Profiling Protein Methylation with Cofactor Analog Containing Terminal Alkyne Functionality

Gil Blum1, Ian R. Bothwell1, Kabirul Islam2, Minkui Luo2

1 Tri‐Institutional Training Program in Chemical Biology, Memorial Sloan‐Kettering Cancer Center, New York, New York, 2 Molecular Pharmacology and Chemistry Program, Memorial Sloan‐Kettering Cancer Center, New York, New York
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120241
Online Posting Date:  March, 2013
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Enzymatic transmethylation from the cofactor S‐adenosyl‐L‐methionine (SAM) to biological molecules has recently garnered increased attention because of the diversity of possible substrates and implications in normal biology and diseases. To reveal the substrates of protein methyltransferases (PMTs), the present article focuses on an alkyne‐containing SAM mimic, Se‐adenosyl‐L‐selenomethionine (ProSeAM), and a cleavable azido‐azo‐biotin probe to profile the targets of endogenous PMTs in cellular contexts. This article describes the stepwise preparation of cell lysates containing active, endogenous PMTs and subsequent target labeling with ProSeAM. The article continues with the enrichment of the ProSeAM‐labeled proteins with the azido‐azo biotin probe as a pulldown reagent and the subsequent reductive elution with sodium dithionate for proteomic analysis. The protocols provided here were formulated for ProSeAM as a profiling reagent but can be applied to other terminal‐alkyne‐containing SAM analog cofactors. Curr. Protoc. Chem. Biol. 5:67‐88 © 2013 by John Wiley & Sons, Inc.

Keywords: Se‐adenosyl‐L‐selenomethionine; ProSeAM; azido‐azo biotin probe; click chemistry

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Labeling Substrates of Endogenous Protein Methyltransferases with ProSeAM
  • Basic Protocol 2: Target Pulldown and Enrichment
  • Support Protocol 1: Synthesis of Se‐Adenosyl‐L‐Selenomethionine (ProSeAM) and Precursors
  • Support Protocol 2: Proteome‐Wide Target Visualization Using In‐Gel Fluorescence
  • Support Protocol 3: Protein Precipitation with Methanol/Chloroform/Water
  • Support Protocol 4: Protein Precipitation with Methanol
  • Support Protocol 5: Large‐Scale Synthesis of Azido‐Azo‐Biotin Probe
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Labeling Substrates of Endogenous Protein Methyltransferases with ProSeAM

  Materials
  • Human embryonic kidney (HEK) 293T cells (ATCC, cat. no. CRL‐11268)
  • Dulbecco's modified Eagle medium (DMEM, Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS)
  • 1.5 mM adenosine‐2′,3′‐dialdehyde (Adox; Sigma, cat. no. A7154) in deionized, distilled H 2O
  • Phosphate‐buffered saline (PBS; Invitrogen, cat. no. 10010‐031)
  • Tris‐HCl buffer (see recipe)
  • 5′‐methylthioadenosine/S‐adenosylhomocesteine nucleosidase (MTAN; Ibanez et al., ; Wang et al., )
  • S‐adenosyl‐homocysteine (SAH; Sigma, cat. no. A9384)
  • Bradford assay reagent (Bio‐Rad; also see )
  • 8 mM ProSeAM in 0.01% (v/v) trifluoroacetic acid in distilled, deionized H 2O; the acidity of the storage solution is important for ProSeAM's stability (with this buffer, this material can be stored at –80°C for <2 months; see protocol 3 for ProSeAM synthesis)
  • 150‐cm2 (T‐150) cell culture flasks (BD Falcon)
  • Refrigerated centrifuge with 4°C, 3000 × g capacity
  • Sonicator (Misonix Ultrasonic Liquid Processor)
  • Refrigerated high‐speed centrifuge with 4°C, 21,000 × g capacity
  • Additional reagents and equipment for determining protein concentration (Simonian and Smith, ) and protein precipitation ( protocol 6)

Basic Protocol 2: Target Pulldown and Enrichment

  Materials
  • 10 mg air‐dried protein pellet from the whole‐cell lysates of 2 × 107 cells labeled with ProSeAM ( protocol 1).
  • 4% (w/v) SDS buffer supplemented with EDTA‐free Roche protease inhibitors (see recipe), with and without 10 mM disodium EDTA, ice cold
  • 5 mM azido‐azo‐biotin in DMSO (see Strategic Planning and protocol 7)
  • 50 mM and 200 mM tris(2‐carboxyethyl)phosphine (TCEP; Sigma‐Aldrich, cat. no. C4706), freshly prepared
  • 2 mM Tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA; Sigma‐Aldrich, cat. no. 678937) in DMSO, freshly prepared
  • 50 mM CuSO 4, freshly prepared
  • Dilution buffer (see recipe)
  • Streptavidin‐Sepharose beads (GE Healthcare, cat. no. 17‐5113‐01)
  • Phosphate‐buffered saline (PBS; Bio‐Rad, cat. no. 161‐0780), ice cold
  • PBS containing 0.2% (w/v) sodium dodecyl sulfate (SDS)
  • ABC buffer (250 mM ammonium bicarbonate), ice cold
  • Reduction buffer (see recipe)
  • 8 M urea
  • 400 mM iodoacetamide, freshly prepared
  • Elution buffer (see recipe)
  • 0.1% (w/v) sodium dodecyl sulfate (SDS) in ABC buffer (250 mM ammonium bicarbonate)
  • 1% (w/v) SDS in H 2O supplemented with 75 mM 2‐mercaptoethanol
  • Liquid N 2
  • 12‐well 4% to 12% Tris‐HCl protein gel (Criterion XT Precast Gel, Bio‐Rad)
  • 1× loading buffer (see recipe)
  • Fluorescent protein molecular weight ladder (15 to 250 kDa, Bio‐Rad)
  • 1× MOPS electrophoresis buffer (Bio‐Rad)
  • 50‐ml conical polypropylene tubes (e.g., BD Falcon)
  • Refrigerated centrifuge with 4°C, 3000 × g capacity
  • End‐over‐end rotator
  • 2‐ml Dolphin microcentrifuge tubes (Sigma‐Aldrich)
  • Amicon ultra 3K 2‐ml centrifugal filter unit (Millipore)
  • Lyophilizer
  • 100°C heating block
  • SDS‐PAGE electrophoresis gel box for Criterion XT Precast Gel (Bio‐Rad)
  • Additional reagents and equipment for protein precipitation ( protocol 6), SDS‐PAGE (Gallagher, ), staining of gels (Sasse and Gallagher, ), and proteomic analysis (Bothwell et al., )

Support Protocol 1: Synthesis of Se‐Adenosyl‐L‐Selenomethionine (ProSeAM) and Precursors

  Materials
  • For selenohomocystine synthesis:
    • Selenomethionine (Sigma‐Aldrich)
    • Liquid ammonia (Matheson Gas Products, cat. no. G1501240)
    • N 2 source
    • Sodium metal
    • 1 M HCl
    • Dowex50WX8 ion‐exchange resin (Sigma‐Aldrich)
    • Eluting solvent: 1:4 (v/v) NH 4OH:H 2O
  • For 2′,3′‐O‐Isopropylidene‐5′‐O‐p‐toluenesulfonyl adenosine synthesis:
    • 2′,3′‐isopropylidene adenosine (Sigma‐Aldrich)
    • Anhydrous pyridine
    • p‐toluenesulfonyl chloride (Sigma‐Aldrich, cat. no. 674583)
    • Ice‐cold saturated NaHCO 3
    • Ice‐cold chloroform
    • Ice‐cold saturated NaHSO 4
    • Hexane
  • For Se‐(5′‐deoxyadenosine‐5′)‐L‐selenohomocysteine (SeAH) synthesis:
    • Selenohomocystine (Sigma‐Aldrich, cat. no. S3132)
    • 100% ethanol
    • Sodium borohydride
    • 2′,3′‐O‐isopropylidene‐5′‐Op‐toluenesulfonyl adenosine
    • 1 M HCl
    • 1.0 M KOH
    • Dichloromethane
    • 1.0 M H 3PO 4
    • Saturated Ba(OH) 2
    • Acetonitrile
    • 0.1% trifluoroacetic acid (TFA), aqueous
  • For ProSeAM synthesis:
    • Formic acid
    • Acetic acid
    • Propargyl bromide (Fluka Analytical, cat. no. 81831; 80% v/v in toluene)
    • Silver perchlorate (AgClO 4; Sigma‐Aldrich, cat. no. 674583)
    • 0.1% trifluoroacetic acid (TFA) in H 2O
    • Ice‐cold diethyl ether
    • Acetonitrile
    • 0.1% trifluoroacetic acid (TFA), aqueous
  • Separatory funnels
  • Vacuum desiccators
  • Ordinary vacuum source
  • High‐vacuum oil pump
  • Preparative reversed‐phase HPLC column (XBridge Prep C18 5 µm OBD 19 × 150 mm; Waters): for preparation of SeAH and ProSeAM
  • Lyophilizer
  • 0.2‐µm syringe filter (Nalgene): for preparation of ProSeAM
  • Spectrophotometer
  • pHydrion Vivid 1‐11 pH paper

Support Protocol 2: Proteome‐Wide Target Visualization Using In‐Gel Fluorescence

  Materials
  • 50 µg pellet of ProSeAM‐treated cell lysates from protocol 1
  • 4% (w/v) SDS buffer supplemented with EDTA‐free Roche protease inhibitors (see recipe), with and without 10 mM disodium EDTA, ice cold
  • 4% (w/v) SDS buffer supplemented with EDTA‐free Roche protease inhibitors (see recipe), with and without 10 mM disodium EDTA, ice cold
  • 5 mM stock of TAMRA‐Azide (Invitrogen, cat. no. T10182) in DMSO
  • 5 mM tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA) in DMSO
  • 100 mM tris(2‐carboxyethyl)phosphine (TCEP) in distilled, deionized H 2O, freshly prepared
  • 100 mM CuSO 4, freshly prepared
  • 5:2:3 (v/v/v) methanol:chloroform:H 2O
  • 1× loading buffer (see recipe)
  • Fluorescent protein molecular weight ladder (15 to 250 kDa, Bio‐Rad)
  • 1× MOPS electrophoresis buffer (Bio‐Rad)
  • 12‐well 4% to 12% Tris‐HCl protein gel (Criterion XT Precast Gel, Bio‐Rad)
  • Fluorescent gel destaining solution: 4:1:5 (v/v/v) methanol/acetic acid/H 2O
  • Coomassie Blue staining reagent (Bio‐Rad; also see Sasse and Gallagher, )
  • Centrifuge
  • 70°C heating block
  • SDS‐PAGE electrophoresis gel box for Criterion XT Precast Gel (Bio‐Rad)
  • Fluorescence gel scanner equipped with the excitation wavelength at 532 nm, <580 nm cut‐off filter and 30 nm band‐pass (Amersham Biosciences Typhoon 9400)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and staining of gels (Sasse and Gallagher, )

Support Protocol 3: Protein Precipitation with Methanol/Chloroform/Water

  Materials
  • 5‐ml cell lysates (protein concentration, 2 mg/ml) in 50‐ml Falcon tubes ( protocol 2)
  • Ice‐cold methanol
  • Ice‐cold chloroform
  • Ice‐cold distilled, deionized water
  • Refrigerated centrifuge with 4°C, 3000 × g capacity

Support Protocol 4: Protein Precipitation with Methanol

  Materials
  • Ice‐cold methanol
  • 5‐ml cell lysates (protein concentration, 2 mg/ml) in 50‐ml Falcon tubes ( protocol 2)
  • Refrigerated centrifuge with 4°C, 3000 × g capacity

Support Protocol 5: Large‐Scale Synthesis of Azido‐Azo‐Biotin Probe

  Materials
  • Sodium nitrite (NaNO 2; Sigma)
  • Acetonitrile (Fisher)
  • Trifluoromethanesulfonic anhydride (Tf 2O, Sigma)
  • 3:7 (v/v) H 2O:acetonitrile
  • Tyramine (Sigma)
  • ZnCl 2 (Sigma)
  • Triethylamine (Sigma)
  • Ethyl acetate (EtOAc, Sigma)
  • MgSO 4, anhydrous (Sigma)
  • Silica gel (Sigma) for flash chromatography (also see Meyers, )
  • 4‐aminobenzoic acid (Sigma)
  • 6 M HCl, ice cold
  • Sodium bicarbonate (NaHCO 3)
  • THF (Sigma), dry
  • Hexane (Sigma)
  • Methanol (MeOH; Sigma)
  • Dichloromethane (DCM)
  • Argon source
  • N,N′‐dicyclohexylcarbodiimide (DCC; Sigma)
  • N‐hydroxylsuccinimide (Sigma)
  • Biotin‐PEG‐NH 2 (Huang et al., )
  • Brine (saturated NaCl)
  • Sodium sulfate, anhydrous
  • 50‐ and 100‐ml round‐bottom flasks
  • Magnetic stirrer and Teflon‐coated stir bar
  • Separatory funnels
  • Rotary evaporator
  • Vacuum pump
  • Additional reagents and equipment for flash chromatography (Meyers, )
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Beroukhim, R., Mermel, C.H., Porter, D., Wei, G., Raychaudhuri, S., Donovan, J., Barretina, J., Boehm, J.S., Dobson, J., Urashima, M., Mc Henry, K.T., Pinchback, R.M., Ligon, A.H., Cho, Y.J., Haery, L., Greulich, H., Reich, M., Winckler, W., Lawrence, M.S., Weir, B.A., Tanaka, K.E., Chiang, D.Y., Bass, A.J., Loo, A., Hoffman, C., Prensner, J., Liefeld, T., Gao, Q., Yecies, D., Signoretti, S., Maher, E., Kaye, F.J., Sasaki, H., Tepper, J.E., Fletcher, J.A., Tabernero, J., Baselga, J., Tsao, M.S., Demichelis, F., Rubin, M.A., Janne, P.A., Daly, M.J., Nucera, C., Levine, R.L., Ebert, B.L., Gabriel, S., Rustgi, A.K., Antonescu, C.R., Ladanyi, M., Letai, A., Garraway, L.A., Loda, M., Beer, D.G., True, L.D., Okamoto, A., Pomeroy, S.L., Singer, S., Golub, T.R., Lander, E.S., Getz, G., Sellers, W.R., and Meyerson, M. 2010. The landscape of somatic copy‐number alteration across human cancers. Nature 463:899‐905.
   Best, M.D. 2009. Click chemistry and bioorthogonal reactions: Unprecedented selectivity in the labeling of biological molecules. Biochemistry 48:6571‐6584.
   Binda, O., Boyce, M., Rush, J.S., Palaniappan, K.K., Bertozzi, C.R., and Gozani, O. 2011. A chemical method for labeling lysine methyltransferase substrates. Chembiochem 12:330‐334.
   Blum, G. Islam, K., and Luo, M. 2013. Bioorthogonal profiling of protein methylation (BPPM) using an azido analog of S‐adenosyl‐L‐methionine. Curr. Protoc. Chem. Biol. 5:45‐66.
   Bothwell, I.R., Islam, K., Chen, Y., Zheng, W., Blum, G., Deng, H., and Luo, M. 2012. Se‐adenosyl‐L‐selenomethionine cofactor analogue as a reporter of protein methylation. J. Am. Chem. Soc. 134:14905‐14912.
   Charron, G., Wilson, J., and Hang, H.C. 2009. Chemical tools for understanding protein lipidation in eukaryotes. Curr. Opin. Chem. Biol. 13:382‐391.
   Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Mol. Biol. 97:10.2A.1‐10.2A.44.
   Herrmann, F., Lee, J., Bedford, M.T., and Fackelmayer, F.O. 2005. Dynamics of human protein arginine methyltransferase 1(PRMT1) in vivo. J. Biol. Chem. 280:38005‐38010.
   Huang, Z., Park, J. I., Watson, D.S., Hwang, P., and Szoka, F.C. Jr. 2006. Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications. Bioconjugate Chem. 17:1592‐1600.
   Ibanez, G., McBean, J.L., Astudillo, Y.M., and Luo, M. 2010. An enzyme‐coupled ultrasensitive luminescence assay for protein methyltransferases. Anal. Biochem. 401:203‐210.
   Ibanez, G., Shum, D., Blum, G., Bhinder, B., Radu, C., Antczak, C., Luo, M., and Djaballah, H. 2012. A high throughput scintillation proximity imaging assay for protein methyltransferases. Comb. Chem. High. Throughput Screen. 15:359‐371.
   Islam, K., Zheng, W., Yu, H., Deng, H., and Luo, M. 2011. Expanding cofactor repertoire of protein lysine methyltransferase for substrate labeling. ACS Chem. Biol. 6:679‐684.
   Islam, K., Bothwell, I., Chen, Y., Sengelaub, C., Wang, R., Deng, H., and Luo, M. 2012. Bioorthogonal profiling of protein methylation using azido derivative of S‐adenosyl‐L‐methionine. J. Am. Chem. Soc. 134:5909‐5915.
   Levy, D., Liu, C.L., Yang, Z., Newman, A.M., Alizadeh, A.A., Utz, P.J., and Gozani, O. 2011. A proteomic approach for the identification of novel lysine methyltransferase substrates. Epigenetics Chromatin 4:19.
   Luo, M. 2012. Current chemical biology approaches to interrogate protein methyltransferases. ACS Chem. Biol. 7:443‐463.
   Meyers, C.L.F. 2000. Column chromatography. Curr. Protoc. Nucl. Acid Chem. 3:A.3E.1‐A.3E.7.
   Peters, W., Willnow, S., Duisken, M., Kleine, H., Macherey, T., Duncan, K.E., Litchfield, D.W., Luscher, B., and Weinhold, E. 2010. Enzymatic site‐specific functionalization of protein methyltransferase substrates with alkynes for click labeling. Angew. Chem. Int. Ed. Engl. 49:5170‐5173.
   Prescher, J.A. and Bertozzi, C.R. 2006. Chemical technologies for probing glycans. Cell 126:851‐854.
   Rathert, P., Dhayalan, A., Ma, H., and Jeltsch, A. 2008a. Specificity of protein lysine methyltransferases and methods for detection of lysine methylation of non‐histone proteins. Mol. Biosyst. 4:1186‐1190.
   Rathert, P., Dhayalan, A., Murakami, M., Zhang, X., Tamas, R., Jurkowska, R., Komatsu, Y., Shinkai, Y., Cheng, X., and Jeltsch, A. 2008b. Protein lysine methyltransferase G9a acts on non‐histone targets. Nat. Chem. Biol. 4:344‐346.
   Sasse, J. and Gallagher, S.R. 2009. Staining proteins in gels. Curr. Protoc. Mol. Biol. 85:10.6.1‐10.6.27.
   Simonian, M.H. and Smith, J.A. 2006. Spectrophotometric and colorimetric determination of protein concentration. Curr. Protoc. Mol. Biol. 76:10.1.1‐10.1A.9.
   Szychowski, J., Mahdavi, A., Hodas, J.J., Bagert, J.D., Ngo, J.T., Landgraf, P., Dieterich, D.C., Schuman, E.M., and Tirrell, D.A. 2010. Cleavable biotin probes for labeling of biomolecules via azide‐alkyne cycloaddition. J. Am. Chem. Soc. 132:18351‐18360.
   Van Der Werf, P. and Koshland, D.E. Jr. 1977. Identification of a gamma‐glutamyl methyl ester in bacterial membrane protein involved in chemotaxis. J. Biol. Chem. 252:2793‐2795.
   Vedadi, M., Barsyte‐Lovejoy, D., Liu, F., Rival‐Gervier, S., Allali‐Hassani, A., Labrie, V., Wigle, T.J., Dimaggio, P.A., Wasney, G.A., Siarheyeva, A., Dong, A., Tempel, W., Wang, S.C., Chen, X., Chau, I., Mangano, T.J., Huang, X.P., Simpson, C.D., Pattenden, S.G., Norris, J.L., Kireev, D.B., Tripathy, A., Edwards, A., Roth, B.L., Janzen, W.P., Garcia, B.A., Petronis, A., Ellis, J., Brown, P.J., Frye, S.V., Arrowsmith, C.H., and Jin, J. 2011. A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nat. Chem. Biol. 7:566‐574.
   Walsh, C.T., Garneau‐Tsodikova, S., and Gatto, G.J. Jr. 2005. Protein posttranslational modifications: The chemistry of proteome diversifications. Angew. Chem. Int. Ed. Engl. 44:7342‐7372.
   Wang, R., Ibanez, G., Islam, K., Zheng, W., Blum, G., Sengelaub, C., and Luo, M. 2011a. Formulating a fluorogenic assay to evaluate S‐adenosyl‐L‐methionine analogues as protein methyltransferase cofactors. Mol. Biosyst. 7:2970‐2981.
   Wang, R., Zheng, W., Yu, H., Deng, H., and Luo, M. 2011b. Labeling substrates of protein arginine methyltransferase with engineered enzymes and matched S‐adenosyl‐L‐methionine analogues. J. Am. Chem. Soc. 133:7648‐7651.
   Willnow, S., Martin, M., Luscher, B., and Weinhold, E. 2012. A selenium‐based click AdoMet analogue for versatile substrate labeling with wild‐type protein methyltransferases. Chembiochem 13:1167‐1173.
   Yang, Y.Y., Ascano, J.M., and Hang, H.C. 2010. Bioorthogonal chemical reporters for monitoring protein acetylation. J. Am. Chem. Soc. 132:3640‐3641.
   Yount, J.S., Moltedo, B., Yang, Y.Y., Charron, G., Moran, T.M., Lopez, C.B., and Hang, H.C. 2010. Palmitoylome profiling reveals S‐palmitoylation‐dependent antiviral activity of IFITM3. Nat. Chem. Biol. 6:610‐614.
   Zhang, L., Ding, X., Cui, J., Xu, H., Chen, J., Gong, Y.N., Hu, L., Zhou, Y., Ge, J., Lu, Q., Liu, L., Chen, S., and Shao, F. 2012. Cysteine methylation disrupts ubiquitin‐chain sensing in NF‐kappaB activation. Nature 481:204‐208.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library