Identification of Tyrosine-Phosphorylated Proteins Associated with Lung Cancer Metastasis using Label-Free Quantitative Analyses Hsin-Yi Wu,† Vincent S. Tseng,‡,# Lien-Chin Chen,§ Hui-Yin Chang,§ I-Chi Chuang,| Yeou-Guang Tsay,*,⊥ and Pao-Chi Liao*,| Institute of Chemistry, Academia Sinica, Taipei, Taiwan, Department of Computer Science and Information Engineering, National Cheng Kung University, Tainan, Taiwan, Institute of Information Science, Academia Sinica, Taipei, Taiwan, Department of Environmental and Occupational Health, College of Medicine, National Cheng Kung University, Tainan, Taiwan, Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan, and Institute of Medical Informatics, National Cheng Kung University, Tainan, Taiwan Received March 31, 2010
Lung cancer is a lethal disease, and early metastasis is the major cause of treatment failure and cancerrelated death. Tyrosine phosphorylated (P-Tyr) proteins are involved in the invasive and metastatic behavior of lung cancer; however, only a limited number of targets were identified. We attempt to characterize P-Tyr proteins and events involved in the metastatic process. In a previous work, we have developed a strategy for identification of protein phosphorylation. Here, this strategy was used to characterize the tyrosine phosphoproteome of lung cancer cells that have different invasive abilities (CL1-0 vs. CL1-5). Using our analytical strategy, we report the identification of 335 P-Tyr sites from 276 phosphoproteins. Label-free quantitative analysis revealed that 36 P-Tyr peptides showed altered levels between CL1-0 and CL1-5 cells. From this list of sites, we extracted two novel consensus sequences and four known motifs for specific kinases and phosphatases including EGFR, Src, JAK2, and TC-PTP. Protein-protein interaction network analysis of the altered P-Tyr proteins illustrated that 11 proteins were linked to a network containing EGFR, c-Src, c-Myc, and STAT, which is known to be related to lung cancer metastasis. Among these 11 proteins, 7 P-Tyr proteins have not been previously reported to be associated with lung cancer metastasis and are of greatest interest for further study. The characterized tyrosine phosphoproteome and altered P-Tyr targets may provide a better comprehensive understanding of the mechanisms of lung cancer invasion/metastasis and discover potential therapies. Keywords: tyrosine phosphorylation • lung cancer metastasis • mass spectrometry • immunoaffinity enrichment • label-free quantitative analysis
Introduction Lung cancer is a common cause of cancer-related death. The overall cure rate of lung cancer is only 13%1 and more than 80% of patients with non-small-cell lung cancer (NSCLC) will develop metastasis within 5 years after potentially curative treatment. The invasiveness and metastasis during cancer progression are the main formidable barriers to successful * To whom correspondence should be addressed. For P.-C.L.: mailing address, Department of Environmental and Occupational Health, National Cheng Kung University College of Medicine, 138 Sheng-Li Road, Tainan 70428, Taiwan; tel, 886-6-2353535ext 5566; fax, 886-6-2743748; e-mail:
[email protected]. For Y.-G.T.: mailing address, Institute of Biochemistry and Molecular Biology, National Yang-Ming University, No.155, Sec. 2, Linong St., Beitou Dist., Taipei City 112, Taiwan; tel, 886-2-28267119; e-mail:
[email protected]. † Institute of Chemistry, Academia Sinica. ‡ Department of Computer Science and Information Engineering, National Cheng Kung University. § Institute of Information Science, Academia Sinica. | College of Medicine, National Cheng Kung University. ⊥ National Yang-Ming University. # Institute of Medical Informatics, National Cheng Kung University.
4102 Journal of Proteome Research 2010, 9, 4102–4112 Published on Web 06/23/2010
treatment. Several lines of evidence indicate that changes in tyrosine phosphorylated (P-Tyr) proteins result in altered cell adhesion interactions, contributing to the invasive and metastatic properties of tumor cells. Zelinski et al. have reported that the levels of protein tyrosine phosphorylation regulate a balance between cell-cell and cell-extracellular matrix (ECM) adhesions among epithelial cells. They showed that enhanced tyrosine kinase activity weakens cell-cell contact and promotes ECM adhesion, which accelerates the metastatic process.2 Enhanced expression of focal adhesion kinase (FAK), a nonreceptor protein tyrosine kinase, correlates with the enhanced motility3 and invasiveness of human tumor cells.4,5 Moreover, P-Tyr FAK was observed in invasive ovarian carcinoma, but it was not detected in normal cells.6 P-Tyr β-catenin enhanced invasion and metastasis in a subset of colorectal adenocarcinomas.7 Previous studies have demonstrated that tyrosine phosphorylation may be important for the development of target-specific drugs or biomarkers for the prevention and diagnosis of metastasis. Treatment of highly metastatic Lewis lung carcinoma cells with sodium orthovanadate, a phospho10.1021/pr1006153
2010 American Chemical Society
Identification of Tyrosine-Phosphorylated Proteins tyrosine phosphatase inhibitor, resulted in suppression of metastatic ability.8 Tyrosine kinase activity of EGFR promotes tumor cell proliferation, cell survival, angiogenesis, invasion, and metastasis.9 Activation of EGFR is reported to enhance cell migration by means of receptor phosphorylation on tyrosine residues and subsequent activation of downstream signaling pathways, such as the ERK/MAP kinase cascade.10,11 A selective drug inhibitor of EGFR tyrosine kinase, gefitinib (ZD1839, Iressa), was approved as a treatment for NSCLC.12,13 Although protein tyrosine phosphorylation is known to be related to the cell metastatic process, only a handful of protein targets have been reported. We hypothesized that there may be some other tyrosine phosphorylation events involved in lung cancer metastasis but not previously discovered. We attempted to identify these protein targets by using comparative phosphoproteome analysis of CL1-0 and CL1-5 cells, which have different metastatic abilities.1
Materials and Methods Cell Culture and Cell Lysis. The human lung adenocarcinoma cell lines CL1-0 and CL1-5 were kindly provided by Dr. P.-C. Yang (Academia Sinica, Taipei, Taiwan) and cultured in RPMI-1640. Medium was supplemented with 10% fetal bovine serum (Gibco BRL, Gaithersburg, MD) and antibiotics. The cells were incubated at 37 °C under 5% CO2. To extract cellular protein, the dispersed cells were centrifuged at 550g at 4 °C for 10 min. Cell pellets were resuspended in ddH2O containing phosphatase inhibitors (2 mM sodium orthovanadate and 10 mM β-glycerolphosphate). Cell rupture was achieved by sonication three times at 20 W for 10 s. After centrifugation at 25 000g for 1 h, the supernatant that contained proteins was collected. A small aliquot was extracted for the estimation of protein concentration. Immunoprecipitation of Phosphotyrosine Proteins. A total of 700 µg of proteins in 300 µL of lysis buffer (50 mM Tris HCl, 150 mM NaCl, 1% NP40, and 0.5% sodium deoxycholate) was incubated with 50 µL of immobilized anti-pTyr antibody (PT66)-agarose beads (Sigma, St. Louis, MO) and 50 µL of immobilized anti-pTyr antibody (4G10)-agarose beads (UpstateMillipore, Billerica, MA) at 4 °C. After overnight incubation, the sample was washed three times with 500 µL of lysis buffer. Proteins were eluted by the addition of 40 µL of 4× sample buffer (250 mM Tris-HCl, 8% sodium dodecyl sulfate (SDS), 40% glycerol, 0.04% bromophenol blue, and 400 mM dithiothreitol (DTT)). To facilitate the following in-gel digestion process, SDS-PAGE comprised of a 5% acrylamide stacking (upper) gel and 20% acrylamide separating (lower) gel was used to concentrate the eluted proteins in one single band. After electrophoresis at 50 V for 1 h, proteins were enriched on the top of the separating gel. After Coomassie blue staining, the protein gel band was excised and subjected to in-gel digestion. Tryptic in-Gel Digestion. Gel pieces were washed twice with solutions containing 50% v/v acetonitrile and 50% v/v acetonitrile/25 mM ammonium bicarbonate. The gel pieces were incubated in 10 mM DTT at 65 °C for 45 min for reduction followed by alkylation with 55 mM iodoacetamide in 25 mM ammonium bicarbonate at room temperature for 1 hr. To digest proteins, 0.1 µg of trypsin (Promega, Madison, WI) was added. Samples were incubated at 37 °C. After overnight incubation, the supernatants were transferred into new tubes. An additional volume of 20 µL of 50% acetonitrile/5% formic acid (v/v) was added to extract the remaining peptides from the gel pieces.
research articles The supernatants were pooled with the previously colleted supernatants. Enrichment of Phosphopeptides by TiO2 Microcolumns and Alkaline Phosphatase Treatment. A small plug of C8 material was stamped out of a C8 solid phase extraction disk (SUPELCO, Bellefonte, PA) to restrict the end of the GELoader tip. One 3-mm TiO2 microcolumn was rinsed with 20 µL of sample loading buffer (2% trifluoroacetic acid (TFA)/65% CH3CN solution saturated with glutamic acid). The peptide mixture (15 µL) was diluted with 185 µL of sample loading buffer and loaded into the microcolumn with a syringe pump set at a flow rate of 10 µL/min. The microcolumn was washed in the following order: 20 µL of sample loading buffer, 20 µL of 65% acetonitrile/0.5% TFA, and 20 µL of 65% acetonitrile/0.1% TFA. To elute the bound peptides, a total of 30 µL of 300 mM NH4OH/50% CH3CN was used. An aliquot of the TiO2-eluted sample was incubated with 0.25 U of alkaline phosphatase (Roche Applied Science, Mannheim, Germany) at 37 °C for 2 h. The 10× dephosphorylation buffer (Roche Applied Science, Mannheim, Germany) was used to adjust the pH value to 8-9. Online Liquid Chromatography Mass Spectrometry (LC-MS) Analysis. Full-scan LC-MS was conducted on Orbitrap in order to obtain a high mass accuracy measurement. Protein tryptic digests were fractionated using a BioBasic C18 300 Å packed PicoFrit column (75 µm I.D. × 10 cm, New Objective, Woburn, MA) with a Finnigan Surveyor highperformance liquid chromatography system (Thermo Finnigan Scientific, Bremen, Germany) coupled to an LTQ/Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific), which was equipped with an electrospray ionization source. The flow rate was set at 200 nL/min. Sample loading transitioned from 100% buffer A (5% acetonitrile/0.1% formic acid) to 10% buffer B (80% acetonitrile/0.1% formic acid) in 2 min. For peptide elution, the following steps were used: transition from 10% buffer B to 60% buffer B in 38 min followed by a further increase to 100% buffer B within 1 min. The buffer condition was then changed to 100% buffer A in the subsequent 9 min and was held for another 20 min. The mass spectrometer was operated in the positive ionization mode with a spray voltage of 1.8 kV. The scan range of each full MS scan was m/z 300-2000. LC-MS data was acquired in the Orbitrap with resolution set at 60 000 (at m/z 400). Identification of Potential Phosphopeptide Signals by DeltaFinder. The procedure used to find potential phosphopeptide signals was previously described.14,15 Briefly, noncommercial ReAdW.exe, which was available at http://tools.proteomecenter.org/software.php, was used to convert Thermo Xcalibur LC-MS files (.raw files) to the open file format mzXML. Another open-source computer program, msInspect (http://proteomics.fhcrc.org/) was utilized to find peptides signals in mzXML files. The data files encoding peak information were saved as .tsv files. An in-house program, LcmsFormatConverter, was used to filter the signal noise and to transform the peak list into a format that is compatible with DeltaFinder program (http://binfo.csie.ncku.edu.tw:8080/ DeltaFinder/) which was used for selecting dephosphorylated and phosphorylated signal pairs within the alkaline phosphatases treated and untreated LC-MS data. In the process of “Find ∆”, signals with a mass shift of 79.966n ( k Da between Journal of Proteome Research • Vol. 9, No. 8, 2010 4103
research articles two LC-MS data sets were extracted as potential phosphopeptide signals and their dephosphorylated forms. Targeted Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Analysis and Database Search. Targeted LC-MS/MS analysis was performed on a nano-HPLC system coupled to an ion trap mass spectrometer (LCQ DECA XP Plus, ThermoFinnigan, San Jose, CA). Tryptic digests were fractionated using a C18 microcapillary column (75 µm I.D. × 15 cm) with a nano-HPLC system (LC Packings, Amsterdam, Netherlands). Peptides were electrosprayed into the LCQ with an application of a 1.3 kV spraying voltage. Each cycle was comprised of one full scan mass spectrum (m/z 300-2000) followed by three tandem mass spectra with collision energy set at 35%. The m/z values of potential phosphopeptide signals that came from DeltaFinder were set in the “inclusion list” function on LCQ, which triggered the MS/MS analysis once these m/z values were observed. All MS/MS files (DTA files) were generated using Bioworks Browser 3.1 (ThermoElectron, San Jose, CA). For DTA file generation, precursor mass tolerance was set to 1.4 Da. Up to 25 MS/MS scans have the same precursor mass and still allow those spectra to be grouped into a single .dta file. Only MS/MS spectra that contained more than 12 peaks were accepted. At least one scan with the same precursor mass and charge state was required to create a .dta file. A Perl script, merge.pl, provided on the Matrix Science Web site was used to concatenate the generated DTA files. The resulting peak lists were searched against the Swiss-Prot database via an in-house Mascot server (Matrix Science Ltd., U.K.). Oxidation on methionine, deamidation on asparagine and glutamine, carboxyamidomethylation on cysteine, and phosphorylation on serine, threonine, and tyrosine residues was set as variable modifications. Trypsin was chosen as enzyme specificity. A maximum of two miscleavages were allowed. The minimal peptide length was specified to be six amino acids. The initial maximal mass deviation of parental ions was set to 1 Da, and for fragment ions it was set to 1 Da. A concatenated forward-reverse database was constructed to calculate the in situ false discovery rate (FDR). The maximum peptide FDR was set to 0.02. Western Blotting. Commercial 1.0 mm ×10 well gradient gels (4% to 12%, NuPAGE Bis-Tris, Invitrogen, Carlsbad, CA) were used to separate proteins. Proteins were separated and transferred onto nitrocellulose membranes followed by blocking in TBST buffer (3.0 g/L Tris, 14.4 g/L glycine, 0.5% Tween 20, pH 8.3) containing 5% nonfat milk at room temperature for 1 h. Before being blotted with antibodies at 4 °C overnight, the membranes were washed three times with TBST. The primary antibodies and concentrations were as follows: anti-teratomaassociated tyrosine kinase (1:1000, Abnova, Taipei, Taiwan), anti-tyrosine-protein kinase TXK (1:100, eBioCenter, California), anti-FYVE, RhoGEF, and PH domain-containing protein 2 (1: 200, ProteinTech Group Inc., Chicago, IL), anti-synaptotagminlike protein 4 (1:200, ProteinTech Group Inc., Chicago, IL), and anti-dipeptidase 2 (1:200, ProteinTech Group Inc., Chicago, IL). Before being incubated with secondary antibodies, membranes were washed three times with TBST. After incubation with HRPconjugated secondary antibodies at a dilution of 1:5000 at room temperature for 1 h, membranes were washed three times with TBST and developed with enhanced chemiluminescence detection. 4104
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Wu et al.
Results and Discussion Strategy for Identifying the Tyrosine Phosphoproteome Involved in Lung Cancer Metastasis. Previous work has shown that protein tyrosine phosphorylation influences the invasive and metastatic potential of tumor cells.16 Chu and co-workers selected progressively greater invasive cancer cell populations from a clonal cell line of human lung adenocarcinoma using a transwell invasion chamber.1 CL1-0 and its more invasive subpopulation, CL1-5, provide good in vitro models for evaluation of the metastatic potential of lung cancer cells. By taking advantage of their innovation and performing comparative proteomic analysis of cells with different invasive abilities (CL1-0 vs. CL1-5), novel tyrosine phosphoprotein targets associated with lung cancer metastasis may be discovered. To identify tyrosine phosphorylation events associated with lung cancer metastasis, qualitative and quantitative analyses were performed. We attempted to characterize the tyrosine phosphoproteome in CL1-0 and CL1-5 cells. Comparative analysis was carried out on the P-Tyr peptides by label-free quantitative analysis. Studies of P-Tyr proteins are hampered by their extremely low abundance.17 To address this issue, an analytical strategy proposed in our previous work was used. Its capability for efficient and confident identification of the tyrosine phosphoproteome by high mass accuracy data has been demonstrated.14,15 Here, as depicted in Figure 1, cell lysates of CL1-0 and CL1-5 were combined and submitted to the following procedures. For the enrichment of P-Tyr proteins by immunoprecipitation, it is suggested that a combination of multiple antibodies may yield better coverage of all possible P-Tyr proteins in a sample.18 A mixture of 4G10 and PT66 antibodies was used for immunoprecipitation. After elution, P-Tyr proteins were concentrated as a single band on the top of the 20% acrylamide separating gel. In this way, unwanted NP40-containing lysis buffer that remained in the protein sample was removed. The gel band was excised and subjected to tryptic digestion. The procedure for phosphopeptide identification was described in our previous work.14,15 Briefly, phosphopeptides were enriched on a TiO2 microcolumn. The TiO2 enrichment procedure was adapted from Wu et al.19 According to that work, it has been demonstrated that the developed TiO2 procedure not only works well for global pSer/pThr enrichment but also for P-Tyr peptides. An aliquot of eluted sample was treated with alkaline phosphatase to remove phosphate groups. Both alkaline phosphatase-treated and untreated samples were subjected to LC-MS analyses. Peak finding was achieved by use of a noncommercial program, msInspect, and an in-house program, LcmsFormatConverter. Another in-house program, DeltaFinder, was used to select signals with 79.966n Da (n represents the number of phosphate groups) mass shift between phosphatase-treated and untreated LC-MS data. This procedure was performed three times to obtain a comprehensive list of potential phosphopeptide signals. Targeted LC-MS/MS analysis was conducted on an untreated sample by focusing on m/z values of the potential phosphopeptide signals. Identification of phosphopeptides was obtained by searching against a human protein database. Six replicate LC-MS analyses of untreated CL1-0 and CL1-5 samples were used for quantitative analysis. A standard phosphopeptide, T6 (1 pmol), was added to each sample prior to LC-MS analysis for the purpose of adjusting signal intensity in the quantitative analysis. Peaks in each LC-MS data set (six CL1-0 vs. six CL1-5) were aligned by use of an in-house
Identification of Tyrosine-Phosphorylated Proteins
Figure 1. Analytical strategy. P-Tyr proteins were immunoprecipitated and concentrated. After in-gel digestion and phosphopeptide enrichment, an aliquot of the sample was treated with alkaline phosphatase to remove the phosphate groups. Samples with or without dephosphorylation reaction were subjected to DeltaFinder program for selecting potential phosphopeptide signal pairs. Phosphopeptide identification was achieved by targeted LC-MS/MS analysis. For quantitative analysis, peaks in 12 replicate LC-MS data sets were aligned. The m/z values identified as tyrosine phosphopeptides were selected. After adjustment of the intensities by that of the standard peptide, P-Tyr peptides with altered levels between CL1-0 and CL1-5 cell lines were determined by statistical analysis.
program, PeakAlign. The peak intensity of the m/z values previously identified as phosphopeptides were obtained from the aligned peak groups. Statistical analysis of the phosphopeptide signals was performed on the peak intensities, which were adjusted by the intensity of T6. The detailed procedures and results are described in the following sections. Identification of Tyrosine Phosphorylated Proteins in CL1-0 and CL1-5 Cells. Samples pooled from individual plates of CL1-0 and CL1-5 cell served as a biological sample. For selecting out potential phosphopeptide signals, three biological replicates were used. After three LC-MS and DeltaFinder program analyses, 347 unique signal pairs (from 421 pairs) were selected. The 347 m/z values that represented the phosphopeptide signals were subjected to targeted LC-MS/MS analysis by using phosphopeptide-containing samples pooled from CL1-0 and CL1-5 cells. Due to the intrinsic limitation of LCQ that only 25 m/z values can be set as precursor ions for each run, we conducted more than 14 targeted LC-MS/MS analyses to obtain most of the identification. We filtered the identification results by lowering the FDR to reduce ambiguous assignment. A mascot score of 31 was taken as a threshold score in this study for the FDR < 2%. To obtain confident identification of the P-Tyr sites, sequencing results were manually inspected. The identification of a P-Tyr peptide or site was considered
research articles
Figure 2. Subcellular locations and functional classification of the 302 identified P-Tyr proteins: (A) the cellular compartment of the 302 identified CL1 cell P-Tyr proteins; (B) molecular functional classification of the 302 identified P-Tyr proteins according to the Gene Ontology database.
acceptable when the following two criteria were fulfilled. First, the peptide score is greater than or equal to 31. Second, more than three b or y ion signals adjacent to or on the assigned P-Tyr site were found. By performing targeted LC-MS/MS analysis on the 347 m/z values, we identified 302 P-Tyr peptides containing 335 P-Tyr sites from 276 proteins. Detailed information of the 302 phosphopeptides is provided in Supporting Information, Table A. To functionally annotate the identified phosphoproteins, 276 proteins were categorized according to their cellular compartment and molecular function as described in Gene Ontology (Figure 2). Most of the P-Tyr proteins were found to be located in the membrane as shown in Figure 2A. As depicted in Figure 2B, many of the 276 proteins were involved in binding interactions. The functions of 41% of the proteins are still unknown. Among 302 P-Tyr peptides, 273 peptides (90.4%) contained one P-Tyr site. Only 26 (8.6%) and 3 (1%) peptides had two and three phosphate groups on tyrosine residues. As for the 335 P-Tyr sites, 74 (22.1%) have been reported in previous works according to a search against PhosphoSitePlus (PSP), which is available on http://www.phosphosite.org. In addition, the identified P-Tyr sites were matched to kinase motifs by using phosphorylation site database (PHOSIDA) at http://www.phosida.com/.20 A total of 102 (30.4%) sites were predicted as potential P-Tyr sites. By combination of these two results, 148 (44.2%) sites were either Journal of Proteome Research • Vol. 9, No. 8, 2010 4105
research articles
Wu et al.
Figure 3. Six phosphorylation consensus sequences extracted by the Motif-X algorithm. Only P-Tyr peptides with altered levels between CL1-0 and CL1-5 cell lines were included. Six amino acids before and after the phosphotyrosine residue were taken into consideration. The kinases that recognize the motif are listed to the right of each motif. Residues [E/D], [A/G/S/T/E/D], [E/D/Y], and [L/I/V] are denoted as B, O, J, and Z, respectively.
reported as known tyrosine phosphorylation sites or predicted as possible substrates for tyrosine kinases. Tyrosine Phosphorylated Proteins Present at Different Levels in CL1-0 and CL1-5 Cells. Immunoprecipitated samples from three plates each of CL1-0 and CL1-5 cells were pooled and subjected to six LC-MS analyses separately, serving as six technical replicates. Quantitative analysis was achieved by using label-free analysis. Peak lists of 12 LC-MS data sets were aligned by an in-house program, PeakAlign, including RT adjustment and peak alignment. First, the user should define one LC-MS data as “reference data”. Since most of the RT shift along the chromatography does not fit the linear regression, a LOESS regression (locally weighted scatterplot smoothing) function was used to better describe each localized RT shift. The second step of the program was to calculate the LOESS regression functions according to 10-20 user-defined peaks at different RT. Those functions were used to represent localized RT shift between one LC-MS data set and the “reference data”. Third, all the remaining RT values in that LC-MS data set were adjusted by fitting the corresponding regression function. After all the other LC-MS data have been processed, the fourth step was to align the peaks. A proposed cluster-based technique based on the distance constraint (m/z and RT range) is applied to align the adjusted peaks. This proposed algorithm showed higher accuracy than using other alignment methods such as dynamic time warping (DTW) and sliding window. (unpublished data) To evaluate the feasibility of PeakAlign, six LC-MS data sets from bovine albumin digest were used. After inspection of 40 m/z values of bovine albumin peptides, all the signals were correctly aligned. We also used another data set that contained bovine albumin peptides and synthetic data to demonstrate the accuracy of the alignment results. After checking the same 40 m/z values, the accuracy was greater than 95%. As for the 12 LC-MS data sets, approximately 5000 peaks were present in each LC-MS data set. After alignment, the peaks were separated into 6358 clusters. To evaluate the alignment result, 50 m/z values were manually checked, of which 48 groups were correctly aligned. The aligned peak information of the previously identified P-Tyr peptides were extracted according to the 302 m/z values (with (0.02 Da). 4106
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Mann-Whitney U tests were carried out on the 12 adjusted intensity of each cluster to examine the significance of phosphopeptide levels between two groups (CL1-0 versus CL1-5). A significance level of 5% was accepted. Once the alteration between two groups was considered significant (p < 0.05), the ratio was calculated by dividing the average value of the peak intensity from the CL1-5 sample by that of the CL1-0 sample. The alignment results of the significantly altered P-Tyr peptides were manually checked and would be excluded once incorrectly aligned. A total of 36 P-Tyr peptides were altered, containing 30 P-Tyr peptides with higher levels in CL1-5 cells and 6 P-Tyr peptides with higher levels in CL1-0 cells. The information is listed in Table 1, together with information such as protein name, peptide sequences, modification sites, and expression ratio (CL1-5/CL1-0). Among the 36 peptides, 23 peptides contained missed cleavage sites. Signals representing the 23 fully digested peptides were inspected to evaluate their impact to the quantification accuracy. Interestingly, most signals were too small to be quantified and sequenced. In Molina and coworkers’ work,21 a high level of missed cleavages in the tryptic digest was also observed, which was thought to be caused by the presence of phosphate groups or highly acidic residues. In their work, on average 1.1 missed cleavages per phosphopeptide was found while 0.8 were observed in our data. To monitor the digestion efficiency, one gel sample containing 0.2 µg of bovine albumin was subjected to gel digestion simultaneously. In this batch, bovine albumin was identified with >60% protein coverage and, on average, 0.2 missed cleavages. Some of the differentially tyrosine phosphorylated proteins have been reported to be relevant to metastasis including lung cancer metastasis. Tyrosine phosphorylation events on focal adhesion kinase 1 (FAK, P19),22–24 β-catenin (P24),25–28 receptor tyrosine-protein kinase, and ErbB2 (P29)29–34 have been reported to be involved in increase of lung cancer cell metastatic ability. Growth factor receptor-bound protein 7 (Grb7, P08) mediates cell migration through a phosphotyrosine-dependent path.35,36 Although the tyrosine phosphorylation events of the receptor tyrosine-protein kinase ErbB4 (P01), G-protein coupled receptor 19 (P07), and tyrosine-protein kinase SYK (P27) have not been reported to be related to cell metastasis, their
Q15760
Q14451
P07
P08
Q4QE82
B7ZMH3 P41732 P42681 P11730
Q7Z6J4
Q05397 P07711 Q5D862
Q15131 Q96C24 P35222 Q14195
Q5GJ75
P43405 Q9BQS8
P04626
A2KCR7 Q9UMZ3
Q13519
Q9H4A9 C5IWV5 Q96PN6 Q13595
P13
P14 P15 P16 P17
P18
P19 P20 P21
P22 P23 P24 P25
P26
P27 P28
P29
P30 P31
P32
P33 P34 P35 P36
ADCY10 TRA2A
DPEP2
PNOC
CDR3a PTPRQ
ErbB2
SYK FYCO1
TNFAIP8L3
CDK10 SYTL4 CTNNB1 CRMP4
FAK CTSL1 FLG2
FGD2
DMXL2 TSPAN7 TXK Camk2g
LmjF17.1090
USP29
TMPRSS12
TNFSF10
GRB7
GPR19
GABRE FRAS1
SCYL1
GOLGA7B FIGNL1
ErbB4
gene name
dipeptidase 2 trypsinogen adenylate cyclase type 10 transformer-2 protein homologue R
cell division protein kinase 10 synaptotagmin-like protein 4 catenin β-1 collapsin response mediator protein 4 tumor necrosis factor, R-induced protein 8-like protein 3 tyrosine-protein kinase SYK FYVE and coiled-coil domain-containing protein 1 receptor tyrosine-protein kinase erbB-2 CDR3a phosphotidylinositol phosphatase PTPRQ nociceptin
transmembrane protease, serine 12 ubiquitin carboxyl-terminal hydrolase 29 ubiquitin hydrolase, putative (cysteine peptidase, clan ca, family c19, putative) DMXL2 protein tetraspanin-7 tyrosine-protein kinase TXK calcium/calmodulin-dependent protein kinase type II γ chain FYVE, RhoGEF and, PH domain-containing protein 2 focal adhesion kinase 1 cathepsin L1 filaggrin-2
teratoma-associated tyrosine kinase cDNA FLJ60826 extracellular matrix protein FRAS1 probable G-protein coupled receptor 19 growth factor receptor-bound protein 7 tumor necrosis factor ligand superfamily member 10 cDNA FLJ78387
receptor tyrosine-protein kinase erbB-4 golgin subfamily A member 7B fidgetin-like protein 1
protein name
AHTMPGTpYAPSTTLSSPpSTQGLQEQAR pYNpSATIDNDIMLIK FLISNSSQVLMpYEGLPGYGK AHTPTPGIpYMGRPTHSGGGGGGGGGGGGGGGGRR
RFSEFMRQpYLVLSMQpSpSQR
CAASVSGGSNpYKLTFG GGHpTpYNIpSVYAVNpSAGAGPKVPMRITMDIK
VKVLGSGAFGTVpYK
MALEEIRPKEVpYLDRK pYSLVHQRLADTLQQCFMNpTK
TVANMLIDDTSSEIFDELpYK
LPLVGQYSLRKQPpYNNLK SSMQSGSSMVSSIRSVVTGMLGpY GNPEEEDVDTSQVLYEWEQGFSQSFTQEQVADIDGQpYAMTR QEVQNLIKDKGVNSFMVpYMAYK
THAVSVSETDDpYAEIIDEEDTYTMPSTR HKRLpYGMNEEGWR QpSpSpYGQHGSGSSQSSGYGQYGpSR
MYSEpYVK
KEpTEIFFQPSQGpYRPPPFSEK HEIKDTFLRTYTDAMQpTpYNGNDER MILSSpYNTIQSVFCCCCCCSVQK HHHHHHpSSGVDLGTENLpYFQCMATCpTR
ISQLGNpYNLGGKSR
AENpSRLPSTQAGVIPQGEpYEGDSLpYRPA
GDSGGPLMCpYLPEYKR
FTISRDDpSKNMLpYMQMNpSLK
ENTKNDKQMVQpYIpYK
VpYSEDGACR
CYRSNApYTITTSpSR
CCTQLVpSYPENEMIpYK GAFSKGQILpYGR
CNTTVCLGKIGSpYLpSApSTR
QLFEETVKTLNGFpYAEAEK KpYHQPQRASGSpSpYGGVK
CAGTENKLSSLSDLEQQpYR
sequence
a
3 3 2 3 3 3 3 2 3 3 3
738.93 854.12 859.43 928.97 822.11 690.98 898.98 753.40 620.34 1162.28 883.51
3 3 3 3
3 3 3
993.84 596.43 767.23 1041.78
2
507.85 1085.79 591.75 892.46
3 3 3 3
891.39 1027.29 951.89 1144.10
3 3
3
627.95 1083.32 530.67
2
2
781.38
3
2
921.34
899.18
2 3
1106.07 459.78
1032.12
3
3 3
767.00 697.40 738.39
3
742.53
m/z
charge state
Y10 T1759, Y1760, S1763, S1769 Y156, S163, S164 Y58, S68 Y127, S129 Y514 Y208
Y735
Y364 Y110, T128
Y149
Y397 Y36 S616, S617, Y618, S636 Y282 Y349 Y86 Y167
Y199
T873,Y883 T130, Y131 Y6 S8, Y19, T27
S898, Y913, Y919 Y109
S96, Y101, S106 Y275
Y209, Y211
Y107
Y353
Y63 Y310,S320, Y321 Y458, S460, S462 S24, Y32 Y3697
Y46
sites
0.09 0.13 0.028 0.27
0.12
247.5 0.1
31.5
61.32 205.5
222.12
136.32 65.82 87.61 175.24
45.62 623.23 89.45
1465.54
13.52 923.32 10.27 66.31
54.41
22.94
56.36
103.42
46.51
96.32
88.27
789.23 113.15
3.8
218.06 15.45
9.64
ratiob (CL1-5/ CL1-0)
0.026 0.03 0.007 0.05
0.015
31.7 0.02
12.1
13.6 50.8
31.6
43.3 28.3 31.9 64.7
12.7 134.8 21.8
174.2
5.8 62.8 2.7 16.6
6.3
8.4
11.6
34.1
12.3
13.3
19.3
84.6 14.7
1.3
40.5 4.2
1.4
SDrc
0.021 0.039 0.015 0.005
0.042
0.035 0.023
0.011
0.011 0.026
0.012
0.023 0.033 0.002 0.008
0.001 0.001 0.021
0.001
0.032 0.022 0.014 0.013
0.013
0.010
0.021
0.013
0.049
0.019
0.031
0.015 0.01
0.012
0.045 0.034
0.023
p valued
18.6/22.1 12.9/19.1 15.6/19.5 8.1/16.7
11.0/5.9
7.2/10.6 10.3/17.1
26.3/28.0
13.8/17.4 16.6/18.3
12.1/7.5
18.9/25.5 26.3/34.0 22.2/28.9 23.9/28.2
14.6/23.7 15.2/15.4 11.8/21.3
9.3/7.4
28.7/31.9 2.3/6.4 14.5/21.9 12.9/21.5
8.3/8.1
22.4/29.0
10.6/17.7
22.3/24.3
16.2/20.9
5.1/12.8
17.6/13.0
5.9/8.9 8.7/9.7
26.5/21.6
12.4/13.9 19.5/18.9
6.3/13.1
CV5/CV0e(%)
down down down down
down
up down
up
up up
up
up up up up
up up up
up
up up up up
up
up
up
up
up
up
up
up up
up
up up
up
altered level in CL1-5
a Modified residues are underlined, indicating phosphorylation (STY), oxidation (M), carboxyamidomethylation (C), and deamidation (N, Q). b Ratio derived from the average value of the peak intensity from CL1-5 sample divided by that of the CL1-0 sample (Ratio ) xj5/jx0). c Standard deviation of the ratio (SDr) was calculated by the following formula, SDr ) Ratio[(S5/jx5)2 + (S0/jx0)2]1/2, where S5 and S0 are the standard deviations of the peak intensities of CL1-5 and CL1-0. d The p value refers to the significance of difference between CL1-0 and CL1-5 cells by using Mann-Whitney U test. e Coefficient of variation from CL1-5 and CL1-0 data.
Q86WS5
Q9HBJ7
P11
P12
P50591
A8MT31 Q86XX4
P05 P06
A8K008
Q96KG9
P04
P09
Q2TAP0 Q6PIW4
P02 P03
P10
Q62956
P01
no.
SwissProt no.
Table 1. P-Tyr Peptides with Altered Levels between CL1-5 and CL1-0 Cell Lines
Identification of Tyrosine-Phosphorylated Proteins
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Journal of Proteome Research • Vol. 9, No. 8, 2010 4107
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Wu et al.
Figure 4. Interaction network analysis of proteins that showed altered levels of tyrosine phosphorylation between CL1-0 and CL1-5 cell lines. Proteins identified in this study (ErbB4, SCYL1, GRB7, TXK, FGD2, FAK, SYTL4, β-catenin, CRMP4, ErbB2, and DPEP2) are marked with a circle and listed in Table 2.
overexpression correlate with higher grade, vascular invasion, and nodal metastases.37–39 Motif Analysis and Kinase Prediction. In order to identify kinases that may play roles in lung cancer metastasis, the 36 P-Tyr sequences that extended six residues before and after the P-Tyr sites (a total length of 13 aa) were submitted to the online motif extractor, Motif-X algorithm.40 The resulting consensus sequences were compared with known kinase recognition motifs using the Human Protein Reference Database PhosphoMotif Finder (http://www.hprd.org/PhosphoMotif_finder). Four tyrosine motifs and two novel consensus
sequences were found (as illustrated in Figure 3). X[E/D]pYX, pY[A/G/S/T/E/D], pYXX[L/I/V], and [E/D/Y]pY are recognized as the substrate motifs of EGFR kinase,41 Src kinase,40 tyrosine kinase Janus kinase 2 (JAK2) kinase,42 and T-cell protein tyrosine phosphatase (TCPTP),43,44 respectively. Interestingly, these four kinases were closely related and most of them are known to be related to cell metastasis. EGFR is involved in invasion and metastasis, including in lung cancer.45–47 Overexpression or hyperactivity of Src family kinases (SFKs) was found to be common in NSCLCs.48 Increased SFK phosphorylation following EGFR activation is required for tumor cell
Table 2. Eleven Identified P-Tyr Proteins That Bind to One Another Either Directly or via Other Proteins in the Extracted Protein-Protein Interaction Network
no.
SwissProt no.
gene name
protein name
P01
Q62956
ErbB4
receptor tyrosine-protein kinase erbB-4 teratoma-associated tyrosine kinase growth factor receptor-bound protein 7 tyrosine-protein kinase TXK FYVE, RhoGEF and PH domain-containing protein 2 focal adhesion kinase 1 synaptotagmin-like protein 4 catenin β-1 collapsin response mediator protein 4 receptor tyrosine-protein kinase erbB-2 dipeptidase 2
P04
Q96KG9
SCYL1
P08
Q14451
GRB7
P16 P18
P42681 Q7Z6J4
TXK FGD2
P19 P23 P24 P25
Q05397 Q96C24 P35222 Q14195
FAK SYTL4 CTNNB1 CRMP4
P29
P04626
ErbB2
P33
Q9H4A9
DPEP2
4108
Journal of Proteome Research • Vol. 9, No. 8, 2010
sequence
localization
literature ratio report of (CL1-5 lung cancer /CL1-0) metastasis
CAGTENKLSSLSDLEQQpYR
membrane
9.64
V
CNTTVCLGKIGSpYLpSApSTR
cytoplasm
3.80
VpYSEDGACR
cytoplasm
96.32
MILSSpYNTIQSVFCCCCCCSVQK MYSEpYVK
cytoplasm cytoplasm
10.27 1465.54
THAVSVSETDDpYAEIIDEEDTYTMPSTR SSMQSGSSMVSSIRSVVTGMLGpY GNPEEEDVDTSQVLYEWEQGFSQSFTQEQVADIDGQpYAMTR QEVQNLIKDKGVNSFMVpYMAYK
cytoplasm membrane cytoplasm cytoplasm
45.62 65.82 87.61 175.24
V
VKVLGSGAFGTVpYK
membrane
31.50
V
AHTMPGTpYAPSTTLSSPpSTQGLQEQAR
membrane
0.09
V
research articles
Identification of Tyrosine-Phosphorylated Proteins 49–51
proliferation and invasion. JAK2 can interact with SFKs in the cell cycle and is involved in cell migration.52,53 TCPTP is an intracellular non-transmembrane tyrosine-specific phosphatase54 that is part of the signal pathway containing SFK and JAK.55 Aside from the known kinases, two extracted consensus sequences did not match any known motifs. One had a glycine residue at position -2 (GXpY), and the other contained a methionine at position +1 (pYM). The unknown consensus sequences may suggest previously uncharacterized kinase activity. Figure 5. Immunoprecipitation (using antiphosphotyrosine antibodies) followed by Western blot analysis of five P-Tyr proteins including teratoma-associated tyrosine kinase, tyrosine-protein kinase TXK, FYVE, RhoGEF, and PH domain-containing protein 2, Synaptotagmin-like protein 4, and dipeptidase 2.
Interaction Network Analysis and Confirmation of Selected Tyrosine Phosphorylated Proteins. A protein-protein interaction network was constructed by using the 36 P-Tyr proteins that were identified with altered tyrosine phosphory-
Figure 6. MS/MS spectra of two identified P-Tyr peptides: (A) CNTTVCLGKIGSpYLpSApSTR belongs to teratoma-associated tyrosine kinase(P04, m/z 738.39, 3+) and (B) MILSSpYNTIQSVFCCCCCCSVQK belongs to tyrosine-protein kinase TXK (P16, m/z 951.89, +3). Journal of Proteome Research • Vol. 9, No. 8, 2010 4109
research articles lation states between the two cell types. The network was generated by Metacore (GeneGo, Inc., St. Joseph, MI). As illustrated in Figure 4, the generated network, laid out according to proteins’ cellular localizations, contained 33 proteins. A total of 11 proteins were identified by our strategy and marked with circles in the network. These 11 proteins (as listed in Table 2) were of greatest interest for the network contained EGFR, c-Src, c-Myc, and STAT, which function as key regulators during lung cancer metastasis.45–47,49–51 In addition, receptor tyrosineprotein kinase ErbB-4 (ErbB4), focal adhesion kinase 1 (FAK), β-catenin, and receptor tyrosine-protein kinase erbB-2 (ErbB2) also have been reported to be involved in lung cancer metastasis,22,24,32 suggesting that the other seven proteins may also play roles in the process. Although further validation of the expression level was hampered by the lack of site-specific antibodies, to confirm the alteration, P-Tyr proteins were first immunoprecipitated with anti-phosphotyrosine antibody followed by Western blotting with antibodies specific to five proteins. Since a proper loading control for this experiment was hardly found, some sample preparation steps before Western blotting were monitored, which may help control the sample loading. (Supporting Information, Figure 1A-C) Small aliquots of the cell extracts resolved by SDS-PAGE showed similar patterns and amount of proteins, indicating that equal amounts of proteins were usedforimmunoprecipitation(SupportingInformation,Figure1A). SDS-PAGE of immunoprecipitated proteins and those followed by blotting using antiphosphotyrosine antibody revealed similar pattern and intensity, suggesting the usage of equal amounts of immuneprecipitated proteins for Western blotting (Supporting Information, Figure 1B,C) As shown in Figure 5, the altered tyrosine phosphorylation between CL1-0 and CL1-5 revealed by immunoprecipitation and Western blotting concurred with that from label-free quantitative analysis. However, the expression level measured from Western blotting may be inconsistent with that from mass spectrometry analysis. Taking P18 (FYVE, RhoGEF, and PH domain-containing protein 2) as an example, the altered ratio measured by mass spectrometry is significantly higher than that by Western blotting. This may result from the antibodies used for Western blotting only recognizing protein epitopes rather than specific P-Tyr sites. The binding between antibodies may be affected by the adjacent residues or other modifications. Besides, if the protein candidate contains multiple P-Tyr sites, the Western blotting results may be inconsistent with the mass spectrometry findings. For the confirmation of the P-Tyr peptide identification, high mass accuracy measurement of the fragment ions was used. Targeted MS/MS analyses of the seven P-Tyr peptides were performed on LTQ/Orbitrap. The accurate mass measurement of the fragment ions generated from CID was achieved by Orbitrap. These results were manually inspected and annotated. Two P-Tyr peptides are provided in Figure 6 as examples. The other five spectra are provided in Supporting Information, Figure 2A-E. Figure 6A presents the P-Tyr peptide CNTTVCLGKIGSpYLpSApSTR (P04), which belongs to the teratomaassociated tyrosine kinase with charge state 3 at m/z 738.39. The fragment ions b132+, b152+, b173+, [y3-HPO3]+, y5+, and y7+ suggested phosphorylation sites on Y458, S460, and S462. Another phosphopeptide (P16) was identified as MILSSpYNTIQSVFCCCCCCSVQK (m/z 951.89, +3) which belongs to tyrosine-protein kinase TXK. The b5+, b6+, y16+, and [y18-HPO3]2+ ions suggested the presence of a phosphate group on the Y6 residue. According to the up-to-date information in the Meta4110
Journal of Proteome Research • Vol. 9, No. 8, 2010
Wu et al. Core database, several proteins remained unlinked to other networks. However, the unlinked proteins may be important candidates for further investigation of metastasis.
Conclusion Signal intensity of P-Tyr peptides is considered relatively small during mass spectrometry analysis, which hampers their identification. Using the phosphopeptide identification strategy developed previously in our laboratory, we identified more than 300 P-Tyr peptides or sites in lung cancer cells. This tyrosine phosphoproteome may provide basic resources for studying roles of protein tyrosine phosphorylation in lung cancer. A total of 36 P-Tyr proteins were considered to be associated with metastasis based on label-free quantitative analysis. The substrate motifs for the EGFR kinase, Src kinase, JAK2 kinase, and TCPTP phosphatase were extracted, while two unknown consensus sequences may provide novel insights into the kinasemotif field. Moreover, 11 of the 36 altered proteins were linked to a protein-protein interaction network that contains EGFR, c-Src, c-Myc, and STAT, which are known to be related to lung cancer metastasis. Seven P-Tyr proteins, including teratomaassociated tyrosine kinase; growth factor receptor-bound protein 7, tyrosine-protein kinase TXK; FYVE, RhoGEF, and PH domain-containing protein 2; synaptotagmin-like protein 4; collapsin response mediator protein 4; and dipeptidase 2, have not been reported in lung cancer metastasis but existed in the network. Further studies of their involvement in the metastatic process may be of great worth.
Acknowledgment. This study was supported by Grant DOH99-TD-G-111-006 from the Department of Health, Executive Yuan, Grant NSC97-2113-M-006-005-MY3 from the National Science Council, and the National Cheng Kung University Project for Promoting Academic Excellence & Developing World Class Research Centers from the Ministry of Education of Taiwan. Supporting Information Available: A complete list of all of the unique P-Tyr peptides identified from CL1-0 and CL1-5 cells, the control experiment for immunoprecipitation and Western blotting, and MS/MS spectra for the other five proteins (P01, P18, P23, P25, P33). This information is available free of charge via the Internet at http://pubs.acs.org. References (1) Chu, Y. W.; Yang, P. C.; Yang, S. C.; Shyu, Y. C.; Hendrix, M. J.; Wu, R.; Wu, C. W. Selection of invasive and metastatic subpopulations from a human lung adenocarcinoma cell line. Am. J. Respir. Cell Mol. Biol. 1997, 17, 353–360. (2) Kinch, M. S.; Clark, G. J.; Der, C. J.; Burridge, K. Tyrosine phosphorylation regulates the adhesions of ras-transformed breast epithelia. J. Cell Biol. 1995, 130, 461–471. (3) Akasaka, T.; van Leeuwen, R. L.; Yoshinaga, I. G.; Mihm, M. C., Jr.; Byers, H. R. Focal adhesion kinase (p125FAK) expression correlates with motility of human melanoma cell lines. J. Invest. Dermatol. 1995, 105, 104–108. (4) Weiner, T. M.; Liu, E. T.; Craven, R. J.; Cance, W. G. Expression of focal adhesion kinase gene and invasive cancer. Lancet 1993, 342, 1024–1025. (5) Owens, L. V.; Xu, L.; Craven, R. J.; Dent, G. A.; Weiner, T. M.; Kornberg, L.; Liu, E. T.; Cance, W. G. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res. 1995, 55, 2752–2755. (6) Grisaru-Granovsky, S.; Salah, Z.; Maoz, M.; Pruss, D.; Beller, U.; Bar-Shavit, R. Differential expression of protease activated receptor 1 (Par1) and pY397FAK in benign and malignant human ovarian tissue samples. Int. J. Cancer 2005, 113, 372–378.
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PR1006153
