the MS/MS data. For quantitation, stable isotope labeling was employed. In this case, after immunoprecipitation and tryptic digestion, peptides from treated and control samples were methyl-esterified by H3- or D3-methanolic HCl, respectively. The two isotope-labeled samples were then mixed prior to IMAC. Enriched phosphopeptides were separated by HPLC and detected by MS. Extracted ion chromatograms of the hydrogen and deuterium forms of the peptide ions were compared to obtain relative quantitation between control and treated samples (see Fig. 1).
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MATERIALS AND METHODS |
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Tryptic Digestion
Proteins were reduced by incubation in the presence of 10 mM DTT at 96 °C for 10 min. After being cooled to room temperature, sulfhydryl groups were alkylated by reacting with 20 mM iodoacetamide in the dark for 30 min. The buffer was changed to 50 mM NH4HCO3 via dialysis using a 10-kDa molecular weight cut-off (MWCO) Slide-A-Lyzer (Pierce). Trypsin (modified sequencing grade; Promega, Madison, WI) digestion was performed at 1:50 to 1:100 trypsin-to-protein ratio (w/w) overnight at 37 °C for 16 h.
Methyl Esterification of Peptides
The tryptic digest was filtered through a spin filter of 10-kDa MWCO (Millipore, Bedford, MA) and speed-vac dried. H3-methanolic HCl or D3-methanolic HCl was prepared by adding 160 µl of acetyl chloride to 1 ml of methyl H3-alcohol or methyl D3-alcohol while stirring. Immunoprecipitated proteins from starting material of 1 x 109 cells were treated with 200 µl of H3/D3-methanolic HCl at room temperature for 30 min and then speed-vac dried.
Immobilized Metal Affinity Chromatography
Poros MC column (2.1 x 30 mm, 100-µl bed volume; Applied Biosystems, Foster City, CA) was activated by the loading of 0.2 M FeCl3. After conditioning with wash buffer 1 (1% acetic acid) and wash buffer 2 (1% acetic acid in 50% ACN), methyl-esterified peptides were solubilized in wash buffer 2 and loaded onto the column. The column was extensively washed with wash buffer 2 prior to the elution of phosphopeptides using 2% NH4OH in 50% ACN (v/v). The eluate was neutralized to pH 5 with the addition of acetic acid and then speed-vac dried.
Global Phosphorylation Study
For the global phosphorylation study of Ser, Thr, and Tyr phosphorylation, cells were treated and lysed as described above. The protein concentration was measured using Bio-Rad DC Protein Assay. Proteins were digested, and the methyl esterification/IMAC procedure was performed as described above. For peptide identification, control and treated samples were analyzed by LC-MS/MS. Quantitative comparison of phosphorylation was achieved through inverse labeling of control and treated samples (13). Specifically, half of each sample was methyl esterified using H3-methanolic HCl and the other half using D3-methanolic HCl. After labeling, the hydrogen form of control sample was mixed with the deuterium form of treated sample, and the deuterium form of control sample was mixed with the hydrogen form of treated sample. After IMAC, the enriched phosphopeptides were analyzed by LC-MS.
HPLC-MS
Peptides eluted off IMAC were separated by RP-HPLC using Pepmap C18 (3 µm, 100 Å, 180 µm x 15 cm; Dionex, Surrey, UK) with a gradient of 540% B in 60 min at a flow rate of 2 µl/min (0.1% formic acid in water as A and 0.1% formic acid in ACN as B). Peptides were directly eluted to a Q-TOF Ultima mass spectrometer (Waters, Manchester, UK) for on-line MS analysis. For peptide sequence and phosphorylation site determination, the data-dependent LC-MS/MS analysis was performed separately on control and treated samples using the following settings: MS survey scan from m/z 4001,800 was followed by MS/MS on the three most intense multiply charged ions (two to four charges) that were above a preset intensity threshold with dynamic exclusion of 90 s. Collision energy was automatically determined by the charge state and the m/z value of a particular precursor ion. MASCOT software (Matrix Science, London, UK) was used to search the MS/MS data against the NCBI data base (human) for peptide sequence/parent protein identification and phosphorylation site determination. The mass tolerance of both the precursor ions and the MS/MS fragment ions was set at ± 0.2 Da. Whereas carbamidomethyl cysteine and methyl esterification of the C terminus and aspartic and glutamic acids were set as static modifications, oxidation of methionine and phosphorylation of serine, threonine, and tyrosine were set as variable modifications. MS/MS spectra were checked manually to verify the sequence assignments. In the quantitative analysis, the mixtures of isotope-labeled control and treated samples were analyzed under similar LC-MS conditions but without data-dependent MS/MS. The ratio of the integrated peak areas of an isotopic pair was used to obtain quantitative comparison of the phosphopeptide between treated and control samples.
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RESULTS |
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Total tyrosine phosphorylation was evaluated by Western blot using the same combination of anti-pTyr antibodies that were used for immunoprecipitation. It was difficult to detect any difference between control and treated samples (Fig. 2A). This result suggested that the induced changes in tyrosine phosphorylation occurred on a small number of specific proteins rather than massively on a large number of proteins such as those induced by sodium pervanadate treatment (a nonspecific tyrosine phosphatase inhibitor, data not shown).
Phosphopeptide Identification
After anti-pTyr immunoprecipitation, tryptic digestion, methyl esterification, and IMAC enrichment, the isolated peptides were analyzed by capillary HPLC on-line with MS using a Q-TOF Ultima instrument as described in "Material and Methods." The MS/MS spectra were used to search the NCBI human data base using MASCOT to identify the amino acid sequence and phosphorylation site(s) (Fig. 3). Each search result of a MS/MS spectrum was manually confirmed. Using this method, we identified 38 tyrosine phosphorylation sites and 19 phosphoserine/threonine sites (Table I). Some of the phosphoserine/threonine-containing peptides were recovered because the protein was tyrosine phosphorylated. For example, three peptides were recovered for LAT protein. While one was tyrosine phosphorylated, two were phosphorylated at Ser sites (EYVNpSQELHPGAAK (Ser-84) and pSPQPLGGSHR (Ser-195)). These two peptides were detected because LAT was tyrosine phosphorylated (EpYVNVSQELHPGAAK (Tyr-191)) (Table I). We also detected several peptides of serine phosphorylation but failed to identify any tyrosine phosphorylation of the corresponding proteins. One possible reason was that the tyrosine phosphorylation site(s) might be in a sequence region that was difficult to map using trypsin digestion and LC-MS. Another possible scenario was that the protein formed a complex with a tyrosine-phosphorylated protein and was co-precipitated with the complex. It was also possible that they represented the nonspecific background in immunoprecipitation. Further investigation is required before a conclusion can be made. In addition, nonphosphorylated peptides from immunoglobin were also detected (Fig. 4D), which was likely the result of antibody leaching off the immuno-beads. In comparison, when general phosphorylation was evaluated without pre-enrichment of pTyr proteins, less than 1% of the peptides identified were pTyr-containing peptides (data not shown), and they corresponded to proteins of relatively high abundance. For example, using 107 cells, Tyr-15 phosphorylation of CDC2 (IGEGTpYGVVYK) was detected using IMAC phosphopeptide enrichment alone without the anti-pTyr immunoprecipitation (data not shown). However, when the double-enrichment method described here was applied, this peptide was not detected at a signal intensity proportional to the amount of starting material (109 cells). Recognizing that antibody capacity was not the limiting factor, the phenomenon suggested that anti-pTyr antibodies, in addition to the recognition of pTyr, had a degree of preference for certain groups/sequences of proteins. Thus, the detection of a tyrosine phosphorylation using this method may not be a true representation of the abundance of pTyr in the cell.
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Quantitative Study of the IFN Treatment Effect
To quantitatively compare changes of tyrosine phosphorylation upon treatment of IFN, we incorporated stable isotope labeling at the methyl esterification step. Treated sample was labeled as hydrogen form and control sample as deuterium form. The key in performing quantitation using isotope labeling was in paring up the isotopic peaks, which proved to be challenging in this case as variable mass differences were produced between the pair (depending on the peptide sequence). In addition, deuterium labeling led to deviation in elution time on RP LC. For peptides for which identification had been established, the isotopic peak correlation was achieved by the predictive calculation of the mass of the deuterium form based on the sequence (i.e. the number of acidic residues). Additionally, the LC elution characteristics (deuterium form elutes slightly earlier than the hydrogen form) (17) were taken into consideration. Alternatively, without the knowledge of the peptide identity, the isotope peak pairing could be achieved by applying the plausible hydrogen and deuterium form mass differences, the C13 isotope patterns, and the LC elution characteristics. Once the isotopic peak pairs were correlated, the integrated peak areas were used to calculate the intensity ratio and to achieve the quantitative comparison of every phosphopeptide between the treatment and control samples (Table I). In this study, the ratio of hydrogen to deuterium form for most peptides fluctuated within the range of 0.751.25 or within 1.5-fold. However, for Tyk2, JAK1, IFNAR2c, and IFNAR1 peptides, hydrogen:deuterium ratios were greater than 5:1, representing a significant change upon treatment (Fig. 4, A and B). These results were consistent with the known involvement of the proteins in IFN
signal transduction. An
-tubulin peptide, IHFPLATpYAPVISAEK (Tyr-271), was also found to be present exclusively in the IFN
-treated sample. Furthermore, we identified a CDC42 GAP-like protein (LOC257106) with high confidence by two phosphoserine-containing peptides, GpSGSLEGEAAGCGR (Ser-630) and GpSGpSLEGEAAGCGR (Ser-630, Ser-632), which showed a hydrogen:deuterium ratio of >5 (Fig. 4C). Our data suggested that this protein was likely involved in the signal transduction of IFN
. Because neither of these two peptides contained a pTyr, we further investigated whether IFN
treatment affected the phosphorylation of the identified phosphoserine sites by a general phosphorylation analysis (IMAC enrichment alone). Only the monophosphorylated pS630 peptide was reliably detected in the general phosphorylation analysis (the doubly phosphorylated peptide was detected at lower intensity when both were detected in the pTyr proteomic analysis). No quantitative difference was observed for this peptide between IFN
-treated and control samples (Fig. 5), suggesting that at least the phosphorylation of Ser-630 was not affected by IFN
treatment.
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DISCUSSION |
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Two proteins (STAT1 and STAT2) with tyrosine phosphorylation sites that were previously reported to be influenced by IFN treatment were not detected in this study. Whereas it is possible that restricted accessibility of antibody to the phosphorylation sites may have limited the recovery by immunoprecipitation, a similar STAT complex was recovered using an anti-pTyr immunoprecipitation procedure (18, 19). More likely, it is possible that the abundance of the STAT1 and STAT2 proteins falls below the detection limits of our experiment. A recent publication in which a phosphotyrosine proteomic study was carried out on Jurkat cells treated with sodium pervanadate (a nonspecific tyrosine phosphatase inhibitor) supports this rationale (20). pTyr sites on STAT1/2 were not detected in the study. In the case of the STAT2 protein, the primary sequence of the tryptic peptide may prevent its detection. The pTyr-690 site has a surrounding sequence of NLQERRKpYLKHRLIV (21). Upon trypsin digestion the pTyr-690 site is located in a hydrophilic trimer peptide (pYLK), which would be difficult to detect under the RP LC/MS condition.
Among identified peptides, less than 8% were nonphosphorylated, all of which were derived from immunoglobin (leached from the antibody column). In contrast, rarely any nonphosphorylated peptide was detected in the general phospho-proteomic study using IMAC alone. It was possible that in an eluate of immunoprecipitation, the leached antibody protein was present at a substantial proportion relative to other protein components and caused a background in the IMAC enrichment. Ficarro et al. (6) have noted that in their experimental setup the multiply phosphorylated peptides were preferentially enriched by IMAC comparing to the singly phosphorylated peptides. That seemed to suggest that the experiments were performed under a condition of limited IMAC capacity. It was proven true by our results in which no preference for multiply phosphorylated peptides was observed. However, the extra capacity of IMAC resin may have contributed to the detection of the nonphosphorylated antibody peptides.
Because different pTyr antibodies have slightly different affinity preferences toward different pTyr-containing proteins (22), the combination of several strains of monoclonal antibodies in immunoprecipitation, as one might have predicted, should offer better coverage than that of a single strain. A combination of two anti-pTyr monoclonal antibodies was employed for immunoprecipitation in this study. Another modification/improvement we made was in sample processing prior to methyl esterification. Dialysis was used for buffer exchange instead of C18 cartridges for de-salting. The use of C18 cartridges presented problems in retaining hydrophilic peptides, including some phosphorylated peptides (data not shown).
When MS is used in comparative proteomics, relative quantitation can be achieved through isotope labeling. Considering the small sample size and the multiple steps involved in the work flow, careful control in experiments for reproducibility is required to achieve reliable quantitation. During our experiments, we found that the intensity ratios of hydrogen:deuterium of peptides varied mostly within ± 0.25. Thus, only the pairs with a hydrogen:deuterium ratio change that exceeded 0.5 (50%) were considered as meaningful changes.
A tyrosine-phosphorylated peptide of -tubulin (Tyr-271) was identified to be present in the treated sample but not in the control sample. It is well known that many signaling events lead to modification of microtubules including
-tubulin (for review, see Ref. 23). However, this specific tyrosine phosphorylation site was not previously documented. The detailed relationship between IFN
signaling and the tyrosine phosphorylation of
-tubulin will require further investigation.
Two serine-phosphorylated peptides of a novel factor LOC257106 were found to be in the immunoprecipitation eluate of IFN-treated samples but not in the control samples. The finding of this CDC42 GAP-like protein is of particular interest. Based on the publicly available microarray data, this gene seems most abundantly expressed in T cells (including Jurkat cells) and to a lesser degree in B cells. The induction of gene expression by IFN
was unlikely because the treatment lasted only for 5 min. The peptides identified were serine phosphorylated. However, in an analysis of total phosphorylation, we failed to observe any quantitative changes caused by IFN
treatment, supporting the notion that the treatment did not induce changes in phosphorylation at this particular site. Another possibility was that LOC257106 was tyrosine phosphorylated at a site that was difficult to detect as a tryptic peptide. By sequence analysis, however, no potential tyrosine phosphorylation site was reliably predicted in LOC257106. Alternatively, rather than asserting the effect through direct phosphorylation, LOC257106 might be involved in IFN
signaling pathway through its interaction with a protein of which the tyrosine phosphorylation was affected by IFN
treatment. Scansite (scansite.mit.edu) analysis indicates that besides the CDC42 GAP domain, LOC257106 also possesses several potential SH3 binding domains, including one Grb2 SH3 binding domain (percentile 0.07%), one Cortactin SH3 binding domain (percentile 0.005%), and one Src SH3 binding domain (percentile 0.026%). These SH3 binding domains are known to be involved in signal transduction via protein-protein interaction (24). There are numerous reports indicating the involvement of Rho-type GAP proteins in signal transductions (25). However the involvement of this protein in IFN
signaling or any other biological pathways has not been reported previously. We speculate that LOC257106 may be particularly important in signal transduction in immune cells. The exact mode of function requires further investigation.
The identification of the involvement of -tubulin and LOC257106 in IFN
signaling demonstrates that the anti-pTyr immunoprecipitation enriches not only proteins with phosphorylated tyrosine but also their interacting partners. This strategy will broaden our view on tyrosine phosphorylation and protein-protein interaction, the two major mechanisms for receptor-mediated signal transduction.
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FOOTNOTES |
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Published, MCP Papers in Press, January 19, 2005, DOI 10.1074/mcp.M400077-MCP200
1 The abbreviations used are: pTyr, phosphotyrosine; GAP, GTPase-activating protein; IFN, interferon; IFNAR2c, interferon /ß receptor ß chain; IFNAR1, interferon
/ß receptor
chain; MWCO, molecular weight cut-off; RP, reverse phase; STAT, signal transducers and activators of transcription.
* 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.
Current address: GlaxoSmithKline, 709 Swedeland Rd., King of Prussia, PA 19406.
¶ To whom correspondence should be addressed: Novartis Institute for Biomedical Research, Inc., 250 Massachusetts Ave., Cambridge, MA 02139. Fax: 617-871-4086; E-mail: karen.wang{at}novartis.com
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REFERENCES |
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