Immunoaffinity Enrichments Followed by Mass Spectrometric Detection for Studying Global Protein Tyrosine Phosphorylation Sara Bergström Lind,*,† Magnus Molin,† Mikhail M. Savitski,‡ Lina Emilsson,† Jonas Åström,§ Ludwig Hedberg,† Chris Adams,‡ Michael L. Nielsen,‡ Åke Engström,| Lioudmila Elfineh,† Eva Andersson,| Roman A. Zubarev,‡ and Ulf Pettersson† Department of Genetics and Pathology, Rudbeck Laboratory, Uppsala University, SE-751 85 Uppsala, Sweden, Department of Cell and Molecular Biology, Molecular Biometry Group, Uppsala Biomedical Centre, Uppsala University, P.O. Box 596, Uppsala, SE-751 24, Sweden, Uppsala BIO, Uppsala Science Park, Dag Hammarskjo¨lds va¨g 60, SE-751 83 Uppsala, Sweden, and Department of Medical Biochemistry and Microbiology, Uppsala Biomedical Centre, Uppsala University, Box 582, Uppsala, SE-751 23, Sweden Received January 22, 2008
Phosphorylation of protein tyrosine residues regulates important cell functions and is, when dysregulated, often crucially involved in oncogenesis. It is therefore important to develop and evaluate methods for identifying and studying tyrosine phosphorylated (P-Tyr) proteins. P-Tyr proteins are present at very low concentrations within cells, requiring highly selective enrichment methods to be detected. In this study, we applied immunoaffinity as enrichment step for P-Tyr proteins. Five selected antiphosphotyrosine antibodies (monoclonal antibodies 4G10, PY100, PYKD1, 13F9 and one polyclonal antiserum) were evaluated with respect to their capability to enrich P-Tyr proteins from cell extracts of the K562 leukemia cell line. The enrichment resulted in the detection of a group of proteins that potentially were tyrosine-phosphorylated (putative P-Tyr proteins). High accuracy identification of actual P-Tyr sites were performed using a highly selective and sensitive liquid chromatography Fourier transform mass spectrometer (LC-FTMS) setup with complementary collision activated dissociation (CAD) and electron capture dissociation (ECD) fragmentations. 4G10 and PY100 antibodies recognized the greatest number of putative P-Tyr proteins in initial screening experiments and were therefore further evaluated and compared in immunoaffinity enrichment of both P-Tyr proteins and peptides. Using the 4G10 antibody for enrichment of proteins, we identified 459 putative P-Tyr proteins by MS. Out of these proteins, 12 were directly verified as P-Tyr proteins by MS analysis of the actual site. Using the PY100 antibody for enrichment of peptides, we detected 67 P-Tyr peptides (sites) and 89 putative P-Tyr proteins. Generally, enrichment at the peptide level made it difficult to reliably determine the identity of the proteins. In contrast, protein identification following immunoaffinity enrichment at the protein level gave greater sequence coverage and thus a higher confidence in the protein identification. By combining all available information, 40 proteins were identified as true P-Tyr proteins from the K562 cell line. In conclusion, this study showed that a combination of immunoaffinity enrichment using multiple antibodies of both intact and digested proteins in parallel experiments is required for best possible coverage of all possible P-Tyr proteins in a sample. Keywords: Tyrosine phosphorylation • anti-phosphotyrosine antibodies • immunoaffinity enrichment • mass spectrometric characterization
Introduction Phosphorylation as a post-translational modification (PTM) is of particular interest because of its ubiquitous nature, playing a pivotal role in, for example, development, cell division and oncogenesis. It is therefore important to develop robust * Corresponding author. Fax: + 46 18 471 4808. E-mail: Sara.Lind@ genpat.uu.se (S. Bergstro¨m Lind). † Department of Genetics and Pathology, Uppsala University. ‡ Department of Cell and Molecular Biology,Uppsala University. § Uppsala BIO. | Department of Medical Biochemistry and Microbiology, Uppsala University. 10.1021/pr8000546 CCC: $40.75
2008 American Chemical Society
methods that selectively detect phosphorylated proteins (or peptides) to study and understand cellular mechanisms. The work presented here focuses on methods specific for tyrosine phosphorylation. Reversible tyrosine phosphorylation determines the fate of human cells by controlling cell survival at the level of, for example, maintenance, differentiation, and apoptosis.1 When normal regulation of tyrosine phosphorylation is lost, the result can be dramatic and even lead to tumor development.2 Dysregulation of protein tyrosine phosphorylation has been suggested to be the underlying cause of more than 80% of Journal of Proteome Research 2008, 7, 2897–2910 2897 Published on Web 06/11/2008
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human cancers. Previous studies have shown that cancer cell lines, depending on origin and malignancy state, have distinct tyrosine phosphorylation profiles.4–6 Among these are cell lines representing Philadelphia chromosome positive leukemia, which are characterized by a t(9:22) translocation. This results in a Bcr/Abl oncogene, with a dysregulated tyrosine kinase activity necessary for development of leukemia, found in 95% of all chronic myeloid leukemia (CML) cases and to a minor extent also in acute lymphoid leukemia (ALL).7 Of total human protein phosphorylation, tyrosine phosphorylation represents 0.05%, while threonine and serine phosphorylation make up 10 and 90%, respectively.8,9 Tyrosinephosphorylated (P-Tyr) proteins are thus comparatively rare and are also present at very low intracellular levels. Their study therefore requires relevant model systems and selective enrichments, as well as sensitive detection and characterization methods. Since protein tyrosine phosphorylation is difficult to measure, treatment with pervanadate, an inhibitor of tyrosine phosphatases, to increase tyrosine phosphorylation is commonly performed. Cells of different origin, however, exhibit different overall levels of tyrosine phosphorylation. In our experience, CML cell lines exhibit tyrosine phosphorylation levels that are higher than other commonly used cell lines (up to 10 times, data not shown), which is advantageous for a study like the one presented here. K562, a CML cell line, was therefore chosen. Antibodies are extremely useful for studying P-Tyr proteins, and have more or less replaced 32P-isotopic labeling, traditionally used to investigate cellular protein phosphorylation. There are numerous publications using anti-phosphotyrosine antibodies in different immunological settings. Anti-phosphotyrosine antibodies are traditionally used in Western blotting experiments for detection and quantification of tyrosine phosphorylation of selected proteins after immunoprecipitation of the interesting protein. For global studies of tyrosine phosphorylation, anti-phosphotyrosine antibodies could be used for another purpose, to enrich for putative P-Tyr proteins before detailed mass spectrometric (MS) characterization. Such P-Tyr immunoaffinity enrichments (precipitation or chromatography) can be performed at the protein level.10–19 In these studies, the monoclonal anti-phosphotyrosine antibody 4G1015 or the PT66 antibody,16,17 combinations of 4G10 and RC2011–13,19 or of 4G10 and PY10014 have been used. Immunoaffinity enrichment at the peptide level has also been reported.5,6,20,21 The binder of choice for these studies has been the PY100 antibody. Enrichment of phosphorylated proteins and peptides can also be performed using chemical methods, such as immobilized metal affinity chromatography (IMAC),22,23 titanium oxide24,25 or phosphoramidate chemistry enrichments.26 These methods have been compared by Bodenmiller et al.26 and were found to be nonselective for tyrosine phosphorylation. However, enriched proteins should not be considered as phosphorylated until at least one P-Tyr site has been directly detected. Currently, the general way of characterizing proteins and peptidesisbyMSanalysisincombinationwithbioinformatics.27–29 Detection of actual P-Tyr sites requires tandem MS analysis (MS/MS). For detection of tyrosine phosphorylation, the most common setup currently used is the LCQ ion trap MS and MS/MS.5,6,16,18 Enriched putative P-Tyr proteins are digested into peptides before MS analysis, while enriched P-Tyr peptides can be analyzed directly. So far, no extensive comparisons of the use of different antiphosphotyrosine antibodies and different strategies for immu2898
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Bergström Lind et al. noaffinity enrichment of P-Tyr proteins have been reported in the literature. In the current investigation, different steps of the early sample preparation procedure for investigating tyrosine phosphorylation on proteins were compared with respect to (i) performance of different anti-phosphotyrosine antibodies and (ii) immunoaffinity enrichment at the protein versus the peptide level. The identity of P-Tyr proteins and sites were determined by nano-liquid chromatography (LC) coupled with a Fourier transform (FT) MS instrument, in combination with two complementary MS-MS fragmentations, collision activated dissociation (CAD) and electron capture dissociation (ECD). This powerful combination made it possible to enrich putative P-Tyr proteins and subsequently confirm the phosphorylation by detection of the actual P-Tyr sites in the investigated peptides.
Experimental Procedures Chemicals. All chemicals were from Sigma-Aldrich Sweden AB, Steinheim, Germany, if not otherwise stated. Modified RIPA (Radio Immuno Precipitation Assay) buffer with EGTA consisted of 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA supplemented with 2 µg/mL aprotinin, 20 µg/mL bestatin, 1 µg/mL leupeptin, 1 µg/mL pepstatin A, 0.5 mg/mL Pefabloc SC (AEBSF), 0.1 mM PMSF, 1 mM Na3VO4, 1 mM NaF, 2 mM β-glycerophosphate, and 1 mM sodium pyrophosphate. Anti-phosphotyrosine antibodies: The 4G10 antibody was kindly provided by Prof. Brian Druker (Oregon Health and Science University, Portland, OR), the PY100 antibody was from Cell Signaling Technologies (Boston, MA), the 13F9 ascites was from Rockland Immunochemicals, Inc. (Gilbertsville, PA), the PYKD1 antibody was kindly provided by Prof. Paul Tempst and co-workers (Memorial Sloan-Kettering Cancer Center, New York, NY) and the Polyclonal antiserum was from Promega (Madison, WI). Details of the antibodies are given in Table 1. Cell Culture. K562 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/ streptomycin. Cells were grown to a density of about 1 × 106 cells/mL before harvesting. Cells were pelleted by centrifugation and then washed three times with PBS and two times with Ca, Mg and NaHCO3 free PBS (Gibco, Paisley, U.K.). Both PBS solutions were supplemented with 1 mM Na3VO4. Cell pellets were snap-frozen on dry ice and then stored at -70 °C until use. Cell Lysis. Frozen cell pellets containing 3-3.5 × 108 K562 cells were resuspended in 1.0 mL of modified RIPA with EGTA through repeated pipetting, followed by low speed vortex. The lysed material was then centrifuged for 15 min at 13 000g at 4 °C. The supernatants were transferred into new tubes and frozen on dry ice. An additional volume of 500 µL of modified RIPA per aliquot pellet was added. After 5 min of vortex, followed by centrifugation for 5 min at 13 000g at 4 °C, the supernatants were pooled with the previously collected supernatants. The cell extracts were stored at -70 °C until use. The protein concentration in the lysates was about 20-25 mg/mL. For antibody comparison experiments, pooled and aliquoted (each aliquot corresponded to 1 × 108 cells) or matched amounts from one and the same cell harvest were used. 2-D SDS-PAGE Western Blotting. To a thawed aliquot of the pooled extract of K562 cells (1 × 108 cells), 4 mL of icecold acetone was added. The tube was inverted 10 times and proteins were precipitated overnight at -25 °C. Proteins were then pelleted by 30 min centrifugation at 4000 rpm, 4 °C. The
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Studying Global Protein Tyrosine Phosphorylation Table 1. Properties of the Different Anti-Phosphotyrosine Antibodies Included in This Study antibody
4G10 PY100
antigena
Phosphotyramine (KLH-conjugated) Phosphotyrosine containing peptides (KLH-conjucated) 13F9 Phosphotyrosine (KLH-conjugated) PYKD1 Phosphotyrosine (KLH-conjugated) Polyclonal Phosphotyrosine (ovalbumin- conjugated) a
produced in
class
type
provided in
Mouse Mouse
IgG2bκ Monoclonal IgG1 Monoclonal
Mouse Mouse Rabbit
IgG1κ IgG1 IgG
0.2 M NaHCO3, 0.5 M NaCl, pH 8.3, 7.2 mg/mL BSA containing buffer, ∼1 mg/mL
Monoclonal Ascites Monoclonal PBS, 8 mg/mL Polyclonal PBS, 1 mg/mL (antiserum)
KLH ) Keyhole Limpet Hemocyanin, used as a carrier for antibody production.
protein precipitation was then washed with a second round of acetone after which the precipitate was air-dried. Prior to 2-D SDS-PAGE, protein pellets were dissolved in 1.5 mL of 8 M urea, 2% Chaps and 75 or 300 µg of protein was loaded onto six isoelectrical focusing strips, pH 3-11. Isoelectrically focused proteins where then further separated on second dimension, SDS-PAGE, and subsequently electroblotted onto PVDF membranes. Membranes were blocked using “StartingBlock T20 (TBS) Blocking Buffer” (Pierce, Rockford, IL), and incubated with the following different antibodies: polyclonal (0.5 µg/mL), 13F9 (ascites diluted 1:1000), 4G10 (1 µg/mL), PYKD1 (1 µg/ mL) and PY100 (∼0.5 µg/mL, antibody diluted 1:2000), respectively, at 4 °C overnight. Membranes were washed four times with TBS-T (TBS with 0.05% Tween 20), followed by incubation for 1 h with secondary antibody “sheep anti-mouse IgG-HRP” (GE Healthcare, Uppsala, Sweden) diluted 100 000 times for all antibodies except for the polyclonal for which “goat antirabbit IgG-HRP (sc-2004)” (Santa Cruz Biotechnology, CA) was used, at 10 000 times dilution. Membranes were again washed four times with TBS-T and then developed using Enhanced Chemiluminiscence (ECL Advance Western Blotting Detection Kit, GE Healthcare) solution and a ChemiDoc XRS system (BioRad Laboratories, Inc., Hercules, CA). To visualize all proteins and all phosphorylated proteins, 75 µg of protein was analyzed, as described above, with 2-D SDS-PAGE, and the gels were stained with Deep purple staining (GE Healthcare) and Pro-Q Diamond (phosphate specific stain, Molecular Probes, Invitrogen, Carlsbad, CA), respectively. Coupling of Antibodies with Enrichment Media. 1. Columns. Antibodies were coupled with N-hydroxysuccinimide (NHS)-activated HiTrap 1 mL-columns (GE Healthcare) according to the manufacturer’s protocol. Briefly, antibody diluted in 0.2 M NaHCO3 and 0.5 M NaCl, pH 8.3, was loaded onto the column and covalently bound by amide linkage. Thereafter, excess active groups were blocked by 0.5 M ethanolamine and 0.5 M NaCl, pH 8.3. Nonspecifically bound ligands were washed out with 0.1 M acetate and 0.5 M NaCl, pH 4.0. Efficient coupling was verified by SDS-PAGE and colloidal Coomassie blue stain of input and wash fractions. For the antibody comparison, 0.3 mg of each antibody was coupled with separate columns at a concentration of 0.5 mg/mL. Before coupling with the column, the 13F9 and PY100 antibodies were purified on HiTrap Protein G columns (GE Healthcare) according to the manufacturer’s instructions. For further investigation of the 4G10 antibody, 1 mg of 4G10 was coupled per column at a concentration of 1 mg/mL. 2. Beads. Protein G sepharose (GE Healthcare) beads were washed twice with binding buffer (20 mM phosphate buffer, pH 7.0). The 4G10 antibody was diluted in binding buffer and coupled noncovalently with the beads at a concentration of 4 mg/mL beads and incubated overnight at 4 °C with rotation.
After coupling, antibody resin was washed twice with PBS (5-10 bead volumes of buffer for each wash). Beads were stored in PBS with 0.1% NaN3. Before use, the beads were washed three times with immunoaffinity purification buffer (PhosphoScan Kit (P-Tyr-100) (Cell Signaling Technologies, Boston, MA)). Efficient coupling was verified by analyzing a small amount of the input antibody solution together with the supernatant (to be discarded after coupling) by SDS-PAGE stained with colloidal Coomassie blue. Affinity Enrichment of Phosphotyrosine Proteins. Cell extracts were thawed on ice and centrifuged at 13 000g for 1 min. Supernatants were filtered through 0.45 µm GHP acrodisc filters (Pall Life Sciences, Ann Arbor, MI). Filtrates were then loaded onto antibody coupled HiTrap columns (1 mL), equilibrated with 4 mL of modified RIPA with EGTA, supplemented with inhibitors. A volume of 0.7-1.0 mL of cell extract (input, denoted I) was loaded onto HiTrap columns, which were then placed on ice for 10 min, after which the remainder of the cell extract was loaded (a maximum of 1.0 mL). Columns were then placed on ice for an additional 35 min followed by a wash step with 10 mL of ice-cold modified RIPA supplemented with inhibitors (wash fractions, denoted W). Phosphotyrosine containing proteins were then eluted in 4 mL of room-tempered 100 mM phenyl phosphate in PBS (elute fractions, denoted E). Columns were subsequently regenerated by washing with 6 mL of modified RIPA with EGTA supplemented with 1.5 M NaCl (regeneration fractions, denoted R), and stored in PBS with 0.1% NaN3 at 4 °C. All affinity enrichments were verified by SDS-PAGE and Western blotting. Elute fractions were pooled and proteins were precipitated in acetone overnight, at -25 °C. After a wash with additional acetone, the protein pellets were left to dry on ice for 4 h. Alternatively, proteins fractions were concentrated using ultracentrifugation filters (Amicon Ultra, 15 mL, followed by Ultrafree, 0.5 mL, Millipore, Bedford, MA), and washed once with 15 mL of MilliQ water and once with modified RIPA according to the manufacturer’s instructions. The final volume was 40 µL. Protein pellets or concentrated proteins were dissolved in 1× Laemmli sample buffer (containing 0.3 M β-mercapto ethanol) at a final volume of ∼80-200 µL, and boiled for 5 min, after which all of it was loaded onto a 10% SDS-PAGE, and run at 150 V for ∼2 h. The gel was fixed (10% methanol, 7% acetic acid) for 45 min and stained with colloidal Coomassie. The lane was then cut into ∼15 pieces and subjected to in-gel tryptic digestion, essentially as described by Shevchenko and co-workers.30 Briefly, the gel pieces were destained, washed, exposed to dithiothreitol reduction and iodoacetamide alkylation, followed by porcine trypsin (modified sequencing grade; Promega, Madison, WI) digestion overnight at 37 °C. Tryptic peptides were extracted from the gel pieces with 2/3 acetonitrile, 5% formic acid and 100% acetonitrile. The Journal of Proteome Research • Vol. 7, No. 7, 2008 2899
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Figure 1. A routine MS/MS spectrum of a phosphotyrosine peptide (NSLETLLyKPVDR) in the breakpoint cluster region (Bcr) or Bcr-Abl protein. The site of phosphorylation is on the tyrosine (Y) residue 644. The peptide sequence is shown in the top right of the panels. Ions derived from the N-terminal of the peptide are denoted b and c, and ions derived from the C-terminal are denoted y and z. Arrows with pY indicate the mass difference of a phosphorylated tyrosine residue.
extracts were evaporated in a vacuum centrifuge and dissolved in 15 µL of 0.1% TFA just before LC-MS analysis. Analyses were performed by pooling peptides from different gel pieces, or by analyzing gel piece by gel piece. Affinity Enrichment of Phosphotyrosine Peptides. Affinity enrichment was essentially performed using the PhosphoScan Kit (P-Tyr-100) (Cell Signaling Technologies).31 All reagents for this analysis were thus provided in the kit (Cell Signaling Technologies). Briefly, cell pellets of 2-3 × 108 cells were lysed in 9 M urea (in 20 mM HEPES buffer supplemented with inhibitors). After carboxyamidomethylation of proteins, the sample was diluted 4-fold to a final concentration of 2 M urea and 20 mM HEPES buffer, pH 8.0. Trypsin (1/100 volume of 1 mg/mL trypsin-TPCK solution) was added and digestion was performed overnight. The digested cell lysates was then acidified with trifluoroacetic acid (TFA) and cleaned by centrifugation. The digestion was verified on a 10% polyacrylamide gel. Peptides were purified on a Sep-Pak C18 column and lyophilized. Peptides were then dissolved in immunoaffinity purification buffer (50 mM MOPS, pH 7.2, 10 mM sodium phosphate, and 50 mM NaCl) and incubated with antibodies coupled with Protein G agarose beads overnight. PY100 antibody was provided coupled with beads via the PhosphoScan Kit, while 4G10 antibody was coupled in-house, as described above. Unbound peptides were removed by washing, and phosphotyrosinecontaining peptides were eluted with 0.15% TFA. Peptides were purified and concentrated on self-made reverse phase C18 Stage Tips, as described by Rappsilber and co-workers,32 using Empore Disks C18 from Varian (Palo Alto, CA). Phosphatase Treatment of P-Tyr Proteins. P-Tyr proteins enriched via the 4G10 antibody were treated with Calf Intestine Alkaline Phosphatase (CIAP) (Fermentas Gmbh, Helsingborg, Sweden). A total of 50 U of enzyme was used per 3.3 µg of proteins. As a control, P-Tyr proteins were treated with water. 2900
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Both sample and control were incubated for 1 h at 37 °C. After treatment, proteins were separated by 1-D SDS-PAGE and subsequently electroblotted onto PVDF membranes. Membranes were labeled with 4G10 antibody, as described above (see 2-D SDS-PAGE Western Blotting). LC-MS Analysis. All experiments were performed on a 7-T hybrid linear ion trap Fourier transform mass spectrometer (LTQ FT, Thermo Electron, Bremen, Germany) modified with a nanoelectrospray ion source (Proxeon Biosystems, Odense, Denmark). The high-performance liquid chromatography setup used in conjunction with the mass spectrometer consisted of a solvent degasser, nanoflow pump, and thermostat controlled microautosampler (Agilent 1100 nanoflow system). A 15-cm fused silica emitter (75-µm inner diameter, 375-µm outer diameter; Proxeon Biosystems) was used as analytical column. The emitter was packed in-house with methanol slurry of reverse-phase, fully end-capped Reprosil-Pur C18-AQ 3-µm resin (Dr. Maisch GmbH, Ammerbuch.Entringen, Germany) using a pressurized “packing bomb” operated at 50-60 bar (Proxeon Biosystems). Mobile phases consisted of 0.5% acetic acid and 99.5% water (v/v) (buffer A) and 0.5% acetic acid and 10% water in 89.5% acetonitrile (v/v) (buffer B). Eight microliters of prepared peptide mixture was automatically loaded onto the column and rinsed for 20 min in 4% buffer B at a flow rate of 500 nL/min followed by a 90-min gradient from 4 to 45% buffer B at a constant flow rate of 200 nL/min. For the data in Figure 4, the analysis was made using 12 h gradients and the material from the whole gel was analyzed in one run. This was done in order to minimize the variance in the data. MS analysis was performed using unattended data-dependent acquisition mode in which the mass spectrometer automatically switches between a high resolution survey scan (resolution ) 100 000; m/z range; 200-1600) followed by lower resolution fragmentation spectra (electron capture dissociation followed
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Figure 2. Total protein extract of K562 cells separated using 2-D SDS-PAGE stained/labeled with (A) Deep Purple for total protein stain, (B) Pro-Q Diamond for phosphoprotein stain, (C) 4G10 antibody, (D) PY100 antibody, (E) 13F9 antibody, (F) PYKD1 antibody, and (G) polyclonal antiserum. The amount of total protein loaded onto gels was 75 µg in panels A, B, and G and 300 µg in panels C-F. The exposure time was 3 min for panels C-F and 2 min for panel G.
Figure 3. Comparison of phosphotyrosine protein enrichments with different antibodies coupled with sepharose columns. (A) Schematics of the enrichment procedure. Selected fractions from the enrichment were analyzed by 1-D SDS-PAGE. (B-F) The result of Western blots labeled with the same antibody as was used for the enrichment. Antibodies in the panels are 4G10 (B), PY100 (C), 13F9 (D), PYKD1 (E) and the polyclonal antiserum (F). Annotations above the separation lanes indicate: I, loading material, W, wash steps, E, elution fractions, R, regeneration fractions.
by collision-activated dissociation; resolution ) 25 000) of the most abundant peptides eluting at a given time.
Peptide and Phosphosite Identification. Acquired RAW files were converted to dta files using Extract_msn through Bioworks Journal of Proteome Research • Vol. 7, No. 7, 2008 2901
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Figure 4. Graphs showing the comparison of the 4G10 and the PY100 antibodies using total Mascot score. (A) Scores of 4G10 enriched proteins plotted against scores of nonenriched proteins of the K562 cell line (y ) 0.619x + 172, R2 ) 0.298). (B) Scores of PY100 enriched proteins plotted against scores of nonenriched proteins from the K562 cell line (y ) 0.460x + 226, R2 ) 0.139). (C) Scores of 4G10 enriched proteins plotted against scores of PY100 enriched proteins (y ) 0.612x + 68.4, R2 ) 0.430). Numbers on the axes indicate total Mascot scores of the proteins.
Browser (Thermo Electron), and complementary pairs were identified as described previously.33,34 Base peptides were identified by searching against the International Protein Index (IPI) human database using Mascot search engine (version 2.2, Matrix Science, London, U.K.). Searches were performed with trypsin specificity, and mass tolerance for monoisotopic peptide identification was set to 5 ppm and (0.02 Da for fragment ions. The instrument setting was “ESI-FTICR”, which permits b, y, b-NH3 and y-H2O fragment ion types. Only base peptides having a Mascot score (M-score) above the significant threshold corresponding to (p < 0.05) were used for protein identification. Parsing of data and statistical analysis of the search results reported by Mascot were performed using the open-source software MSQuant. Proteins were identified by at least two peptides with a significant score. The score of the peptides were combined to a total Mascot score for a protein. All identified phosphotyrosine sites reported by Mascot were manually verified in the MS/MS spectra (either CAD or ECD or both). To be accepted as a phosphotyrosine site, b, y, c or z- ions detected on both sides of the phosphorylated sites were identified with a mass tolerance of (0.02 Da. Safety Considerations. Chemicals used in the sample preparation procedure might be toxic and/or carcinogenic and should be handled with great care. Also, biological material should also be handled with care and according to the local laboratory instructions.
Results and Discussion Successful investigation of tyrosine phosphorylation, on a global scale, requires both a selective separation procedure to enrich P-Tyr proteins or peptides, and a reliable subsequent characterization of the enriched compounds. We here make a comprehensive study of both the enrichment step, based on anti-phosphotyrosine antibodies, and the characterization step by high performance MS. When considering the enrichment of P-Tyr proteins and peptides, it is important to remember that each antibody has its own unique properties and vary in specificity. The selected antibodies for this study (Table 1) have either previously been used for immunoaffinity enrichments of P-Tyr proteins followed by MS detection (4G10, PY100 and PYKD1) 5,6,11–15,20,21,35 or are new to this application (13F9 and polyclonal antiserum were both primarily developed for Western blot experiments). The 13F9 monoclonal antibody was provided as an anti2902
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phosphotyrosine ascites solution. The PYKD1 monoclonal antibody was originally developed for use in a prototype antibody microarray platform to monitor changes of tyrosine phosphorylation.35 Among other anti-phosphotyrosine antibodies not included in this study are PY99 (Santa Cruz Biotechnology), PY20 (BD Transduction Laboratories), PY102 (Cell Signaling Technologies) and SP4 (Abcam) antibodies. Table 1 summarizes the properties of the antibodies used in this study. The antibodies investigated were produced using slightly different antigens, which imply potential differences in specificity and performance. Also, the clone selection procedure, which is important for the selectivity of an antibody, might differ (this parameter was not reported for all antibodies). In this work, antibodies were used for enrichment of putative P-Tyr proteins from K562 cells. This leukemia cell line was chosen as a model system due to its biological relevance for studying tumor development as well as its relatively high overall levels of tyrosine phosphorylation. Initial investigation of the different antibodies was performed by Western blotting experiments, followed by experiments where the antibodies were used for immunoaffinity enrichments. Determination of which proteins that actually were tyrosine-phosphorylated was subsequently performed using MS/MS analysis. We here for the first time report on the routine analysis and identification of individual P-Tyr peptides, and their corresponding proteins, using a novel combination of MS methods. Both CAD and ECD fragmentation, which are orthogonal fragmentation techniques, were applied during acquisition of all MS/MS spectra recorded. This increased the number of peptides that could be de novo sequenced.33,34,36 In Figure 1, an MS/MS spectrum of a phosphorylated peptide fragmented using both CAD and ECD is shown. The peptide originated from the Breakpoint cluster region (Bcr) protein or Bcr-Abl protein (K562 cells expresses to our knowledge both Bcr and Bcr-Abl proteins, and it was thus not possible to determine the exact origin of the peptide). The peptide was dissociated into 6 and 9 detectable b-and y-ions, respectively, in the CAD spectrum and into 3 and 7 detectable c-and z-ions, respectively, in the ECD spectrum. The phosphorylation on the tyrosine residue was detected between the b7- and b8-ions and y5- and y6-ions. For many peptides, the tyrosine phosphorylation could be detected in both the CAD and the ECD spectra. The FTMS instrument combines high mass accuracy, at the ppm-level, with high resolution. The data generated allowed the search
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Studying Global Protein Tyrosine Phosphorylation Table 2. Comparison of 4G10 and PY100 Antibodies Used for Enrichment of P-Tyr Peptides protein
ABL1 ABL1 ABL1 ACTN1 BCR BCR BCR BCR CBL CDC2 CDC2 DYRK1B
EEF1A1 GAB2
GRLF1 GSK3B H41 HCK HSPCB HSPCB INPPL1 LKP MAPK1 MAPK14 PRPF4B SRC TJP2 VASP VIM VIM
IPI accession
IPI protein annotation
sitea
reported 4G10 PY100 for K562c
phosphotyrosine peptideb
IPI00216970 Isoform IA of Proto-oncogene 226 R.NKPTVyGVSPNYDKWEMER.T tyrosine-protein kinase ABL1 IPI00216969 Isoform IA of Proto-oncogene 393 R.LMTGDTyTAHAGAK.F tyrosine-protein kinase ABL1 IPI00216969 Isoform IA of Proto-oncogene 469 R.MERPEGCPEKVyELMR.A tyrosine-protein kinase ABL1 IPI00013508 Alpha-actinin-1 246 K.AIMTYVSSFyHAFSGAQK.A IPI00004497 Breakpoint cluster region 58 R.MIyLQTLLAK.E protein IPI00004499 Breakpoint cluster region 177 K.GHGQPGADAEKPFyVNVEFHHER.G protein IPI00004497 Breakpoint cluster region 644 K.NSLETLLyKPVDR.V protein IPI00004500 Breakpoint cluster region 910 K.LQTVHSIPLTINKEDDESPGLyGFLNVIVHSATGFK.Q protein IPI00027269 E3 ubiquitin-protein ligase CBL 674 K.IKPSSSANAIySLAARPLPVPK.L IPI00026689 Hypothetical protein 15 K.IGEGtyGVVYK.G DKFZp686L20222 IPI00026691 Hypothetical protein 19 K.IEKIGEGtYGVVyK.G DKFZp686L20222 IPI00000352 Isoform 1 of Dual specificity 273 R.IYQyIQSR.F tyrosine-phosphorylationregulated kinase 1B IPI00396485 Elongation factor 1-alpha 1 29 K.STTTGHLIyK.C IPI00186990 Isoform 1 of 324 R.EFGDLLVDNMDVPATPLSAyQIPR.T GRB2-associated-binding protein 2 IPI00334715 Isoform 1 of Glucocorticoid 1105 R.NEEENIySVPHDSTQGK.I receptor DNA-binding factor 1 IPI00028570 Isoform 1 of Glycogen synthase 216 R.GEPNVSyICSR.Y kinase-3 beta IPI00014197 Hypothetical protein 190 R.KTPQGPPEIySDTQFPSLQSTAK.H IPI00029769 Isoform p59-HCK of 390 R.VIEDNEyTAR.E Tyrosine-protein kinase HCK IPI00334775 85 kDa protein 484 K.SIyYITGESK.E IPI00334775 85 kDa protein 596 R.LVSSPCCIVTSTyGWTANMER.I IPI00016932 Inositol polyphosphate 1135 K.TLSEVDyAPAGPAR.S phosphatase-like 1 IPI00643920 Transketolase 275 K.NMAEQIIQEIySQIQSK.K IPI00003479 Mitogen-activated protein 187 R.VADPDHDHTGFLTEyVATR.W kinase 1 IPI00002857 Mitogen-activated protein 182 R.HTDDEMTGyVATR.W kinase 14 isoform 2 IPI00013721 Serine/threonine-protein kinase 849 K.LCDFGSASHVADNDITPyLVSR.F PRP4 homologue IPI00328867 OTTHUMP00000030931 425 R.LIEDNEyTAR.Q IPI00003843 Isoform A1 of Tight junction 1118 R.IEIAQKHPDIyAVPIK.T protein ZO-2 IPI00301058 Vasodilator-stimulated 38 R.VQIyHNPTANSFR.V phosphoprotein IPI00418471 Vimentin 52 R.SLyASSPGGVYATR.S IPI00418472 Vimentin 60 R.SLYASSPGGVyATR.S
a Site indicates the number of the phosphorylated tyrosine residue. et al.6
b
The y indicates the phosphorylated tyrosine residue.
parameters of the Mascot software to be set only to identify peptides of a high probability, thus, lowering the number of false positive protein identifications. The protein identification was furthermore performed using at least two peptides, both having a probability better than (p < 0.05). Through the combined use of immunoaffinity enrichment and mass spectrometric analysis, we performed an evaluation of the alternatives for investigating the phosphotyrosine proteome. Antibody Comparison. 1. Western Blotting Experiments. As a first screen, the ability of different antibodies to recognize
c
x
Yes
x
No
x
x
x
No
x x
No No
x
Yes
x
Yes
x
No
x x
Yes Yes
x
Yes
x
Yes
x x
Yes
x
x
Yes
x
x
Yes
x
Yes No
x
x x x
No No Yes
x x
x
No Yes
x
x
Yes x
Yes
x
No Yes
x
Yes
x
No No
x
x x
Reported for K562 by Goss
putative P-Tyr proteins when used as primary antibodies during Western blotting experiments was investigated. Total cell lysates were separated using 2-D SDS-PAGE. One of the gels was stained with Deep purple for visualization of total proteins (Figure 2A), while another gel was stained with Pro-Q Diamond to visualize all the proteins having a phosphorylated amino acid residue (Figure 2B). When comparing panels A and B of Figure 2, it was observed that the total protein staining only detected a small number of phosphorylated proteins spots, most likely due to their low concentration in the cell lysate. To specifically Journal of Proteome Research • Vol. 7, No. 7, 2008 2903
research articles
Figure 5. Results from the enrichment of phosphotyrosine proteins using the 4G10 antibody. (A) Enriched phosphotyrosine proteins separated using 1-D SDS-PAGE. (B) Correlation of the gel piece number and the molecular mass of identified proteins.
investigate P-Tyr proteins, the separated proteins (300 µg) on five 2-D gels were transferred to membranes and each membrane was labeled with different anti-phosphotyrosine antibodies (Table 1, Figure 2C-G) according to the provider’s recommendations. The PY100 and the 13F9 antibodies were used in a BSA-containing solution or as an ascites fluid, respectively, while the other antibodies were protein G purified. When comparing the P-Tyr protein patterns (Figure 2C-G) to the total phosphorylation pattern (Figure 2B), no obvious correlation could be discovered. We infer this may be because tyrosine phosphorylation only constitutes about 0.05% of human protein phosphorylation.8 When comparing the results of the Western blotting using the different anti-phosphotyrosine antibodies, it was found that the 4G10 antibody detected a greater number of protein spots than any of the other antibodies (Figure 2C). The PY100, the 13F9, and the PYKD1 antibodies (Figure 2D-F) all exhibited a similar pattern but with fewer protein spots compared to the 4G10 antibody. When loading 300 µg of proteins, the polyclonal antiserum gave too high of a background to detect individual spots; thus, the amount had to be lowered to 75 µg (Figure 2G). There is always a danger in using antibodies for the detection of P-Tyr proteins, that is, to assume that all detected spots on a Western blot are phosphorylated proteins. Some factors are background from contaminating proteins (e.g., if the antibody is provided in ascites, as with the 13F9 antibody, Figure 2E), and nonspecific reactivity of the antibodies toward unrelated proteins. To investigate to what extent the 4G10 antibody bound to unphosphorylated proteins, Western blot analysis of a phosphatase treated cell extract was performed (data not shown). The result showed that this particular antibody exclusively detected phosphorylated proteins. When using antibodies as in this study mainly for enrichment, a small number of false positive events can be tolerated since the MS analysis subsequently will reveal the true P-Tyr proteins. We concluded that all investigated antibodies were found to detect putative P-Tyr proteins to such extent that they were interesting to be further evaluated in immunoaffinity enrichment experiments. 2. Immunoaffinity Enrichments of Proteins and Peptides. To further investigate the antibodies, 300 µg of each antibody (pure antibodies, see Experimental Procedures) was coupled with individual HiTrap columns. Extracts from pooled K562 cells extracts were applied to the columns and subjected to immunoaffinity enrichments. The enrichment fractions were 2904
Journal of Proteome Research • Vol. 7, No. 7, 2008
Bergström Lind et al. investigated for enriched P-Tyr proteins by SDS-PAGE and Western blotting analysis, using the same antibody as was used in each enrichment column. As seen in the elute fractions, visualized in Figure 3, 4G10, PY100 and to a lesser extent also 13F9 antibodies enriched phosphotyrosine proteins in solution (Figure 3B-D). The PYKD1 antibody and the polyclonal antiserum did not, under these conditions, enrich proteins from the extracts (Figure 3E-F), even though they recognized phosphotyrosine groups in denaturated proteins during Western blot experiments (Figure 2F,G). In Figure 3, P-Tyr proteins were observed to pass through the column during the load and wash steps. This most likely is due to overloading of the columns (a relatively low amount of each antibody was used), but could also be an effect of detergents present in the RIPA buffer. Out of the investigated antibodies, 4G10 and PY100 antibodies were selected for further experiments and the elution fractions from enrichments using these antibodies were pooled and separated using 1-D SDS-PAGE. The protein bands were cut out and peptides extracted after tryptic in-gel digestion. The resulting protein identity lists, generated by LC-MS/MS analysis of the extracted peptides, were compared to each other and to a nonenriched protein sample (100 µg of total proteins). Identified P-Tyr containing proteins should have a high Mascot score after enrichment but a lower score in the nonenriched sample. By plotting the total Mascot scores of the enriched P-Tyr proteins against the total Mascot scores of the proteins in the nonenriched sample, affinity graphs for the two antibodies were created (Figure 4A,B). A correlation close to 0 indicates that an antibody has a selective affinity, and does not randomly bind proteins. Since the correlation of the graphs was 0.298 and 0.139 for the 4G10 and the PY100 antibodies, respectively, it could be concluded that the antibodies do indeed enrich specific proteins from the total protein population. The total number of identified proteins for the 4G10 and the PY100 antibodies was 253 and 231, respectively. When correlating the proteins enriched by the two different antibodies (Figure 4C), the correlation was found to be 0.430. This showed that they, to some extent, overlap in specificity and function in the enrichment step, but that the overlap is far from complete. It is important to remember that the automated acquisition may influence the selection of individual peptides during the MS analysis, and thereby affect the final protein identification. Out of the protein identities, 68 and 46 proteins were unique for 4G10 and PY100, respectively, and 185 protein identities overlapped. No P-Tyr sites were directly detected, which can be due to the fact that P-Tyr peptides were not present at concentrations high enough using these enrichments conditions (300 µg of antibodies). As discussed above in the case of 2-D SDS-PAGE and following Western blotting, using different anti-phosphotyrosine antibodies, there is a risk of nonspecific binding. In the immunoaffinity enrichment, proteins might bind nonspecifically to the antibody or to the chromatography media. It is also possible that proteins binding to P-Tyr proteins are co-purified, once again pointing to the importance of confirming phosphorylation. From these results, the 4G10 antibody was concluded to be the best choice for P-Tyr protein enrichment. To further compare 4G10 and PY100, two experiments were done to test their ability to enrich P-Tyr peptides from (i) a tryptic digest of K562 cells, obtained from a single cell culture where the cell pellet was divided into two aliquots and (ii) control peptides from the PhosphoScan kit.31 These experi-
research articles
Studying Global Protein Tyrosine Phosphorylation
Table 3. P-Tyr Peptides Identified by Immunoaffinity Enrichment at the Protein Level Using the 4G10 Antibody gel piece protein IPI accession
1A
ABL 1
1B
ABL 1
1B
BCR
2A
INPPL1
3B 3B
BCAR 1 INPPL1
3B
INPPL1
4B
CBL
6B 6B 6B
CTTN CTTN GAB2
7B 7B
PXN SH3
8B
LYN
10B LASP1 10B MAPK1
protein name
sitea
phosphotyrosine peptideb
IPI00216969 Splice isoform 1A of 393 LMTGDTyTAHAGAK prpto-oncogene tyrosine-protein kinase ABL1 IPI00216969 Splice isoform 1A of 185 LyVSSESR prpto-oncogene tyrosine-protein kinase ABL1 IPI00472302 Breakpoint cluster region 644 NSLETLLyKPVDR isoform 2 IPI00016932 Inositol polyphosphate 1135 TLSEVDyAPAGPAR 5-phosphate IPI00011998 CRK-associated substrate 234 VGQGYVYEAAQPEQDEyDIPR IPI00016932 Inositol polyphosphate 986 NSFNNPAyYVLEGVPHQLLPPEPPSPAR 5-phosphate IPI00016932 Inositol polyphosphate 1135 TLSEVDyAPAGPAR 5-phosphate IPI00027269 CBL E3 ubiquitin protein 674 IKPSSSANAIySLAARPLPVPK ligase IPI00029601 Src substrate cortactin 421 LPSSPVyEDAASFK IPI00029601 Src substrate cortactin 446 GPVSGTEPEPVySmEAADYR IPI00186990 Splice isoform 1 of 324 EFGDLLVDNMDVPATPLSAyQIPR GRB2-associated binding protein 2 IPI00220030 Splice isoform alpha of paxillin 88 FIHQQPQSSSPVyGSSAK IPI00431025 Spectrin SH3 domain binding 213 TLEPVKPPTVPNDyMTSPAR protein 1 IPI00298625 V-yes-1 Yamaguchi sarcoma 397 VIEDNEyTAR viral related oncogene homologue IPI00000861 Splice isoform 1 of LIM and 171 RPLEQQQPHHIPTSAPVyQQPQQQPVAQSYGGYKEPAAPVSIQR SH3 domain protein 1 IPI00003479 Mitogen-activated protein 187 VADPDHDHPGFLTEyVATR kinase 1
ions protein scorec scored
62
1078
45
1904
67
2092
57
416
30 72
1144 2441
116
2441
39
1277
99 87 29
2233 2233 578
67 67
279 607
64
219
53
809
136
349
a Site indicates the number of the phosphorylated tyrosine residue. b The y indicates the phosphorylated tyrosine residue. c Ion score denotes the individual ion score for the peptide from the Mascot software. d Protein score denotes the sum of significant (p < 0.05) peptide scores for the protein.
ments provided an interesting comparison of the enrichment of P-Tyr proteins, since the selectivity of the antibodies might differ for proteins and peptides, respectively. The antibodies, coupled with Sepharose protein G media (PY100 coupled by the manufacturer, 4G10 coupled in-house), were incubated with the samples. A summary of the P-Tyr peptides identified, by LC-MS/MS analysis, out of the captured material from the K562 sample, is presented in Table 2. In total, 30 phosphotyrosine sites were discovered from K562 extracts. Out of these, 10 and 13 were uniquely identified by 4G10 and PY100, respectively, while 7 were identified by both antibodies. This is in agreement with a preliminary study of the two antibodies, both coupled in-house with Sepharose protein G media, where the 4G10 antibody identified 2 sites and the PY100 antibody identified 9 sites (data not shown). Of the 30 sites, 12 were considered new, that is, not previously reported by Goss et al.6 The result clearly indicated that the coverage of the P-Tyr proteome was far from complete. Analysis of the control peptide sample detected a total of 100 phosphorylation sites. Of them, 15 and 58 were identified using the 4G10 and the PY100 antibodies, respectively, while 27 sites were identified by both antibodies (data not shown). The control peptides of the PhosphoScan kit (derived from Jurkat cells) should produce 35 high scoring P-Tyr peptides during LC-MS/MS analysis after immunoaffinity enrichment. We could detect 8 and 18 of these with 4G10 and PY100, respectively. Theoretically, the data resulting from the immunoaffinity enrichments should only contain phosphotyrosine peptides, but nonphosphorylated peptides were detected as well. This could be due to the loss of the phosphate group during the analysis, or more likely, to unspecific binding of peptides by the antibodies or to the Sepharose protein G media. When
comparing the fraction of P-Tyr peptides to the total number of detected peptides in the K562 sample, the PY100 antibody gave a ratio of 0.15, while the corresponding value for the 4G10 antibody was found to be 0.04. We thus concluded that there was a higher selectivity using the PY100 antibody. The large fraction of nonphosphorylated peptides identified using the 4G10 antibody could decrease the efficiency of the ESI-MS analysis of phosphopeptides (ion suppression). When analyzing the PhosphoScan kit control peptides, the ratio was considerably higher, 0.40 and 0.31 for the PY100 and the 4G10 antibody, respectively. The higher level of phosphorylated peptides in the commercial control sample produced from Jurkat cells, compared to the K562 sample, is most likely due to phosphatase inhibitor treatment of these cells. Taken together, these results showed that both the 4G10 and the PY100 antibodies enriched P-Tyr proteins and peptides, but with different efficiencies. The PY100 antibody was more efficient for enrichment of P-Tyr peptides than 4G10. Interestingly, the reverse was observed for immunoaffinity enrichment at the protein level, where the 4G10 antibody was the most efficient. This could be because the PY100 antibody was produced using P-Tyr peptides as antigen, and the 4G10 antibody was produced using phosphotyramine. Tyrosine Phosphorylation Studies in K562 Cells. To detect as many putative P-Tyr proteins as possible, experiments using the 4G10 antibody were set up where 1 mg of antibody was coupled with a HiTrap column and K562 cell extracts were loaded onto the column. The 4G10 antibody was chosen since it was found to be most efficient for P-Tyr protein enrichments. The estimated enrichment efficiency during these conditions (about 35 mg of total proteins loaded onto a column with 1 mg of 4G10 antibody) was approximately 35-40%. This was Journal of Proteome Research • Vol. 7, No. 7, 2008 2905
2906
IPI00216969
IPI00216972
IPI00216970
IPI00216971
IPI00013508 IPI00004497 IPI00004499 IPI00004497 IPI00004498 IPI00004500 IPI00075248 IPI00027269 IPI00302925
IPI00412771 IPI00026689 IPI00026691 IPI00031681 IPI00031682 IPI00220421 IPI00024067 IPI00004840 IPI00004839 IPI00004841 IPI00182469 IPI00215637 IPI00015287 IPI00000352
IPI00396485 IPI00186290 IPI00029263 IPI00216008
IPI00186991 IPI00186990 IPI00334715
IPI00028570 IPI00014198 IPI00014197 IPI00029769
IPI00464963 IPI00215965
IPI00171903
IPI00002966 IPI00334775 IPI00334776 IPI00016934 IPI00016932 IPI00016933
ABL1
ABL1
ABL1
ACTN1 BCR BCR BCR BCR BCR CALM1 CBL CCT8
CD2AP CDC2 CDC2 CDK2 CDK2 CENTD2 CLTC CRKL CRKL CRKL CTNND1 DDX3X DOK1 DYRK1B
EEF1A1 EEF2 FER G6PD
GAB2 GAB2 GRLF1
GSK3B H41 H41 HCK
HEMGN HNRPA1
HNRPM
HSPA4 HSPCB HSPCB INPPL1 INPPL1 INPPL1
IPI accession
ABL1
protein
Isoform IA of Proto-oncogene tyrosine-protein kinase ABL1 Isoform IA of Proto-oncogene tyrosine-protein kinase ABL1 Isoform IA of Proto-oncogene tyrosine-protein kinase ABL1 Isoform IA of Proto-oncogene tyrosine-protein kinase ABL1 Alpha-actinin-1 Breakpoint cluster region protein Breakpoint cluster region protein Breakpoint cluster region protein Breakpoint cluster region protein Breakpoint cluster region protein Calmodulin E3 ubiquitin-protein ligase CBL Chaperonin containing TCP1, subunit 8 (Theta) variant CD2-associated protein Hypothetical protein DKFZp686L20222 Hypothetical protein DKFZp686L20222 Cell division protein kinase 2 Cell division protein kinase 2 Isoform 2 of Centaurin-delta 2 Clathrin heavy chain 1 Crk-like protein Crk-like protein Crk-like protein Isoform 1AB of Catenin delta-1 ATP-dependent RNA helicase DDX3X Isoform 1 of Docking protein 1 Isoform 1 of Dual specificity tyrosine-phosphorylation-regulated kinase 1B Elongation factor 1-alpha 1 Elongation factor 2 Proto-oncogene tyrosine-protein kinase FER Isoform Long of Glucose-6-phosphate 1-dehydrogenase Isoform 1 of GRB2-associated-binding protein 2 Isoform 1 of GRB2-associated-binding protein 2 Isoform 1 of Glucocorticoid receptor DNA-binding factor 1 Isoform 1 of Glycogen synthase kinase-3 beta Hypothetical protein Hypothetical protein Isoform p59-HCK of Tyrosine-protein kinase HCK Hemogen Heterogeneous nuclear ribonucleoprotein A1 isoform b Heterogeneous nuclear ribonucleoprotein M isoform a Heat shock 70 kDa protein 4 85 kDa protein 85 kDa protein inositol polyphosphate phosphatase-like 1 Inositol polyphosphate phosphatase-like 1 Inositol polyphosphate phosphatase-like 1
IPI protein annotation
Journal of Proteome Research • Vol. 7, No. 7, 2008 336 484 596 986 1135 1162
64
479 347
216 190 244 390
249 266 1105
29 442 714 447
88 15 19 15 15 497 634 207 251 198 and 207 96 461 449 273
246 58 177 591 644 910 99 674 30
469
393
226
172
sitea
K.LKKEDIyAVEIVGGATR.I K.SIyYITGESK.E R.LVSSPCCIVTSTyGWTANMER.I K.NSFNNPAyYVLEGVPHQLLPPEPPSPAR.A K.TLSEVDyAPAGPAR.S R.GLPSDyGRPLSFPPPR.I
R.GGNRFEPyANPTKR.Y
K.EKPKEEPGIPAILNESHPENDVySYVLF.R.SSGPYGGGGQyFAKPR.N
R.GEPNVSyICSR.Y R.KTPQGPPEIySDTQFPSLQSTAK.H K.LQLDNQyAVLENQK.S R.VIEDNEyTAR.E
K.LAQGNGHCVNGISGQVHGFySLPKPSR.H R.DSTyDLPR.S R.NEEENIySVPHDSTQGK.I
K.STTTGHLIyK.C K.EDLyLKPIQR.T R.QEDGGVySSSGLK.Q R.VQPNEAVyTK.M
R.ISTyGLPAGGIQPHPQTK.N K.IEKIGEGTyGVVYK.G K.IEKIGEGtYGVVyK.G K.IGEGTyGVVYK.A K.VEKIGEGTyGVVYK.A R.LDSmKPLEKHySVVLPTVSHSGFLYK.T R.ALEHFTDLyDIKR.A R.NSNSYGIPEPAHAyAQPQTTTPLPAVSGSPGAAITPLPSTQNGPVFAK.A K.RVPCAyDKTALALEVGDIVK.V R.NSNSyGIPEPAHAyAQPQTTTPLPAVSGSPGAAITPLPSTQNGPVFAK.A K.LNGPQDHSHLLySTIPR.M K.KGADSLEDFLyHEGYACTSIHGDR.S K.SHNSALySQVQK.S R.IYQyIQSR.F
K.AIMTYVSSFyHAFSGAQK.A R.MIyLQTLLAK.E K.GHGQPGADAEKPFyVNVEFHHER.G K.LASQLGVyR.A K.NSLETLLyKPVDRVTR.S K.LQTVHSIPLTINKEDDESPGLyGFLNVIVHSATGFK.Q R.VFDKDGNGyISAAELR.H K.IKPSsSANAIySLAARPLPVPK.L K.HFSGLEEAVyR.N
R.MERPEGCPEKVyELmR.A
R.LMTGDTyTAHAGAK.F
R.NKPTVyGVSPNYDKWEmER.T
R.YEGRVyHYR.I
phosphotyrosine peptideb
Table 4. P-Tyr Peptides Identified by Immunoaffinity Enrichment at the Peptide Level Using the PY100 Antibody
30 32 45 83 101 37
53
50 37
51 89 84 52
81 29 91
46 26 63 32
102 90 27 92 99 46 27 64 137 39 52 27 63 65
30 21 84 32 53 65 53 40 76
80
128
17
28
ion scorec
30 276 276 339 339 339
88
50 229
51 173 173 52
110 110 91
712 194 63 32
102 90 87 191 191 46 36 255 255 255 52 88 63 65
30 144 342 342 342 342 54 40 76
208
208
208
208
protein scored
No No No Yesg Yes No
No
No No
Yes No No Yes
No No No
No No No No
No Yes No Yes Yes Yes No No No No No No No No
No No No No No No No No No
No
No
No
No
Phosidae
No No No Yes Yes No
No
No No
Yes Yes No No
No No Yes
Yes No No No
Yes Yes Yes Yes Yes No No No No No No No No Yes
No No Yes Yes Yes No No Yes No
No
Yes
Yes
No
reported for K562f
research articles Bergström Lind et al.
research articles
IPI00216423 IPI00003479 IPI00002857 IPI00293312 IPI00007935 IPI00031388
IPI00013721 IPI00219622 IPI00220030 IPI00008438 IPI00021326
IPI00431025 IPI00030783 IPI00003843 IPI00218344 IPI00218343 IPI00022353 IPI00301058 IPI00418471 IPI00418471
ITSN2 MAPK1 MAPK14 NHN1 PDLIM5 PIK3CB
PRPF4B PSMA2 PXN RPS10 SHC1
SSH3BP STAT5A TJP2 TUBA6 TUBA6 TYK2 VASP VIM VIM
a Site indicates the number of the phosphorylated tyrosine residue. b The y indicates the phosphorylated tyrosine residue. c Ion score denotes the individual ion score for the peptide from the Mascot software. Protein score denotes the sum of significant (p < 0.05) peptide scores for the protein. e Site reported in phosphorylation database Phosida (www.phosida.com). f Site reported for K562 by Goss et al.6 g Found with another IPI accession number in database. d
No Yes Yes No No No Yes No No No Yes No No No No No No No 56 63 55 604 604 56 86 388 94 K.TLEPVKPPTVPNDyMTSPAR.L K.AVDGyVKPQIK.Q R.IEIAQKHPDIyAVPIK.T R.QLFHPEQLITGKEDAANNyAR.G R.IHFPLATyAPVISAEK.A R.LLAQAEGEPCyIR.D R.VQIyHNPTANSFR.V R.SLyASSPGGVYATR.S R.SLYASSPGGVyATR.S 213 694 1118 103 272 292 38 52 60
56 63 55 25 99 56 86 102 63
Yes No No No Yes Yes No Yes No Yes 117 159 83 90 102 K.LCDFGSASHVADNDITPyLVSR.F K.HIGLVySGmGPDYR.V R.VGEEEHVySFPNKQK.S R.IAIyELLFK.E R.ELFDDPSyVNVQNLDK.A 849 75 118 12 428
117 88 83 61 62
No Yes Yes No Yes No No Yes Yes No No No 41 45 75 32 86 29 41 45 75 32 86 29 K.LIyLVPEK.Q R.VADPDHDHTGFLTEyVATR.W R.HTDDEMTGyVATR.W R.GGQyENFR.V R.YTEFyHVPTHSDASK.K R.ERVPFILTyDFIHVIQQGK.T 552 187 182 409 250 962
Isoform 4 of Intersectin-2 Mitogen-activated protein kinase 1 Mitogen-activated protein kinase 14 isoform 2 CDNA FLJ32070 fis, clone OCBBF1000119 PDZ and LIM domain protein 5 Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta isoform Serine/threonine-protein kinase PRP4 homologue Proteasome subunit alpha type 2 Isoform Alpha of Paxillin 40S ribosomal protein S10 SHC (Src homology 2 domain containing) transforming protein 1 Abl-interactor 1 isoform a Signal transducer and activator of transcription 5A Isoform A1 of Tight junction protein ZO-2 Tubulin alpha-6 chain Tubulin alpha-6 chain Nonreceptor tyrosine-protein kinase TYK2 Vasodilator-stimulated phosphoprotein Vimentin Vimentin
IPI accession protein
Table 4. Continued
phosphotyrosine peptideb sitea IPI protein annotation
ion scorec
protein scored
Phosidae
reported for K562f
Studying Global Protein Tyrosine Phosphorylation
an expected yield since the column was overloaded in order to achieve maximal recovery of P-Tyr proteins. The eluted P-Tyr proteins were, as before, separated using 1-D SDS-PAGE. The resulting gel lane was cut into 15 pieces, and the proteins were in-gel digested with trypsin and subsequently analyzed using LC-MS/MS. The result of the experiment is summarized in Figure 5. As expected, the proteins identified from each gel piece had an average molecular mass that decreased the further the protein had migrated in the gel (Figure 5B). This trend verified that the overall identification success of the proteins by the Mascot software was high. On average, about 20 proteins were identified per gel piece. Two independent experiments were pooled and a list of 459 unique putative P-Tyr proteins was created. These proteins will be studied further for their biological relevance (protein identities provided upon request). The overlap of identified proteins between the two runs was 50%. The 50% overlap between the two analyses was expected37 and due to the fact that many proteins were present in the sample at threshold concentrations and their appearance in the mass spectrometric analysis were therefore intermittent. A phosphatase (Calf Intestine Alkaline Phosphatase, CIAP) treatment of the enriched putative P-Tyr proteins was performed to ensure that the proteins enriched indeed were phosphoproteins. After separation on 1-D SDS-PAGE followed by Western blotting labeled with the 4G10 antibody, intensity of all CIAP treated protein bands was reduced or undetectable, compared to a control treated with water. Among the 459 proteins, 12 were directly verified as P-Tyr proteins by the detection of the corresponding phosphorylated peptide (Table 3) in the MS/MS spectrum. Using immunoaffinity enrichment at the protein level, 50-70 tyrosine phosphorylated peptides have previously been reported from Jurkat and HeLa cells.17,18 In these experiments, pervanadate treatment was applied and the overall levels of tyrosine phosphorylation were greatly increased. In K562, 30 P-Tyr peptides were detected when applying immunoaffinity enrichment at the protein level, followed by digestion and IMAC purification of resulting P-Tyr peptides after digestion.16 In initial testing of pervanadate treatment, we found a significant increase in overall levels of tyrosine phosphorylation. Taking this into account, the number of P-Tyr peptides (12) detected in this study is in agreement with what can be expected since neither pervanadate treatment nor further purification of phosphorylated peptides have been applied. Furthermore, in order to detect as many P-Tyr peptides as possible, the PY100 antibody was chosen since it was found most efficient for P-Tyr peptide enrichment. According to CST, the enrichment efficiency for a typical sample was 30%, which is in the same region as achieved for immunoaffinity enrichment at the protein level. In this way, 67 unique P-Tyr peptides were identified (Table 4) from two experiments. This result is in direct comparison with a study from Goss et al.6 Of the 67 peptides, 38 were considered new, that is, not reported by Goss et al.6 or by Phosida (web-based compilation of phosphorylated proteins) (Table 4). Using all available peptide sequence data, we identified a total of 89 putative P-Tyr proteins, based on at least two peptides of significant score. Of the 89 proteins, 17 were confirmed as P-Tyr proteins by direct detection of one or more P-Tyr peptides. This should be compared to immunoaffinity enrichment at the protein level, where 459 were identified, and 12 were confirmed. Immunoaffinity enrichment starting at the protein level was expected, and confirmed, to give a statistically better identificaJournal of Proteome Research • Vol. 7, No. 7, 2008 2907
research articles
Bergström Lind et al.
Table 5. Summary of True Tyrosine Phosphorylated Proteins Identified in the K562 Cell Line, Describing How the Actual Tyrosine Phosphorylated Site Was Discovered and How the Protein Identification Was Determined P-Tyr site detected from enrichment at the protein
P-Tyr protein identity determined by data from enrichment at the
IPI accession
IPI protein annotation
peptide level
protein level
peptide level
protein level
ABL1
IPI00216969
X
X
X
X
BCAR BCR CBL CCT8
IPI00011998 IPI00004497 IPI00027269 IPI00302925
X X X
X
CDC2 CDK2 CENTD2 CLTC CRKL CTNND1 CTTN DDX3X DOK1 EEF1A1 EEF2 G6PD
IPI00026689 IPI00031681 IPI00220421 IPI00024067 IPI00004839 IPI00182469 IPI00029601 IPI00215637 IPI00015287 IPI00396485 IPI00186290 IPI00216008
GAB2 H41 HEMGN HNRPA1
IPI00186990 IPI00014197 IPI00464963 IPI00215965
HNRPM
IPI00171903
HSPA4 HSPCB INPPL1 LASP1
IPI00002966 IPI00334775 IPI00016932 IPI00000861
LYN
IPI00298625
MAPK1 MAPK14 PDLIM5 PIK3CB
IPI00003479 IPI00002857 IPI00007935 IPI00031388
PSMA2 PXN RPS10 SH3 SHC1
IPI00219622 IPI00220030 IPI00008438 IPI00431025 IPI00021326
SSH3BP TJP2 TUBA6 VIM
IPI00431025 IPI00003843 IPI00218343 IPI00418471
Isoform IA of Proto-oncogene tyrosine-protein kinase ABL1 CRK-associated substrate Breakpoint cluster region protein E3 ubiquitin-protein ligase CBL Chaperonin containing TCP1, subunit 8 (Theta) variant Hypothetical protein DKFZp686L20222 Cell division protein kinase 2 Isoform 2 of Centaurin-delta 2 Clathrin heavy chain 1 Crk-like protein Isoform 1AB of Catenin delta-1 Scr substrate cortactin ATP-dependent RNA helicase DDX3X Isoform 1 of Docking protein 1 Elongation factor 1-alpha 1 Elongation factor 2 Isoform Long of Glucose-6-phosphate 1-dehydrogenase Isoform 1 of GRB2-associated-binding protein 2 Hypothetical protein Hemogen Heterogeneous nuclear ribonucleoprotein A1 isoform b Heterogeneous nuclear ribonucleoprotein M isoform a Heat shock 70 kDa protein 4 85 kDa protein Inositol polyphosphate phosphatase-like 1 Splice isoform 1 of LIM and SH3 domain protein 1 V-yes Yamaguchi sarcoma viral related oncogene homologue Mitogen-activated protein kinase 1 Mitogen-activated protein kinase 14 isoform 2 PDZ and LIM domain protein 5 Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta isoform Proteasome subunit alpha type 2 Isoform Alpha of Paxillin 40S ribosomal protein S10 Spectrin SH3 domain binding protein 1 SHC (Src homology 2 domain containing) transforming protein 1 Abl-interactor 1 isoform a Isoform A1 of Tight junction protein ZO-2 Tubulin alpha-6 chain Vimentin
tion of the analyzed proteins due to greater sequence coverage. This is due to the fact that the digestion of proteins into peptides followed by LC-MS/MS analysis included all peptides from the enriched protein. As a result, this approach could also provide additional information regarding serine and threonine phosphorylation sites as well as other PTMs. In addition (depending on the sample preparation used), one could also expect sequence information from proteins that interact with and bind to P-Tyr proteins. Immunoaffinity enrichment starting with tryptic peptides, on the other hand, resulted in direct enrichment of the P-Tyr peptides. A considerable part of the non-P-Tyr peptides was thereby removed before the LC-MS/ MS analysis, reducing sequence coverage. This explained some of the difficulty in protein identification when performing immunoaffinity enrichment at the peptide level. On the other hand, more P-Tyr sites (67) were detected when performing the experiments at the peptide level using the PY100 antibody, compared to 16 when performing the enrichment at the protein 2908
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level using the 4G10 antibody. By combining the data, additionally, 15 proteins that could not be considered P-Tyr proteins from neither the peptide enrichment nor the protein enrichment alone were verified. In total, by combining P-Tyr enrichments at the peptide level, with the PY100 antibody, and at the protein level using the 4G10 antibody, 40 P-Tyr proteins were identified from K562 cells line (Table 5). Also, when comparing the results from the immunoaffinity enrichment at the peptide level (Table 2) and at the protein level (Table 3) using the 4G10 antibody, only six sites were overlapping. This showed that several aspects, such as the choice of antibody as well as enrichment strategy, need to be carefully considered when performing immunoaffinity enrichment of phosphotyrosine containing polypeptides.
Conclusions The results from this study provide us, and other investigators, with valuable information on antibodies and sample
research articles
Studying Global Protein Tyrosine Phosphorylation preparation methods suitable for studying tyrosine phosphorylation in the context of the human proteome. Using the CML cell line K562, the 4G10 antibody detected the most number of protein spots during Western blotting. Furthermore, the 4G10 and the PY100 antibodies were found to be most efficient for enriching phosphotyrosine proteins and peptides, respectively. The selected antibodies were thus found to be the most efficient when used in combination with each other. It is important to remember, when comparing the outcome of different enrichment approaches, that more reliable protein identification is observed when starting at the protein level, but more P-Tyr sites were detected when starting at the peptide level. Regarding how to choose experimental procedure from the aspect of biological relevance, our work with the K562 cell line indicates that the protein-based approach should be preferred. This is, in our view, because signal transduction in the cell is mediated by protein interactions controlled by phosphorylation, thus, highlighting the need for protein identification during analysis.
Acknowledgment. The authors are grateful to Dr. Gisela Barbany for kindly providing the K562 cells. Prof. Brian Druker and Prof. Tempst are acknowledged for kindly providing the 4G10 and the PYKD1 antibody, respectively. Prof. Per Andre´n and Mårten Linde´n are acknowledged for valuable discussions. Financial support from Kjell and Ma¨rta Beijer Foundation and the Swedish Research Council is gratefully acknowledged. S.B.L. has a postdoc position at the Swedish Cancer Foundation. Uppsala BIO is acknowledged for support. References (1) Zwick, E.; Bange, J.; Ullrich, A. Receptor tyrosine kinase signalling as a target for cancer intervention strategies. Endocr. Relat. Cancer 2001, 8, 161–73. (2) Kolibaba, K. S.; Druker, B. J. Protein tyrosine kinases and cancer. Biochim. Biophys. Acta 1997, 1333, F217–48. (3) Levitzki, A. Protein tyrosine kinase inhibitors as novel therapeutic agents. Pharmacol. Ther. 1999, 82, 231–9. (4) Machida, K.; Mayer, B. J.; Nollau, P. Profiling the global tyrosine phosphorylation state. Mol. Cell. Proteomics 2003, 2, 215–33. (5) Rush, J.; Moritz, A.; Lee, K. A.; Guo, A.; Goss, V. L.; Spek, E. J.; Zhang, H.; Zha, X. M.; Polakiewicz, R. D.; Comb, M. J. Immunoaffinity profiling of tyrosine phosphorylation in cancer cells. Nat. Biotechnol. 2005, 23, 94–101. (6) Goss, V. L.; Lee, K. A.; Moritz, A.; Nardone, J.; Spek, E. J.; MacNeill, J.; Rush, J.; Comb, M. J.; Polakiewicz, R. D. A common phosphotyrosine signature for the Bcr-Abl kinase. Blood 2006, 107, 4888– 97. (7) Pane, F.; Intrieri, M.; Quintarelli, C.; Izzo, B.; Muccioli, G. C.; Salvatore, F. BCR/ABL genes and leukemic phenotype: from molecular mechanisms to clinical correlations. Oncogene 2002, 21, 8652–67. (8) Druker, B. J.; Mamon, H. J.; Roberts, T. M. Oncogenes, growth factors, and signal transduction. N. Engl. J. Med. 1989, 321, 1383– 91. (9) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127, 635–48. (10) Schumacher, J. A.; Crockett, D. K.; Elenitoba-Johnson, K. S.; Lim, M. S. Evaluation of enrichment techniques for mass spectrometry: identification of tyrosine phosphoproteins in cancer cells. J. Mol. Diagn. 2007, 9, 169–77. (11) Pandey, A.; Podtelejnikov, A. V.; Blagoev, B.; Bustelo, X. R.; Mann, M.; Lodish, H. F. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 179–84. (12) Pandey, A.; Fernandez, M. M.; Steen, H.; Blagoev, B.; Nielsen, M. M.; Roche, S.; Mann, M.; Lodish, H. F. Identification of a novel immunoreceptor tyrosine-based activation motif-containing molecule, STAM2, by mass spectrometry and its involvement in growth
(13)
(14)
(15)
(16)
(17)
(18)
(19)
(20)
(21)
(22)
(23)
(24)
(25)
(26)
(27)
(28)
(29)
(30)
(31)
factor and cytokine receptor signaling pathways. J. Biol. Chem. 2000, 275, 38633–9. Pandey, A.; Blagoev, B.; Kratchmarova, I.; Fernandez, M.; Nielsen, M.; Kristiansen, T. Z.; Ohara, O.; Podtelejnikov, A. V.; Roche, S.; Lodish, H. F.; Mann, M. Cloning of a novel phosphotyrosine binding domain containing molecule, Odin, involved in signaling by receptor tyrosine kinases. Oncogene 2002, 21, 8029–36. Blagoev, B.; Ong, S. E.; Kratchmarova, I.; Mann, M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics. Nat. Biotechnol. 2004, 22, 1139–45. Ficarro, S.; Chertihin, O.; Westbrook, V. A.; White, F.; Jayes, F.; Kalab, P.; Marto, J. A.; Shabanowitz, J.; Herr, J. C.; Hunt, D. F.; Visconti, P. E. Phosphoproteome analysis of capacitated human sperm. Evidence of tyrosine phosphorylation of a kinase-anchoring protein 3 and valosin-containing protein/p97 during capacitation. J. Biol. Chem. 2003, 278, 11579–89. Salomon, A. R.; Ficarro, S. B.; Brill, L. M.; Brinker, A.; Phung, Q. T.; Ericson, C.; Sauer, K.; Brock, A.; Horn, D. M.; Schultz, P. G.; Peters, E. C. Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 443–8. Brill, L. M.; Salomon, A. R.; Ficarro, S. B.; Mukherji, M.; StettlerGill, M.; Peters, E. C. Robust phosphoproteomic profiling of tyrosine phosphorylation sites from human T cells using immobilized metal affinity chromatography and tandem mass spectrometry. Anal. Chem. 2004, 76, 2763–72. Amanchy, R.; Kalume, D. E.; Iwahori, A.; Zhong, J.; Pandey, A. Phosphoproteome analysis of HeLa cells using stable isotope labeling with amino acids in cell culture (SILAC). J. Proteome Res. 2005, 4, 1661–71. Steen, H.; Kuster, B.; Fernandez, M.; Pandey, A.; Mann, M. Tyrosine phosphorylation mapping of the epidermal growth factor receptor signaling pathway. J. Biol. Chem. 2002, 277, 1031–9. Gu, T. L.; Goss, V. L.; Reeves, C.; Popova, L.; Nardone, J.; Macneill, J.; Walters, D. K.; Wang, Y.; Rush, J.; Comb, M. J.; Druker, B. J.; Polakiewicz, R. D. Phosphotyrosine profiling identifies the KG-1 cell line as a model for the study of FGFR1 fusions in acute myeloid leukemia. Blood 2006, 108, 4202–4. Walters, D. K.; Goss, V. L.; Stoffregen, E. P.; Gu, T. L.; Lee, K.; Nardone, J.; McGreevey, L.; Heinrich, M. C.; Deininger, M. W.; Polakiewicz, R.; Druker, B. J. Phosphoproteomic analysis of AML cell lines identifies leukemic oncogenes. Leuk. Res. 2006, 30, 1097– 104. Andersson, L.; Porath, J. Isolation of phosphoproteins by immobilized metal (Fe3+) affinity chromatography. Anal. Biochem. 1986, 154, 250–4. Ficarro, S. B.; McCleland, M. L.; Stukenberg, P. T.; Burke, D. J.; Ross, M. M.; Shabanowitz, J.; Hunt, D. F.; White, F. M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae. Nat. Biotechnol. 2002, 20, 301–5. Larsen, M. R.; Thingholm, T. E.; Jensen, O. N.; Roepstorff, P.; Jorgensen, T. J. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell. Proteomics 2005, 4, 873–86. Pinkse, M. W.; Uitto, P. M.; Hilhorst, M. J.; Ooms, B.; Heck, A. J. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns. Anal. Chem. 2004, 76, 3935–43. Bodenmiller, B.; Mueller, L. N.; Mueller, M.; Domon, B.; Aebersold, R. Reproducible isolation of distinct, overlapping segments of the phosphoproteome. Nat. Methods 2007, 4, 231–7. Mann, M.; Hendrickson, R. C.; Pandey, A. Analysis of proteins and proteomes by mass spectrometry. Annu. Rev. Biochem. 2001, 70, 437–73. Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probabilitybased protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551–67. Venable, J. D.; Dong, M. Q.; Wohlschlegel, J.; Dillin, A.; Yates, J. R. Automated approach for quantitative analysis of complex peptide mixtures from tandem mass spectra. Nat. Methods 2004, 1, 39– 45. Shevchenko, A.; Chernushevich, I.; Wilm, M.; Mann, M. De Novo peptide sequencing by nanoelectrospray tandem mass spectrometry using triple quadrupole and quadrupole/time-of-flight instruments. Methods Mol. Biol. 2000, 146, 1–16. Cell Signaling Technology, Phosphoscan Kit (P-Tyr-100), #7900, 2006.
Journal of Proteome Research • Vol. 7, No. 7, 2008 2909
research articles (32) Rappsilber, J.; Ishihama, Y.; Mann, M. Stop and go extraction tips for matrix-assisted laser desorption/ionization, nanoelectrospray, and LC/MS sample pretreatment in proteomics. Anal. Chem. 2003, 75, 663–70. (33) Nielsen, P. A.; Olsen, J. V.; Podtelejnikov, A. V.; Andersen, J. R.; Mann, M.; Wisniewski, J. R. Proteomic mapping of brain plasma membrane proteins. Mol. Cell. Proteomics 2005, 4, 402–8. (34) Savitski, M. M.; Nielsen, M. L.; Zubarev, R. A. New data baseindependent, sequence tag-based scoring of peptide MS/MS data validates Mowse scores, recovers below threshold data, singles out modified peptides, and assesses the quality of MS/MS techniques. Mol. Cell. Proteomics 2005, 4, 1180–8.
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Journal of Proteome Research • Vol. 7, No. 7, 2008
Bergström Lind et al. (35) Gembitsky, D. S.; Lawlor, K.; Jacovina, A.; Yaneva, M.; Tempst, P. A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation. Mol. Cell. Proteomics 2004, 3, 1102–18. (36) Savitski, M. M.; Nielsen, M. L.; Kjeldsen, F.; Zubarev, R. A. Proteomics-grade de novo sequencing approach. J. Proteome Res. 2005, 4, 2348–54. (37) Old, W. M.; Meyer-Arendt, K.; Aveline-Wolf, L.; Pierce, K. G.; Mendoza, A.; Sevinsky, J. R.; Resing, K. A.; Ahn, N. G. Comparison of label-free methods for quantifying human proteins by shotgun proteomics. Mol. Cell. Proteomics 2005, 4, 1487–502.
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