Selective Enrichment of Thiophosphorylated Polypeptides as a Tool for the Analysis of Protein Phosphorylation

Abstract

A chemoselective alkylation method is described for the isolation and subsequent identification of thiophosphorylated peptides/proteins. The method involves thiophosphorylation of proteins using adenosine 5′-O-(thiotriphosphate) (ATPγS) followed by selective in situ alkylation of the newly thiophosphorylated proteins resulting in a stable covalent bond. The chemoselective alkylation exploits the relatively high nucleophilicity at low pH of the sulfur in thiophosphate residues, whereas the nucleophilicities of phosphates, amines, and other functionality of amino acids are negligible or significantly suppressed. Modified alkylation reagents linked to biotin or solid supports (e.g. glass or Sepharose beads) with or without a photocleavable linker facilitate the isolation of the thiophosphorylated peptide/proteins. This approach is demonstrated through the localization of phosphorylation sites on myosin regulatory light chain. We anticipate that this technique will be useful for isolation and subsequent identification of newly thiophosphorylated proteins, produced either in vivo or in vitro, thus facilitating the dissection of protein phosphorylation networks. A chemoselective alkylation method is described for the isolation and subsequent identification of thiophosphorylated peptides/proteins. The method involves thiophosphorylation of proteins using adenosine 5′-O-(thiotriphosphate) (ATPγS) followed by selective in situ alkylation of the newly thiophosphorylated proteins resulting in a stable covalent bond. The chemoselective alkylation exploits the relatively high nucleophilicity at low pH of the sulfur in thiophosphate residues, whereas the nucleophilicities of phosphates, amines, and other functionality of amino acids are negligible or significantly suppressed. Modified alkylation reagents linked to biotin or solid supports (e.g. glass or Sepharose beads) with or without a photocleavable linker facilitate the isolation of the thiophosphorylated peptide/proteins. This approach is demonstrated through the localization of phosphorylation sites on myosin regulatory light chain. We anticipate that this technique will be useful for isolation and subsequent identification of newly thiophosphorylated proteins, produced either in vivo or in vitro, thus facilitating the dissection of protein phosphorylation networks. Protein phosphorylation can profoundly affect the function of proteins and is often associated with cellular regulation, inter alia, such as transcription, replication, apoptosis, and signal transduction (1Hunter T. Signaling—2000 and beyond.Cell. 2000; 100: 113-127Google Scholar). Analysis of the human genome reveals that ∼2% of human genes code for protein kinases or protein phosphatases (2Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. Zhang Q. Kodira C.D. Zheng X.H. Chen L. Skupski M. Subramanian G. Thomas P.D. Zhang J. Gabor Miklos G.L. Nelson C. Broder S. Clark A.G. Nadeau J. McKusick V.A. Zinder N. Levine A.J. Roberts R.J. Simon M. Slayman C. Hunkapiller M. Bolanos R. Delcher A. Dew I. Fasulo D. Flanigan M. Florea L. Halpern A. Hannenhalli S. Kravitz S. Levy S. Mobarry C. Reinert K. Remington K. Abu-Threideh J. Beasley E. Biddick K. Bonazzi V. Brandon R. Cargill M. Chandramouliswaran I. Charlab R. Chaturvedi K. Deng Z. Di Francesco V. Dunn P. Eilbeck K. Evangelista C. Gabrielian A.E. Gan W. Ge W. Gong F. Gu Z. Guan P. Heiman T.J. Higgins M.E. Ji R.R. Ke Z. Ketchum K.A. Lai Z. Lei Y. Li Z. Li J. Liang Y. Lin X. Lu F. Merkulov G.V. Milshina N. Moore H.M. Naik A.K. Narayan V.A. Neelam B. Nusskern D. Rusch D.B. Salzberg S. Shao W. Shue B. Sun J. Wang Z. Wang A. Wang X. Wang J. Wei M. Wides R. Xiao C. Yan C. et al.The sequence of the human genome.Science. 2001; 291: 1304-1351Google Scholar). While many kinases are known to be involved in various cellular functions and disease progression, their substrates and phosphorylation sites remain largely unknown. Since characterization of protein phosphorylation networks requires a detailed knowledge of kinase/phosphatase substrates and their specificities as well as localization of phosphorylation sites, efficient techniques for the isolation of newly phosphorylated peptides/proteins would expedite progress in these areas. A variety of methods have been described for the isolation of phosphate-containing peptides/proteins, including metal ion affinity purification (3Ficarro 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: 301-305Google Scholar), tagging phosphorylated residues through chemical reactions (4Oda Y. Nagasu T. Chait B.T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome.Nat. Biotechnol. 2001; 19: 379-382Google Scholar, 5Zhou H. Watts J.D. Aebersold R. A systematic approach to the analysis of protein phosphorylation.Nat. Biotechnol. 2001; 19: 375-378Google Scholar), and affinity purification by immobilized anti-phosphoryl-residue antibodies (6Pandey 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: 179-184Google Scholar). Many of the preceding methods have been used for the identification of protein phosphorylation sites, for the isolation of phosphorylated signal proteins, and for global surveys of protein phosphorylations (3Ficarro 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: 301-305Google Scholar, 4Oda Y. Nagasu T. Chait B.T. Enrichment analysis of phosphorylated proteins as a tool for probing the phosphoproteome.Nat. Biotechnol. 2001; 19: 379-382Google Scholar, 5Zhou H. Watts J.D. Aebersold R. A systematic approach to the analysis of protein phosphorylation.Nat. Biotechnol. 2001; 19: 375-378Google Scholar, 6Pandey 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: 179-184Google Scholar). While powerful, these methods are not able to distinguish newly phosphorylated proteins from steady-state phosphorylated proteins. Since one-third of proteins in a cell are estimated to be phosphorylated (7Hunter T. The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease.Philos. Trans. R Soc. Lond. B Biol. Sci. 1998; 353: 583-605Google Scholar, 8Mann M. Ong S.E. Gronborg M. Steen H. Jensen O.N. Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome.Trends Biotechnol. 2002; 20: 261-268Google Scholar), isolation of newly phosphorylated proteins from unphosphorylated and/or steady-state phosphorylated proteins would simplify the identification of newly phosphorylated proteins responding to diverse signal stimulations. Herein we report a novel method for the isolation of thiophosphorylated peptides/proteins. The method involves phosphorylation of proteins using ATPγS and the selective in situ alkylation of the resultant thiophosphorylated proteins resulting in a stable covalent bond. The alkylation reaction exploits the relatively high nucleophilicity of the phosphoryl sulfur at low pH, whereas the nucleophilicity of phosphates, amines, and other functionality of amino acid residues are negligible or significantly suppressed. The thiophosphate-specific alkylating reagent can be linked to biotin or solid support (e.g. glass or Sepharose beads) with or without a photocleavable linker to facilitate convenient, high yield isolation of phosphorylated peptide/proteins. This approach was demonstrated through the localization of phosphorylation sites in myosin regulatory light chain. In addition, the foregoing technique can be refined and extended to the study of protein phosphorylation networks. The following reagents were obtained from commercial companies and used directly. Casein kinase I, casein kinase I phosphopeptide substrate (CKI 1The abbreviations used are: CKIcasein kinase IBSAbovine serum albuminHPLChigh performance liquid chromatographyMALDImatrix-assisted laser desorption ionizationMS/MSmass spectrometry/mass spectrometryRLCsmooth muscle myosin regulatory light chainTOFtime of flightATPγSadenosine 5′-O-(thiotriphosphate)MOPS4-morpholinepropanesulfonic acidPEO iodoacetyl biotin(+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamineGTPγSguanosine 5′-3-O-(thio)triphosphate. peptide), and casein kinase I buffer were from New England BioLabs (Beverly, MA); synthetic peptide (KKFFCAIS, C-peptide) was from BioSynthesis Co. (Dallas, TX); ATPγS, bovine serum albumin (BSA), potassium chloride, sodium chloride, n-octyl β-d-glucopyranoside, l-cysteine, α-cyano-4-hydroxy-cinnamic acid, and dithiothreitol were from Sigma; ammonium bicarbonate, monobasic sodium phosphate, and dibasic sodium phosphate were from Fisher Scientific; (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO iodoacetyl biotin), ImmunoPure d-biotin, and ImmunoPure immobilized monomeric avidin were from Pierce; trifluoroacetic acid was from Fluka (Buchs, Switzerland); acetic acid was from Aldrich; and HPLC grade solvents were from EM Science (Gibbstown, NJ). Smooth muscle myosin regulatory light chain and myosin light chain kinase were kindly provided by Drs. Kris Kamm and Gang Zhi, Department of Physiology, University of Texas Southwestern Medical Center at Dallas. casein kinase I bovine serum albumin high performance liquid chromatography matrix-assisted laser desorption ionization mass spectrometry/mass spectrometry smooth muscle myosin regulatory light chain time of flight adenosine 5′-O-(thiotriphosphate) 4-morpholinepropanesulfonic acid (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine guanosine 5′-3-O-(thio)triphosphate The thiophosphorylation reaction for CKI substrate peptide (KRRRALpSVASLPGL where pS represents phosphorylated serine) was carried out in 50 μl of CKI reaction buffer containing 200 μm CKI phosphopeptide substrate, 1000 units of CKI, and 4 mm ATPγS at 30 °C for 5 h. Both CKI and CKI reaction buffers were obtained from New England BioLabs. For kinase reactions for smooth muscle myosin regulatory light chain (RLC), 2 μg of RLC were incubated with 0.12 μg of myosin light chain kinase at 30 °C for 2 h in a kinase reaction buffer (50 mm MOPS (pH 7.2), 10 mm MgCl2, 3 μm calmodulin, 10 mm Ca2+, 2.5 mm dithiothreitol, and 1 mm ATPγS). The protein was dissolved in 50 mm NH4HCO3 (pH 8.0) and reduced with 5 mm dithiothreitol at 37 °C for 2 h. The resulting solution was heated at 95 °C for 5 min, cooled at 4 °C for 5 min, and then subjected to proteolytic digestion with sequence grade modified trypsin (Promega Co., Madison, WI) at an enzyme to substrate ratio of 1:50 at 37 °C overnight. One hundred picomoles of PEO iodoacetyl biotin and 30 pmol of peptides (or protein tryptic digest) in 30 μl of reaction buffer at either pH 3.5 (0.1 m sodium acetate buffer) or pH 7.0 (0.1 m potassium phosphate buffer) were incubated at room temperature in the dark for a suitable amount of time. The reaction was quenched by adding 100 mml-cysteine. The buffer pH was adjusted to pH 7.0 by adding 0.5 m phosphate buffer (pH 7.4) when the alkylation reaction was performed at pH 3.5. To the resulting solution were added 3 μl of 5% n-octyl β-d-glucopyranoside and 3 μl of agarose beads immobilized with monomeric avidin, and the mixture was incubated at 4 °C for 30 min. The suspension was collected by centrifugation at 15,000 × g for 3 min, and the supernatant containing unbound peptides was removed. The beads were washed twice with 200 μl of washing buffer A (100 mm KCl, 0.5% n-octyl β-d-glucopyranoside, and 50 mm phosphate buffer, pH 7.4) and briefly with 200 μl of washing buffer B (2 mm NH4HCO3). The biotinylated peptides on avidin beads were either eluted by 10 μl of 10 mmd-biotin phosphate-buffered saline (pH 7.4) solution or used directly for MALDI-TOF mass spectrometric analysis. One microliter of the avidin beads containing biotinylated peptides was mixed with 0.5 μl of acetonitrile and 1 μl of saturated matrix solution (α-cyano-4-hydroxycinnamic acid in water:acetonitrile:trifluoroacetic acid (66:33:1, v/v/v)). The sample was transferred to a MALDI sample plate (Applied Biosystems, Foster City, CA) and dried. Mass spectra were measured on a Voyager DE MALDI-TOF mass spectrometry system (Applied Biosystems) in the positive polarity mode. HPLC/MS/MS analyses were performed in an LCQ DECA ion-trap mass spectrometer (ThermoFinnigan, San Jose, CA) equipped with a nanoelectrospray ionization source. The electrospray source was coupled on line with a Waters CapLC system (Waters, Milford, MA) with MAGIC Variable splitter (Michrom BioResource, Pleasanton, CA). Two microliters of the peptide solution in buffer A (5% acetonitrile, 94.9% water, 0.1% acetic acid (v/v/v)) were manually injected and separated in a capillary HPLC column (50-mm length × 75-μm inner diameter, 5-μm particle size, 300-Å pore diameter) packed in-house with Luna C18 resin (Phenomenex, St. Torrance, CA). The peptides were eluted from the column with a gradient (25–80% buffer B (90% acetonitrile, 9.9% water, 0.1% acetic acid (v/v/v)) in buffer A over 10 min at a flow rate of about 1 μl/min; the eluted peptides were electrosprayed directly into the LCQ mass spectrometer. The MS/MS spectra were acquired in a data-dependent mode, which determined the masses of the parent ions and fragments of the strongest ion. Phosphorothioic acid has three apparent proton dissociation constants: pK1 < 2; pK2 = 5.75; pK3 = 10.4 (9Neumann H. Steinberg I.Z. Katchalski E. Reducing properties of phosphorothioate.J. Am. Chem. Soc. 1965; 87: 3841-3848Google Scholar). We reasoned that pK1 for the phosphorothioic acid in the thiophosphorylated residue is similar to that of the free phosphorothioic acid, i.e. ≤3.0. Therefore, when the solution pH is adjusted to below 4.0, a significant proportion of the phosphorothioic acid on thiophosphoresidues still exists in ionized form, while the side chains of all the basic amino residues (e.g. Arg and Lys) and the amine at the N terminus are protonated, while that of cysteine residue is neutralized. The protonated N-terminal amine and protonated side chains of basic residues are non-nucleophilic. Neutral cysteine thiols still retain some nucleophilicity; their reactivity is significantly lower than that of the anionic sulfur on thiophosphorylated residues. These differences allow chemoselective alkylation of thiophosphorylated residues in the presence of other amino acid residues in natural state (Fig. 1). To test the feasibility of chemoselective alkylation of thiophosphorylated peptides and subsequent enrichment, two model peptides were alkylated: the first peptide (KKFFCAIS, or C-peptide) containing cysteine and lysine residues and the second one (the thiophosphorylated CKI peptide, KRRRAL(pS)VA(p-SS)LPGL, or T-peptide) including thiophosphorylated serine (p-SS), phosphorylated serine (pS), arginine, and lysine residues. The T-peptide was produced by thiophosphorylation of the CKI peptide in the presence of ATPγS. Iodoacetamide was used as the alkylation agent to survey the effect of pH on selectivity. At pH 7.0, both peptides were 100% alkylated after 1 h of alkylation reaction (Fig. 2C and D, ). The alkylated peptide has a mass increase of 57 Da. However, when the pH was decreased to 3.5, a negligible amount of C-peptide was alkylated, while >70% of the T-peptide was modified (Fig. 2). MS/MS experiments showed that the alkylation reaction occurred at the sulfur atom in the cysteine residue for C-peptide or in the thiophosphorylated residue for T-peptide (data not shown). These results confirm that at low pH, the alkylation favors the thiophosphorylated residue. Next we used PEO iodoacetyl biotin to alkylate and simultaneously tag the thiophosphorylated peptide. PEO iodoacetyl biotin contains an iodoacetamidyl moiety that has an electrophilic property similar to that of iodoacetamide. Alkylation using this reagent linked a water-soluble biotin to the thiophosphorylated residue, allowing affinity enrichment by solid beads containing immobilized monomeric avidin. Non-alkylated peptides present in solution were washed away. The enriched biotinylated peptides were mixed with α-cyano-4-hydroxycinnamic acid matrix solution and loaded onto the sample plate for MALDI-TOF mass spectrometric analysis. Alternatively the enriched peptides were eluted by 10 mmd-biotin phosphate-buffered saline solution, and the masses of the eluted peptides were determined. When the alkylation reaction was carried out at pH 7.0, both the C-peptide and T-peptide (Fig. 3A) were biotinylated and subsequently affinity-enriched (Fig. 3B). On the other hand, when the reaction was carried out at pH 3.5, only the T-peptide was biotinylated and affinity-enriched afterward, while the C-peptide was not (Fig. 3C). Thus, chemoselective biotinylation of the thiophosphorylated peptide allows selective enrichment of the thiophosphorylated peptides. To further demonstrate the utility of the approach for the isolation of thiophosphorylated peptides, we mixed the T-peptide with BSA tryptic digest. BSA was selected due to its high number of cysteine residues (total, 25), which have low nucleophilicity and might potentially with the chemoselective We mixed 30 pmol of T-peptide with 30 pmol of BSA tryptic peptides (Fig. and the resulting peptide mixture was subjected to biotinylation reaction at pH 3.5 and affinity enrichment as described T-peptide was affinity-enriched (Fig. while all the BSA tryptic peptides were washed the affinity This to the that the alkylating agent used in this study at low pH with of peptides. Next we the utility of this technique for protein phosphorylation RLC was thiophosphorylated by in kinase reaction in the presence of ATPγS and myosin light chain peptides of RLC were produced by digestion of the protein with trypsin (Fig. Alkylation and subsequent affinity enrichment in the isolation of a biotinylated peptide with a mass of which the mass of a peptide from residue to residue of The of biotinylated was used to HPLC/MS/MS analysis for the of of unphosphorylated and thiophosphorylated the thiophosphorylation occurred at either or serine residue in RLC of ion with a mass of the thiophosphorylation occurred at serine J. of in an ion mass Am. Soc. Mass 1998; Scholar). ion mass spectrometric analysis of the tryptic only one peptide not shown). are with the report that showed that serine in RLC was phosphorylated by myosin light chain kinase J.C. C.A. Kamm phosphorylated in myosin light chain in smooth Biol. Chem. Scholar, M. M. H. of human is associated with protein kinase phosphorylation of myosin light Scholar). are a in the described method for the isolation of thiophosphorylated proteins. technique is able to distinguish newly thiophosphorylated proteins steady-state phosphorylated proteins. While isolation of thiophosphorylated peptides was described in this method can be extended to the isolation of thiophosphorylated proteins and subsequent identification of the proteins. is estimated that about one-third of proteins in the cell are phosphorylated (7Hunter T. The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease.Philos. Trans. R Soc. Lond. B Biol. Sci. 1998; 353: 583-605Google Scholar, 8Mann M. Ong S.E. Gronborg M. Steen H. Jensen O.N. Pandey A. Analysis of protein phosphorylation using mass spectrometry: deciphering the phosphoproteome.Trends Biotechnol. 2002; 20: 261-268Google Scholar). of newly phosphorylated proteins from the steady-state phosphorylated proteins and other proteins in will the of the protein subsequent protein identification and analysis In addition, the technique is The isolation of thiophosphorylated proteins on the alkylation reaction thiophosphorylated residues and an alkylation as the thiophosphate is to solution, can be to their and anionic the phosphorylated residues are of proteins and for The described technique a method for protein phosphorylation of protein phosphorylation sites is carried out in a mass spectrometry and requires in The described method allows the isolation of thiophosphorylated peptides only a of the proteins are Enrichment of will subsequent mass spectrometric analysis and In addition, the of is to its the thiophosphorylation has been used in to study protein phosphorylation when the phosphorylated state is to be Since the is separated from the in a in is to the substrate specificities of other than ATPγS have been used to study protein phosphorylation and kinase substrates K. A chemical for substrates reveals of a signaling Biol. 2002; Scholar). We that some kinases might ATPγS than However, showed that of the for in kinase reactions (e.g. adding metal phosphorylation ion and into the of protein kinase Scholar). technique can potentially be used to in vivo thiophosphorylated proteins. The of such a technique the isolation of newly phosphorylated proteins responding to in cellular The proteins, diverse signal will be by a approach and further by mass protein thiophosphorylation was demonstrated in by and In vivo thiophosphorylation of proteins. and analysis of and Biol. Chem. Scholar, purification of newly phosphorylated protein and of and and the high protein using adenosine and protein Biol. Chem. Scholar). showed that after were with sodium for 3 h ATPγS, and thiophosphorylated proteins were While these experiments further for the results the system is able to to ATPγS and subsequently thiophosphorylation in Alternatively ATPγS can be directly into that are by Dunn P.T. in Protein New Scholar, G. D. D. of is not by 2001; Scholar, by ATPγS from a cell line Scholar). Many signal transduction remain after the are with cell G. D. D. of is not by 2001; Scholar, by ATPγS from a cell line Scholar). In we a method for the isolation of newly thiophosphorylated peptides/proteins. The method a tool for protein phosphorylation sites as as a of the proteins are The technique can potentially be to the isolation of newly thiophosphorylated proteins, produced either in vivo or in vitro, thus facilitating of protein phosphorylation We Drs. Kris and for various and We are to Drs. Kris Kamm and Gang for some reagents used in the