Bioorthogonal Profiling of Protein Methylation (BPPM) Using an Azido Analog of S‐Adenosyl‐L‐Methionine

Gil Blum1, 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.ch120240
Online Posting Date:  March, 2013
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Abstract

Protein methyltransferases (PMTs) utilize S‐adenosyl‐L‐methionine (SAM) as a cofactor and transfer its sulfonium methyl moiety to diverse substrates. These methylation events can lead to meaningful biological outcomes, from transcriptional activation/silencing to cell cycle regulation. This article describes recently developed technology based on protein engineering in tandem with SAM analog cofactors and bioorthogonal click chemistry to unambiguously profile the substrates of a specific PMT. The protocols encapsulate the logic and methods of selectively profiling the substrates of a candidate PMT by (1) engineering the selected PMT to accommodate a bulky SAM analog; (2) generating a proteome containing the engineered PMT; (3) visualizing the proteome‐wide substrates of the designated PMT via bioorthogonal labeling with a fluorescent tag; and finally (4) pulling down the proteome‐wide substrates for mass spectrometric analysis. Curr. Protoc. Chem. Biol. 5:45‐66 © 2013 by John Wiley & Sons, Inc.

Keywords: G9a; protein engineering; synthetic cofactor; click chemistry

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Identification of Active G9a Mutants by Mass Spectrometry
  • Support Protocol 1: Synthesis, Purification, and Characterization of Ab‐SAM
  • Support Protocol 2: Expression and Purification of Native and Variant G9a Protein Methyltransferases
  • Basic Protocol 2: Preparation of G9a Variant Cell Lysates
  • Basic Protocol 3: Proteome‐Wide BPPM Using In‐Gel Fluorescence
  • Basic Protocol 4: Proteome‐Wide BPPM Using Proteome Pulldown for MS Analysis
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Identification of Active G9a Mutants by Mass Spectrometry

  Materials
  • 50 mM Tris‐Cl buffer, pH 8.0 (see recipe)
  • 100 µM H3K9 peptide substrate (aa 1‐21, ARTKQTARKSTGGKAPRKQLA) in 0.1% (v/v) aqueous trifluoroacetic acid (TFA) (synthesized according to standard solid‐phase peptide synthesis methods; can be stored for >6 months at −80°C)
  • 1 mM Ab‐SAM in 0.01% (v/v) TFA (see protocol 2)
  • 1 mM S‐adenosyl‐L‐methionine (SAM, Sigma) in 0.01% (v/v) TFA (store up to 3 months at −80°C)
  • G9a stock solutions (see protocol 3): exogenously expressed and purified native G9a (aa 913‐1193) and corresponding variants (Y1067A, Y1085A, R1109A, F1152A, Y1154A, and F1158A)
  • Saturated α‐cyano‐hydroxycinnamic acid (Protea Biosciences) in 1:1 (v/v) acetonitrile/DD water
  • 1.5‐ml microcentrifuge tubes
  • C18 cartridge pipet tips (Waters; optional)
  • MALDI sample plate (ABSciex)
  • MALDI‐TOF mass spectrometer (Voyager‐DE STR, Applied Biosystems, with 2.0‐m flight tube)

Support Protocol 1: Synthesis, Purification, and Characterization of Ab‐SAM

  Materials
  • (E)‐1,4‐Dibromobut‐2‐ene
  • Tetrahydrofuran (THF)
  • Sodium azide
  • Deionized distilled (DD) water
  • Diethyl ether
  • Saturated NaCl solution
  • Anhydrous Na 2SO 4
  • Silica gel (pore size 60 Å, 70‐230 mesh, 63‐200 µm; Sigma‐Aldrich)
  • Petroleum ether
  • Dichloromethane (DCM)
  • S‐Adenosyl‐L‐homocystine (SAH)
  • Formic acid
  • Acetic acid
  • AgClO 4
  • Acetonitrile
  • 0.01% (v/v) aqueous trifluoroacetic acid (TFA)
  • Diethyl ether
  • 25‐ml round‐bottom flask
  • Glass chromatography column (Chemglass, cat. no. CG‐1196‐03)
  • 4‐ml glass vial with cap
  • Analytical reversed‐phase HPLC column (e.g., XBridge C18, 5 µm, 4.6 × 150 mm)
  • 0.2‐µm syringe filter (Nalgene)
  • Preparative reversed‐phase HPLC column (e.g., XBridge Prep C18, 5 µm OBD, 19×× 150 mm)
  • SpeedVac evaporator
  • Lyophilizer
  • UV spectrophotometer
  • Additional reagents and equipment for silica gel column chromatography and reversed‐phase HPLC

Support Protocol 2: Expression and Purification of Native and Variant G9a Protein Methyltransferases

  Materials
  • pET28a‐LIC vector containing N‐terminal His6‐tagged methyltransferase SET domain of human G9a, aa 913‐1193 (Dr. Jingrong Min, University of Toronto, jr.min@utoronto.ca)
  • QuikChange site‐directed mutagenesis system (Stratagene)
  • E. coli Rosetta‐2(DE3) strain (Novagen)
  • pET28a‐LIC vector (Novagen)
  • ZnSO 4
  • 0.5 mM isopropyl β‐D‐1‐thiogalactopyranoside (IPTG)
  • Ni‐NTA agarose resin (Qiagen)
  • Superdex‐75 exclusion chromatography gel (GE Healthcare)
  • 25 mM Tris‐Cl, pH 8, containing 200 mM NaCl and 15% (v/v) glycerol
  • Amicon Ultra‐15 centrifugal filter (Millipore)

Basic Protocol 2: Preparation of G9a Variant Cell Lysates

  Materials
  • Human embryonic kidney 293T (HEK293T) cells
  • Dulbecco's modified Eagle medium (DMEM, GIBCO) with 10% (v/v) fetal calf serum (FCS)
  • Lipofectamine 2000 (Invitrogen)
  • Opti‐MEM reduced‐serum medium (GIBCO)
  • pCDNA3‐FLAG plasmid vector carrying engineered full‐length G9a variant (Islam et al., ) or mock pCDNA3 plasmid
  • 1.5 mM adenosine‐2′,3′‐dialdehyde (Adox, Sigma, A7154) in deionized distilled (DD) water
  • 1× phosphate‐buffered saline (PBS, GIBCO)
  • Modified RIPA lysis buffer (see recipe)
  • 50 mM Tris‐Cl buffer (see recipe)
  • T‐25 or T‐75 cell culture flask (353108 or 353136, BD Falcon)
  • 37°C, 5% CO 2 cell culture incubator
  • 1.5‐ml microcentrifuge tubes
  • Sonicator (e.g., Misonix Ultrasonic Liquid Processor)
  • Refrigerated high speed centrifuge
  • 2‐ or 4‐ml detergent removal spin columns (Pierce, cat. no. 87778 or 87779)
  • Additional reagents and equipment for Bradford protein assays (e.g., Bio‐Rad) and western blot analysis (Gallagher et al., )

Basic Protocol 3: Proteome‐Wide BPPM Using In‐Gel Fluorescence

  Materials
  • 2 mg/ml cell lysate, freshly prepared (from 2 × 106 cells in a T‐25 flask, see protocol 4)
  • 8 mM Ab‐SAM in 0.01% (v/v) TFA (see protocol 2)
  • 50 mM Tris‐Cl buffer (see recipe)
  • 5 mM TAMRA‐DIBO alkyne (Invitrogen, cat. no. C10410) in dimethyl sulfoxide (DMSO)
  • 3:1:2 (v/v/v) methanol/chloroform/DD water, ice cold
  • Methanol, ice cold
  • 1× protein loading buffer (see recipe)
  • Fluorescent protein molecular weight ladder (15‐250 kDa, Bio‐Rad)
  • 12‐well, 4% to 12% Tris‐Cl protein gel (Criterion XT Precast Gel, Bio‐Rad)
  • 1× MOPS electrophoresis buffer (Bio‐Rad)
  • Destaining solution: 4:1:5 (v/v/v) methanol/acetic acid/DD water
  • Coomassie Blue staining reagent (Bio‐Rad)
  • 1.5‐ml microcentrifuge tube
  • 2‐ml detergent removal spin column (Pierce, cat. no. 87778)
  • 70°C heating block
  • SDS‐PAGE electrophoresis gel dock for Criterion XT Precast Gel (Bio‐Rad)
  • Fluorescence gel scanner, excitation wavelength 532 nm, equipped with <580 nm cut‐off filter and 30‐nm bandpass filter (Amersham Biosciences Typhoon 9400)
  • Additional reagents and equipment for SDS‐PAGE (e.g., Gallagher, )
NOTE: Steps 1‐9 should be carried out with minimal time intervals; otherwise, labeling efficiency typically decreases for unknown reasons.

Basic Protocol 4: Proteome‐Wide BPPM Using Proteome Pulldown for MS Analysis

  Materials
  • 2 mg/ml cell lysate (from 1 × 107 cells in a T‐75 flask, see protocol 4), freshly prepared
  • 8 mM Ab‐SAM in 0.01% (v/v) TFA (see protocol 2)
  • Methanol, ice cold
  • 0.5% (w/v) SDS dilution buffer (see recipe), ice cold
  • 5 mM Click‐iT biotin‐DIBO alkyne (Invitrogen, cat. no. C10412) in dimethyl sulfoxide (DMSO)
  • Streptavidin agarose beads (GE healthcare, cat. no. 17‐5113‐01)
  • Phosphate‐buffered saline (PBS, GIBCO), ice cold, with and without 0.2% (w/v) SDS
  • 250 mM ammonium bicarbonate (ABC) buffer, ice cold
  • Reduction buffer (see recipe)
  • 1× protein loading buffer (see recipe)
  • Fluorescent protein molecular weight ladder (15‐250 kDa, Bio‐Rad)
  • 12‐well, 4% to 12% Tris‐Cl protein gel (Criterion XT Precast Gel, Bio‐Rad)
  • 1× MOPS electrophoresis buffer (Bio‐Rad)
  • Coomassie Blue staining reagent (Bio‐Rad)
  • 50‐ml conical polypropylene tubes
  • Turn‐over‐turn mixer
  • 2‐ml Dolphin microcentrifuge tubes
  • 100°C heating block
  • 1.5‐ml microcentrifuge tubes
  • SDS‐PAGE electrophoresis gel dock for Criterion XT Precast Gel (Bio‐Rad)
  • Additional reagents and equipment Bradford protein assays (e.g., Bio‐Rad) and for SDS‐PAGE (e.g., Gallagher, )
NOTE: Steps 1‐9 should be carried out with minimal time intervals; otherwise, labeling efficiency tends to decrease for unknown reasons.
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