Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes

Christina M. Woo1, Carolyn R. Bertozzi2

1 Department of Chemistry, Stanford University, Stanford, California, 2 Howard Hughes Medical Institute, Stanford, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch150185
Online Posting Date:  March, 2016
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Abstract

Protein glycosylation plays many critical roles in biological function and creates the most diversity of all post‐translational modifications (PTMs). Glycan structural diversity is directly correlated with difficulty in characterizing the intact glycoproteome by mass spectrometry (MS). In this protocol, we describe a novel mass‐independent chemical glycoproteomics platform for characterizing intact, metabolically labeled glycopeptides from complex proteomes, termed Isotope Targeted Glycoproteomics (IsoTaG). To use IsoTaG, cell culture samples are metabolically labeled with an azido‐ or alkynyl‐sugar. Metabolically labeled glycoproteins are then tagged using Click chemistry and enriched with an isotopic recoding biotin probe. Intact glycopeptides are recovered by cleavage of the probe, analyzed with directed MS, and assigned by targeted mass‐independent data analysis. The outlined procedure is well defined in cell culture and has been executed with over 15 cell lines. © 2016 by John Wiley & Sons, Inc.

Keywords: glycoproteomics; bioorthogonal chemistry; metabolic labeling; chemical proteomics; mass spectrometry

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of Metabolically Labeled Cell Lysates and Conditioned Medium
  • Basic Protocol 2: Chemical Glycoproteomics Enrichment Procedure
  • Basic Protocol 3: Mass‐Independent Mass Spectrometry
  • Support Protocol 1: Synthesis of the IsoTaG Alkynyl Biotin Probe
  • Support Protocol 2: Synthesis of the IsoTaG Azido Biotin Probe
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Metabolically Labeled Cell Lysates and Conditioned Medium

  Materials
  • Sugar precursor:
  • N‐Azidoacetylgalactosamine‐tetraacetylated (Ac 4GalNAz, C 16H 22N 4O 10, 500 mM in DMSO, Pierce) or
  • N‐Azidoacetylglucosamine‐tetraacetylated (Ac 4GlcNAz, C 16H 22N 4O 10, 500 mM in DMSO, Pierce) or
  • N‐Azidoacetylmannosamine‐tetraacetylated (Ac 4ManNAz, C 16H 22N 4O 10, 500 mM in DMSO, Pierce) or
  • N‐(4‐pentynoyl)mannosamine‐tetraacetylated [Ac 4ManNAl, C 19H 25NO 10, 500 mM in DMSO, prepared in house according to the procedure of Chang and co‐workers (Chang et al., )]
  • Dimethyl sulfoxide (DMSO, C 2H 6OS; Sigma Aldrich)
  • Suspension of cells
  • Medium A (see recipe)
  • Phosphate‐buffered saline (PBS; pH 7.4, Life Technologies)
  • Medium B (see recipe)
  • Triton X‐100 (0.1% in PBS; Sigma Aldrich)
  • Trypsin, EDTA free (0.25%, HyClone)
  • Lysis buffer (see recipe)
  • Ice
  • RapiGest (10% in PBS, Waters)
  • BCA protein quantification assay (Pierce)
  • Disposable sterile pipets (5, 10, 25, 50 ml, Fisher Scientific)
  • Tissue culture dish (150 mm; Corning)
  • Tissue culture incubator (37°C, humidified, 5% CO 2)
  • 150‐cm2 tissue culture flasks (Corning)
  • 50‐ml centrifuge tubes (Fisher)
  • Centrifugal filter units (10 kDa, 15 ml, Amicon)
  • 2‐ml microcentrifuge tubes
  • 5‐ml Dounce homogenizer (Wheaton)
  • Probe tip sonicator (Misionix)
  • Absorbance plate reader

Basic Protocol 2: Chemical Glycoproteomics Enrichment Procedure

  Materials
  • Metabolically labeled cell lysates (see protocol 1)
  • Click chemistry reagents:
  • IsoTaG alkynyl biotin probe, (10 mM stock in DMSO, see protocol 4)
  • OR IsoTaG azido biotin probe, (10 mM stock in DMSO, see protocol 5)
  • Copper(II) sulfate pentahydrate (CuSO 4•5H 2O, 50 mM stock in water, Sigma Aldrich)
  • 3‐(4‐((bis((1‐tert‐butyl‐1 H‐1,2,3‐triazol‐4‐yl)methyl)amino)methyl)‐1 H‐1,2,3,triazol‐1‐yl)propanol [BTTP, 30 mM stock in DMSO, prepared according to the procedure of Wang and co‐workers (Wang et al., )]
  • Sodium ascorbate (C 6H 7NaO 6, 100 mM fresh stock in PBS; Sigma Aldrich)
  • Methanol (MeOH CH 3OH, LC/MS grade; Sigma Aldrich)
  • RapiGest (10% stock in PBS; Waters)
  • Streptavidin–agarose (50% slurry; Pierce)
  • Phosphate‐buffered saline (PBS; Life Technologies), pH 7.4
  • Urea (CH 4N 2O, 6 M fresh stock in water; Sigma Aldrich)
  • Dithiothreitol (DTT; C 4H 10O 2S 2, 100 mM fresh stock in PBS; Sigma Aldrich)
  • Iodoacetamide (C 2H 4INO, 500 mM fresh stock in PBS; Sigma Aldrich)
  • Trypsin resuspension buffer (50 mM acetic acid; Pierce)
  • Trypsin (sequencing grade; Pierce)
  • Formic acid (CH 2O 2, LC/MS grade; Pierce)
  • Acetonitrile (CH 3CN, LC/MS grade; Pierce)
  • Water (MilliQ filtered; generated in house)
  • Immunoblot reagents including:
  • Loading buffer (see recipe)
  • 4% to 12% Criterion XT Bis‐Tris gel (26 well; Bio‐Rad)
  • MES SDS running buffer (20×, Life Technologies)
  • Tris/glycine transfer buffer (10×, Bio‐Rad)
  • Bovine serum albumin (BSA; Sigma Aldrich)
  • Ponceau stain (see recipe)
  • High sensitivity streptavidin–HRP (Pierce)
  • Super Signal West Pico Chemiluminescence Substrate (Thermo Scientific)
  • Low‐bind 1.5‐ml microcentrifuge tubes (Eppendorf)
  • Vortex mixer
  • Centrifuge
  • Eppendorf rotator
  • –80°C freezer
  • Probe tip sonicator
  • Speedvac
  • Incubator (37°C)
  • ZipTip P10 tips (Millipore)
  • 95°C water bath
  • Immunoblot equipment including:
  • Electrophoresis chamber
  • Nitrocellulose membranes (Life Technologies)
  • Immunoblot chamber
  • Immunoblot cassette
  • X‐Ray developer (Kodak)
  • 500‐μl Low‐bind microcentrifuge tubes (Eppendorf)

Basic Protocol 3: Mass‐Independent Mass Spectrometry

  Materials
  • Desalted samples (see protocol 2)
  • Formic acid (1‐ml ampules, Thermo Pierce)
  • Water (MilliQ filtered, obtained in house)
  • Acetonitrile (Optima grade, Fisher)
  • Vortex mixer
  • Centrifuge
  • Autosampler vials (Polypropylene, Agilent)
  • Septa caps (Agilent)
  • LTQ Orbitrap (XL or newer, Thermo)
  • NanoLC system (Dionex Ultimate3000 or Easy Nano 1000, Thermo)
  • C18 analytical column (Acclaim PepMap 300, 150‐mm length × 0.075‐mm inner diameter, 5‐μm particles, 300 Å pores; Thermo)
  • PC Computer
  • ReAdW.exe or MSConvert (ProteoWizard)
  • XCalibur (Thermo)
  • Proteome Discoverer with SEQUEST (Thermo)
  • Byonic (Protein Metrics)
  • Isotopic Signature Transfer an Mass Pattern Prediction (IsoStamp, Bertozzi laboratory)

Support Protocol 1: Synthesis of the IsoTaG Alkynyl Biotin Probe

  Materials
  • Propargyl bromide (C 3H 3Br, 80% in toluene; Sigma Aldrich)
  • (E)‐2,3‐dibromobut‐2‐ene‐1,4‐diol (C 4H 6Br 2O 2; Sigma Aldrich)
  • Potassium hydroxide (KOH; Sigma Aldrich)
  • N,N‐Dimethylformamide (DMF, C 3H 7NO; Sigma Aldrich)
  • Silica (60 Å, Silicycle)
  • Ethyl acetate (EtOAc, C 4H 8O 2; Sigma Aldrich)
  • Hexanes (C 6H 14; Sigma Aldrich)
  • Water (H 2O, deionized on site)
  • Triethylamine (C 6H 15N, distilled over calcium hydride; Sigma Aldrich)
  • Dichlorodiphenylsilane [(C 6H 5) 2SiCl 2; Sigma Aldrich]
  • Biotin–CA(PEG) 4–alcohol [C 25H 46N 4O 8S; prepared in house according to the procedure of Szychowski and co‐workers (Szychowski et al., )]
  • Dichloromethane (CH 2Cl 2; Sigma Aldrich)
  • Sodium bicarbonate (NaHCO 3; Sigma Aldrich)
  • Rotovap (Büchi)
  • Column chromatography equipment

Support Protocol 2: Synthesis of the IsoTaG Azido Biotin Probe

  Materials
  • 3‐azidopropyl 4‐methylbenzenesulfonate [C 10H 13N 3O 3S; synthesized in‐house according to the procedure of Phetsang and coworkers (Phetsang et al., )]
  • (E)‐2,3‐dibromobut‐2‐ene‐1,4‐diol (C 4H 6Br 2O 2; Sigma Aldrich)
  • Potassium hydroxide (KOH; Sigma Aldrich)
  • N,N‐Dimethylformamide (DMF, C 3H 7NO; Sigma Aldrich)
  • Water (H 2O; deionized on site)
  • Silica (60 Å, Silicycle)
  • Ethyl acetate (EtOAc, C 4H 8O 2; Sigma Aldrich)
  • Hexanes (C 6H 14; Sigma Aldrich)
  • Triethylamine (C 6H 15N, distilled over calcium hydride; Sigma Aldrich)
  • Dichlorodiphenylsilane [(C 6H 5) 2SiCl 2;Sigma Aldrich]
  • Biotin–CA(PEG) 4–alcohol [C 25H 46N 4O 8S; prepared in house according to the procedure of Szychowski and co‐workers(Szychowski et al., )]
  • Sodium bicarbonate (NaHCO 3; Sigma Aldrich)
  • Dichloromethane (CH 2Cl 2; Sigma Aldrich)
  • Rotovap (Büchi)
  • Column chromatography equipment
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Figures

Videos

Literature Cited

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Internet Resources
  http://bertozzigroup.stanford.edu/isostamp/
  The latest version of IsoStamp is available as a Web application and for download. Selected examples of data collected with this procedure and applications are also provided.
Key Reference
  Woo et al., 2015. See above.
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