Stable Isotope Labeling of Phosphoproteins for Large-scale Phosphorylation Rate Determination
2014; Elsevier BV; Volume: 13; Issue: 4 Linguagem: Inglês
10.1074/mcp.o113.036145
ISSN1535-9484
AutoresRosalynn C. Molden, Jonathan Goya, Zia Khan, Benjamin A. García,
Tópico(s)Mitochondrial Function and Pathology
ResumoSignals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [γ-18O4]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min−1 to 0.001 min−1. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680. Signals that control responses to stimuli and cellular function are transmitted through the dynamic phosphorylation of thousands of proteins by protein kinases. Many techniques have been developed to study phosphorylation dynamics, including several mass spectrometry (MS)-based methods. Over the past few decades, substantial developments have been made in MS techniques for the large-scale identification of proteins and their post-translational modifications. Nevertheless, all of the current MS-based techniques for quantifying protein phosphorylation dynamics rely on the measurement of changes in peptide abundance levels, and many methods suffer from low confidence in phosphopeptide identification due to poor fragmentation. Here we have optimized an approach for the stable isotope labeling of amino acids by phosphate using [γ-18O4]ATP in nucleo to determine global site-specific phosphorylation rates. The advantages of this metabolic labeling technique are increased confidence in phosphorylated peptide identification, direct labeling of phosphorylation sites, measurement phosphorylation rates, and the identification of actively phosphorylated sites in a cell-like environment. In this study we calculated approximate rate constants for over 1,000 phosphorylation sites based on labeling progress curves. We measured a wide range of phosphorylation rate constants from 0.34 min−1 to 0.001 min−1. Finally, we applied stable isotope labeling of amino acids by phosphate to identify sites that have different phosphorylation kinetics during G1/S and M phase. We found that most sites had very similar phosphorylation rates under both conditions; however, a small subset of sites on proteins involved in the mitotic spindle were more actively phosphorylated during M phase, whereas proteins involved in DNA replication and transcription were more actively phosphorylated during G1/S phase. The data have been deposited to the ProteomeXchange with the identifier PXD000680. Protein phosphorylation is crucial for modulating protein structure, protein localization, and the protein–protein interactions that form the basis of many cell-signaling networks. Phosphorylation-based signaling often takes the form of a cascade in which sequential protein phosphorylations lead to changes in protein stability, function, and localization. Protein kinases, the enzymes that propagate these signals, catalyze the transfer of γ phosphate from ATP onto serine, threonine, or tyrosine residues of substrate proteins. The sites of protein phosphorylation and phosphorylation dynamics are important in determining the biological outcome of a signaling event (1Kholodenko B.N. Cell-signalling dynamics in time and space.Nat. Rev. Mol. Cell Biol. 2006; 7: 165-176Crossref PubMed Scopus (1025) Google Scholar). For instance, protein phosphorylation drives many of the changes during the cell cycle (2Potapova T.A. Sivakumar S. Flynn J.N. Li R. Gorbsky G.J. Mitotic progression becomes irreversible in prometaphase and collapses when Wee1 and Cdc25 are inhibited.Mol. Biol. Cell. 2011; 22: 1191-1206Crossref PubMed Scopus (125) Google Scholar, 3Kõivomägi M. Valk E. Venta R. Iofik A. Lepiku M. Morgan D.O. Loog M. Dynamics of Cdk1 substrate specificity during the cell cycle.Mol. Cell. 2011; 42: 610-623Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). During mitosis, kinases are activated at precise times to direct the course of chromosome segregation and cell division. For example, CDK1 activation at the beginning of mitosis leads to phosphorylation of NUP98 during prophase, which in turn promotes nuclear envelope disassembly (4Laurell E. Beck K. Krupina K. Theerthagiri G. Bodenmiller B. Horvath P. Aebersold R. Antonin W. Kutay U. Phosphorylation of Nup98 by multiple kinases is crucial for NPC disassembly during mitotic entry.Cell. 2011; 144: 539-550Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). Additionally, an increased protein phosphorylation rate combined with constitutive activation of signaling networks due to hyperactivated kinases is considered a hallmark of cancer (5Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (42748) Google Scholar, 6Levina E. Oren M. Ben-Ze'ev A. Downregulation of [beta]-catenin by p53 involves changes in the rate of β-catenin phosphorylation and Axin dynamics.Oncogene. 2004; 23: 4444-4453Crossref PubMed Scopus (81) Google Scholar). Because the rate of substrate phosphorylation is a straightforward readout of kinase activity, there is growing interest in measuring phosphorylation rates in order to better understand phosphorylation-based signaling networks and potentially design more effective cancer treatments (7Heinrich R. Neel B.G. Rapoport T.A. Mathematical models of protein kinase signal transduction.Mol. Cell. 2002; 9: 957-970Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 8Hornberg J.J. Bruggeman F.J. Westerhoff H.V. Lankelma J. Cancer: a systems biology disease.Biosystems. 2006; 83: 81-90Crossref PubMed Scopus (310) Google Scholar). Many techniques have been developed to study phosphorylation-based signaling dynamics. Some of the most commonly applied techniques for following changes in phosphorylation levels are the use of site-specific antibodies to probe phosphorylated proteins from cell extracts and quantitative mass spectrometry methods such as stable isotope labeling of amino acids in cell culture to identify and quantify phosphorylated peptides (9Olsen 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: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2810) Google Scholar, 10Andreu-Perez P. Esteve-Puig R. de Torre-Minguela C. Lopez-Fauqued M. Bech-Serra J.J. Tenbaum S. Garcia-Trevijano E.R. Canals F. Merlino G. Avila M.A. Recio J.A. Protein arginine methyltransferase 5 regulates ERK1/2 signal transduction amplitude and cell fate through CRAF.Sci. Signal. 2011; 4: ra58Crossref PubMed Scopus (101) Google Scholar, 11Busillo J.M. Armando S. Sengupta R. Meucci O. Bouvier M. Benovic J.L. Site-specific phosphorylation of CXCR4 is dynamically regulated by multiple kinases and results in differential modulation of CXCR4 signaling.J. Biol. Chem. 2010; 285: 7805-7817Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). In comparison with antibody-based methods, quantitative mass spectrometry techniques have the added advantage that thousands of phosphorylation site changes can be measured in a single experiment (9Olsen 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: 635-648Abstract Full Text Full Text PDF PubMed Scopus (2810) Google Scholar). Both of these techniques provide useful information on whether the total amount of phosphorylated protein is increasing or decreasing over time; however, they do not directly measure new phosphorylation events or phosphorylation rates (12Wu R. Dephoure N. Haas W. Huttlin E.L. Zhai B. Sowa M.E. Gygi S.P. Correct interpretation of comprehensive phosphorylation dynamics requires normalization by protein expression changes.Mol. Cell. Proteomics. 2011; 10 (M111.009654)Abstract Full Text Full Text PDF Scopus (143) Google Scholar). MS techniques and fluorescence techniques have been developed to measure phosphorylation rates on synthetic peptides with known kinase consensus motifs in cell lysates (13Yu Y. Anjum R. Kubota K. Rush J. Villen J. Gygi S.P. A site-specific, multiplexed kinase activity assay using stable-isotope dilution and high-resolution mass spectrometry.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 11606-11611Crossref PubMed Scopus (67) Google Scholar, 14Shults M.D. Janes K.A. Lauffenburger D.A. Imperiali B. A multiplexed homogeneous fluorescence-based assay for protein kinase activity in cell lysates.Nat. Methods. 2005; 2: 277-284Crossref PubMed Scopus (182) Google Scholar). These techniques provide a read-out of kinase activity in cell lysates under different biological conditions. Nonetheless, the use of peptides rather than intact endogenous protein might not reflect actual in vivo phosphorylation rates because of the loss of sequence context. In addition, there is often a loss of kinase specificity in peptide-based assays because the intact protein may contain additional kinase docking sites or be part of a larger protein complex (15Wong W. Scott J.D. AKAP signalling complexes: focal points in space and time.Nat. Rev. Mol. Cell Biol. 2004; 5: 959-970Crossref PubMed Scopus (850) Google Scholar). Approaches used to directly label protein phosphorylation sites for detection by mass spectrometry using chemical approaches or other ATP analogs such as ATPγS have been previously reported. Thiol phosphate approaches have been successfully used in combination with engineered kinases to directly label kinase substrates (16Blethrow J.D. Glavy J.S. Morgan D.O. Shokat K.M. Covalent capture of kinase-specific phosphopeptides reveals Cdk1-cyclin B substrates.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 1442-1447Crossref PubMed Scopus (239) Google Scholar, 17Allen J.J. Li M. Brinkworth C.S. Paulson J.L. Wang D. Hubner A. Chou W.H. Davis R.J. Burlingame A.L. Messing R.O. Katayama C.D. Hedrick S.M. Shokat K.M. A semisynthetic epitope for kinase substrates.Nat. Methods. 2007; 4: 511-516Crossref PubMed Scopus (227) Google Scholar). However, most endogenous kinases utilize ATP more efficiently than ATPγS, and thus these reactions do not give an accurate picture of in vivo kinase activity (18Parker L.L. Schilling A.B. Kron S.J. Kent S.B.H. Optimizing thiophosphorylation in the presence of competing phosphorylation with MALDI-TOF-MS detection.J. Proteome Res. 2005; 4: 1863-1866Crossref PubMed Scopus (11) Google Scholar, 19Kwon S.W. Kim S.C. Jaunbergs J. Falck J.R. Zhao Y. Selective enrichment of thiophosphorylated polypeptides as a tool for the analysis of protein phosphorylation.Mol. Cell. Proteomics. 2003; 2: 242-247Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Another approach is to use radioactively labeled 32P-ATP or 32Pi to directly label and measure protein phosphorylation rates (20Aponte A.M. Phillips D. Hopper R.K. Johnson D.T. Harris R.A. Blinova K. Boja E.S. French S. Balaban R.S. Use of P-32 to study dynamics of the mitochondrial phosphoproteome.J. Proteome Res. 2009; 8: 2679-2695Crossref PubMed Scopus (36) Google Scholar). 32P labeling is highly specific and sensitive. It has been used in the past in combination with mass spectrometry and Edman sequencing (21Wettenhall R.E.H. Aebersold R.H. Hood L.E. [15] solid-phase sequencing of 32P-labeled phosphopeptides at picomole and subpicomole levels.in: Tony Hunter B.M.S. Methods in Enzymology. Washington, D.C, Academic Press1991: 186-199Google Scholar, 22Campbell D.G. Morrice N.A. Identification of protein phosphorylation sites by a combination of mass spectrometry and solid phase Edman sequencing.J. Biomol. Technol. 2002; 13: 119-130PubMed Google Scholar) to identify phosphorylation sites; however, extra precautions need to be used with radioisotopes, and radiolabeled samples cannot be stored for long because of the short half-life of 32P. We have developed a quantitative mass spectrometry technique using stable-isotope-labeled [γ-18O4]ATP to directly label phosphorylation sites and quantify phosphorylation changes over time on hundreds of native proteins in nucleo. Stable isotope labeling of phosphate by [γ-18O4]ATP (SILAP) 1The abbreviations used are:SILAPstable isotope labeling of amino acids with phosphateACNacetonitrileATPadenosine triphosphateAGCautomatic gain controlCDKcyclin dependent kinaseGOGene Ontology. 1The abbreviations used are:SILAPstable isotope labeling of amino acids with phosphateACNacetonitrileATPadenosine triphosphateAGCautomatic gain controlCDKcyclin dependent kinaseGOGene Ontology. can be used to specifically label phosphorylation sites without a radioisotope, and it has the added advantage that many phosphorylation sites can be identified and quantified at once using mass spectrometry. [γ-18O4]ATP labeling has previously been used in reactions with purified kinases to increase the confidence in phosphorylation site assignments and in reactions with purified kinases to characterize different kinase inhibitors (23Zhou M. Meng Z. Jobson A.G. Pommier Y. Veenstra T.D. Detection of in vitro kinase generated protein phosphorylation sites using γ[18O4]-ATP and mass spectrometry.Anal. Chem. 2007; 79: 7603-7610Crossref PubMed Scopus (24) Google Scholar, 24Fu C. Zheng X. Jiang Y. Liu Y. Xu P. Zeng Z. Liu R. Zhao Y. A universal and multiplex kinase assay using [gamma]-[18O4]-ATP.Chem. Commun. 2013; 49: 2795-2797Crossref PubMed Scopus (10) Google Scholar). These studies demonstrated that [γ-18O4]ATP is stable and has properties very similar to those of ATP with natural isotope abundances. In the experiments described here, we demonstrated that [γ-18O4]ATP can be used to label and confidently identify over 1,000 phosphorylation sites in a single experiment in isolated nuclei to measure the phosphorylation rates for proteins in asynchronous and G1/S and M phase synchronized cell nuclei, and to subsequently determine the most actively phosphorylated sites under these conditions. stable isotope labeling of amino acids with phosphate acetonitrile adenosine triphosphate automatic gain control cyclin dependent kinase Gene Ontology. stable isotope labeling of amino acids with phosphate acetonitrile adenosine triphosphate automatic gain control cyclin dependent kinase Gene Ontology. Human embryonic kidney (HEK293) cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% newborn calf serum (Invitrogen) and penicillin–streptomycin solution diluted 1:100 (10,000 units penicillin G and 10 mg streptomycin per milliliter) (Fisher) until they reached 80% confluency. HeLa cells were maintained in Joklik's modified Eagle's medium supplemented with 10% newborn calf serum (Invitrogen) and penicillin–streptomycin solution diluted 1:100 (10,000 units penicillin G and 10 mg streptomycin per milliliter) (Fisher). Cells at 3 × 105 confluency were synchronized by a double thymidine block. 1.5 × 108 cells were collected immediately after the block. The remaining cells were released for 4 h and finally transferred into media containing 100 ng/ml nocodazole for 4 h. These cells were mitotic at harvesting. The efficiency of cell synchronization was analyzed via propidium iodide staining of fixed cells using flow cytometry analysis. In addition to the cells kept for cell cycle analysis, the double thymidine blocked cells and nocodazole blocked cells were split into two samples for two technical replicates of the stable isotope labeling time course experiments. HEK293 or cell-cycle-synchronized HeLa cells were washed three times with ice-cold PBS and were then lysed in ice-cold hypotonic lysis buffer (10 mm KCl, 1.5 mm MgCl2, 10 mm HEPES-KOH pH 7.5, 1x HALT protease and phosphatase inhibitor mixture). The extent of lysis was monitored using trypan blue staining. Intact nuclei were collected by centrifugation and re-suspended in 37 °C ATP reaction buffer (35 mm NaCl, 10 mm KCl, 5 mm MgCl2, 2 μm CaCl2, 10 mm Tris-HCL pH 7.5) with 1x HALT and 5 mm [γ-18O4]ATP (97% purity; Cambridge Isotope Laboratories, Tewksbury, MA). We used 5 mm [γ-18O4]ATP in our assay to maintain intracellular ATP concentrations (1–5 mm ATP) (25Beis I. Newsholme E.A. The contents of adenine nucleotides, phosphagens and some glycolytic intermediates in resting muscles from vertebrates and invertebrates.Biochem. J. 1975; 152: 23-32Crossref PubMed Scopus (361) Google Scholar). The nuclei were incubated in the [γ-18O4]ATP-containing buffer in a 37 °C water bath, and aliquots were collected after 5, 15, 30, 60, 120, and 240 min for HEK293 cells or after 0, 5, 10, 20, 40, 80, and 160 min for synchronized HeLa cells. After collection, the samples were immediately denatured using five volumes of urea buffer (9 mm urea, 10 mm Tris-HCl pH 8) and then snap-frozen. After all of the time points were collected, the samples were sonicated to break apart the chromatin, derivatized with dithiothreitol and iodoacetamide, and digested overnight with trypsin as previously described (26Villen J. Gygi S.P. The SCX/IMAC enrichment approach for global phosphorylation analysis by mass spectrometry.Nat. Protoc. 2008; 3: 1630-1638Crossref PubMed Scopus (498) Google Scholar). After trypsin digestion, the peptides were desalted using SepPac tC18 columns (Waters, Milford, MA) and then lyophilized. Phosphopeptides were enriched using titanium dioxide (27Li Q.R. Ning Z.B. Tang J.S. Nie S. Zeng R. Effect of peptide-to-TiO2 beads ratio on phosphopeptide enrichment selectivity.J. Proteome Res. 2009; 8: 5375-5381Crossref PubMed Scopus (108) Google Scholar, 28Kettenbach A.N. Gerber S.A. Rapid and reproducible single-stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments.Anal. Chem. 2011; 83: 7635-7644Crossref PubMed Scopus (129) Google Scholar). Lyophilized peptides were re-suspended in loading buffer (2 m lactic acid, 50% acetonitrile (ACN)). Titanium beads (GL Sciences, Tokyo, Japan) were mixed with the peptides at a ratio of 4 mg of beads to 1 mg of peptide and rotated at room temperature for 30 min. The beads were rinsed once with loading buffer and twice with wash buffer (65% ACN, 0.1% TFA) and then were eluted in a basic elution buffer (50% ACN, 50 mm KH2PO4, pH to 10 with ammonium hydroxide), immediately dried, desalted using reversed-phase C18 stop-and-go extraction tips (29Rappsilber 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. 2002; 75: 663-670Crossref Scopus (1796) Google Scholar), and stored in a −80 °C freezer until analysis. Metabolites were extracted as detailed previously (30Bennett B.D. Yuan J. Kimball E.H. Rabinowitz J.D. Absolute quantitation of intracellular metabolite concentrations by an isotope ratio-based approach.Nat. Protoc. 2008; 3: 1299-1311Crossref PubMed Scopus (291) Google Scholar). Briefly, nuclei were pelleted at 600 × g and the supernatant was removed. Then 2 ml of 80% methanol (−80 °C) was added and the samples were stored on dry ice for 15 min. The supernatant was collected and the metabolites were re-extracted with 2 ml of 80% methanol (−80 °C). The samples were dried under nitrogen and analyzed using liquid chromatography coupled to an Exactive Orbitrap (Thermo Scientific) as previously described (31Vastag L. Koyuncu E. Grady S.L. Shenk T.E. Rabinowitz J.D. Divergent effects of human cytomegalovirus and herpes simplex virus-1 on cellular metabolism.PLoS Pathog. 2011; 7: e1002124Crossref PubMed Scopus (233) Google Scholar). We analyzed the phosphopeptide-enriched samples and the flow-through samples on an LTQ-Orbitrap XL (HEK293 cells) or an LTQ-Orbitrap Elite and Q-Exactive (synchronized HeLa samples) mass spectrometer (Thermo Scientific) attached to an Eksigent AS2 autosampler and an Eksigent Nano-LC Ultra 2D Plus system run at 250 nL/min. The samples were loaded on a pulled-tip fused silica column with a 100-μm inner diameter packed in-house with 12 cm of 3-μm C18 resin (Reprosil-Pur C18-AQ) that served both as a resolving column and as a nanospray ionization emitter. Phosphopeptides were resolved on a gradient from 0% ACN to 30% ACN in 0.5 mm acetic acid over 115 min. Flow-through peptides were resolved on a two-step gradient from 5% ACN to 40% ACN over 100 min, then from 40% ACN to 60% ACN over 25 min, in 0.5 mm acetic acid. The mass spectrometers were operated in the data-dependent mode with dynamic exclusion enabled (repeat count, 1; exclusion duration, 0.5 min). For the LTQ-Orbitrap XL MS, every cycle we collected one full MS scan (m/z 350 to 1650) at a resolution of 30,000 at an AGC target value of 7 × 106, and then nine MS2 scans of the most intense peptide ions using collisionally activated dissociation (normalized collision energy = 40%, isolation width = 3 m/z) at an AGC target value of 3 × 104. For the LTQ-Orbitrap Elite MS, every cycle we collected one full MS scan (m/z 350 to 1650) at a resolution of 60,000 at an AGC target value of 1 × 106, and then 15 MS2 scans of the most intense peptide ions using collisionally activated dissociation (normalized collision energy = 35%, isolation width = 2 m/z) at an AGC target value of 1 × 104 or Higher-energy collisional dissociation (normalized collision energy = 36%, isolation width = 2 m/z, resolution = 15,000) at an AGC target value of 5 × 104. Ions with a charge state of 1 and a rejection list of common contaminant ions (exclusion width = 10 ppm) were excluded from the analysis. The data were searched using Mascot (version 2.2.07, Matrix Science, London, UK) and X!Tandem (version 2013.02.01.1, The GPM, Alberta, Canada) against the human UniProt database (canonical and isoform sequence data retrieved March 16, 2012; 140,795 sequences), using a mass tolerance of 20 ppm for precursor ions and 0.5 Da for fragment ions. Serine, threonine, and tyrosine phosphorylation; methionine oxidation; and N-terminal acetylation were set as variable modifications, and cysteine carbamidomethylation was set as a fixed modification. Up to two missed cleavages were allowed for a trypsin digest search. Scaffold (version 4.0.5, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. All search results were loaded into Scaffold in a Mud-Pit-type setup. Peptide identifications were accepted at over 95% probability and protein identifications were accepted at over 99% probability according to the PeptideProphet and ProteinProphet algorithms (32Keller A. Nesvizhskii A.I. Kolker E. Aebersold R. Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.Anal. Chem. 2002; 74: 5383-5392Crossref PubMed Scopus (3886) Google Scholar). The filtered peptide identifications from Mascot and X!Tandem were loaded into in-house-developed software that used a naïve Bayes model of co-eluting peptides identified in each run to predict retention times of peptides that were not identified in all runs. This is especially important for time course analysis because it fills in time point measurements from runs containing essentially the same peptides but different sets of MS2 identifications. Phosphorylated peptides from the database searches and our in-house-developed software were loaded into Quantitator, 2J. Goya, D. Wolfgeher, K. Kristjansdottir, D. Perlman, O. Troyanskaya, S. Volchenboum, and S. Kron, manuscript in preparation. which models the isotope distribution for each peptide based on its chemical formula in order to calculate the expected intensity distributions for relevant isotopic labeling states. Isotopic labeling states were defined based on their shared isotopic perturbation, namely, "light" phosphorylated peptides with no incorporated 18O versus "heavy" phosphorylated peptides with between one and three incorporated 18O atoms. The signal intensities of the raw isotopologue peaks were partitioned between the isotopic labeling states using linear regression. Finally, we determined phosphorylation site localization scores using AScore (33Beausoleil S.A. Villen J. Gerber S.A. Rush J. Gygi S.P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization.Nat. Biotechnol. 2006; 24: 1285-1292Crossref PubMed Scopus (1206) Google Scholar) and used a score cutoff of 20 for assigning confident phosphorylation sites. We used the maximum phosphorylation site localization score to filter peptides identified by more than one MS2 spectrum. Processed and raw data have been made available in the ProteomeXchange Consortium (34Vizcaino J.A. Cote R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Perez-Riverol Y. Reisinger F. Rios D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-D1069Crossref PubMed Scopus (1595) Google Scholar) with the dataset identifier PXD000680 and DOI 10.6019/PXD000680. A signal-to-noise cutoff of 10 (where the signal was the fitted intensity for the light isotopic labeling state and the noise was the standard error of the fitted intensity) was used to filter unlabeled phosphopeptides for analysis. This ensured that the signal was high enough to be used to confidently measure the ratio of unlabeled to labeled phosphopeptide. Phosphorylation progress curves (increase in heavy labeled/light labeled phosphopeptide over time) were fit using the nonlinear least squares "SSAsympOrig" function in the R 2.15.0 environment. To test the viability of SILAP as a labeling technique, we incubated asynchronous HEK293 nuclei with [γ-18O4]ATP for varying amounts of time prior to protein digestion, phosphopeptide enrichment, and LC-MS/MS analysis (Fig. 1A). We performed the [γ-18O4]ATP labeling experiments in nucleo because isolated nuclei maintain structural integrity and intranuclear protein concentrations while being permeable to molecules that cannot cross the plasma membrane, such as ATP (35Fischle W. In nucleo enzymatic assays for the identification and characterization of histone modifying activities.Methods. 2005; 36: 362-367Crossref PubMed Scopus (10) Google Scholar). We expected that the endogenous kinases in the isolated nuclei would catalyze the transfer of P18O3 from [γ-18O4]ATP onto the hydroxyl group of substrate residues, producing P18O3-labeled phosphorylation sites that were 6 Da heavier than the unlabeled sites, as well as [β-18O1]ADP (Fig. 1B). Labeling with [γ-18O4]ATP created the expected 6.012-Da shift in the MS1 between new "heavy" P18O3-labeled phosphorylation sites and preexisting "light" phosphorylation sites (Fig. 2A). The heavy and light labeled phosphorylated peptides co-eluted, making the identification and relative quantification of labeled species straightforward (Fig. 2B). We confirmed that P18O3 labeling of phosphorylation sites caused the 6-Da shift by comparing tandem mass spectra for labeled peptides and their unlabeled counterparts. As an example, the phosphorylated b (b3, b7, b8, b9, b15, b16) and y (y16, y17) ions from the MS2 of the heavy-labeled AHNAK S216 peptide had a 6-Da shift in mass relative to an MS2 spectrum for the light species (Fig. 2C). The identification of phosphorylation sites by means of collisionally activated dissociation tandem mass spectrometry is notoriously difficult because of poorer fragmentation, so the further confirmation of heavy phosphorylation at the MS1 level and the residue-specific information at the MS2 level are added advantages of using this method.Fig. 2Confirmation by LC-MS/MS analysis that phosphorylated peptides are labeled by P18O3.A, MS1 spectra for "light" and "heavy" labeled phosphorylated AHNAK (S216). The difference in mass between the heavy and light forms is 6.012 Da, corresponding to the mass difference between phosphorylated peptides with three 18O and with a normal isotope distribution. B, extracted ion chromatograms indicating the heavy and light labeled AHNAK (S216). C, tandem mass spectra for the heavy and light labeled peptides containing AHNAK (S216). A 6-Da shift is present in all phosphorylated fragment ions (labeled in blue for light and red for heavy).View Large Image Figure ViewerDownl
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