Glycomic Analysis of Glycans Released from Glycoproteins Using Chemical Immobilization and Mass Spectrometry

Shuang Yang1, Hui Zhang1

1 Department of Pathology, Johns Hopkins University, Baltimore, Maryland
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140085
Online Posting Date:  September, 2014
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Protein glycosylation is one of most common protein modifications and is involved in many biological activities. N‐linked and O‐linked glycosylation not only represent abundant glycan modifications, but also are structurally diverse. Mass spectrometry has emerged as a major method for glycomic analysis. However, glycan extraction from proteins and glycan modification are two critical steps in glycomic analysis of glycans using mass spectrometry. In this protocol, we describe a novel and high‐throughput method for isolation and modification of glycans from glycoproteins using a chemoenzymatic approach on solid‐phase. Proteins are first immobilized to a solid support and unconjugated molecules are washed away; glycans, while still linked to glycoproteins on the solid support, can be treated enzymatically or chemically on solid phase for glycan derivatization. Glycans are then released from the solid support for analysis by mass spectrometry. The procedures outlined are robust and useful for high‐throughput glycomic analysis from complex biological or clinical samples. Curr. Protoc. Chem. Biol. 6:191‐208 © 2014 by John Wiley & Sons, Inc.

Keywords: solid‐phase; protein immobilization; glycan derivatization; chemoenzymatic processes; MALDI; GIG; mass spectrometry; glycomics

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Table of Contents

  • Introduction
  • Basic Protocol 1: Glycoprotein Immobilization for Glycan Extraction
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Glycoprotein Immobilization for Glycan Extraction

  • Protein [e.g., peptide‐N‐glycosidase F (PNGase F; enzyme for N‐glycan; New England Biolabs) or PNGase F (glycerol‐free): Add PNGase F in the bead solution (See Release of Glycan for solution used) right before release of N‐glycans; according to protocol provided by New England BioLabs, 1 μl of PNGase F is applied for 10 μg glycoprotein]; alternatively, peptide samples can be used
  • Binding buffer (see recipe)
  • Protein denaturing buffer (10×; New England Biolabs, cat. no. P0705L)
  • RIPA lysis buffer: Dilute the protein inhibitor cocktail (Roche) 1:100 with 1× RIPA lysis buffer (Millipore)
  • Ice
  • DI water (generated in the laboratory)
  • Bovine serum albumin (BSA; protein standards; 2 mg/ml; Sigma Aldrich) solutions (including 0.0625, 0.125, 0.25, 0.5, 1, 2 mg/ml)
  • BCA protein assay kit (BCA reagent A:BCA reagent B = 50:1; BSA standard, 2 mg/ml; Thermo Scientific)
  • Aminolink plus coupling resin (aldehyde‐activated 4% beaded agarose, slurried in water with sodium azide; Thermo Scientific)
  • Peptide standards: e.g., Angiotensin I human acetate salt hydrate (AG; 1296.48 Da; peptide for internal standard; Sigma‐Aldrich) and/or Neurotensin (NT; 1672.92 Da; >90%; peptide for internal standard; Sigma‐Aldrich)
  • Sodium borocyanohydride (NaCNBH 3; 98%; Sigma Aldrich)
  • Phosphate‐buffered saline (PBS; 10×, pH 7.4; Life Technologies)
  • Reducing buffer (see recipe)
  • Trizma hydrochloride (Tris·Cl; >99.0%; pH 7.6; Sigma Aldrich)
  • Blocking buffer (pH 7.4): add 31 mg NaCNBH 3 to 10 ml of 1×Tris·Cl, pH 7.6, buffer (or 50 mM solution) (used to block the resin active sites)
  • 1 M NaCl (Sigma Aldrich)
  • Hydrochloric acid (HCl; 36.5% to 38.0%; Sigma Aldrich)
  • p‐toluidine (pT; >99.7%; Sigma Aldrich)N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide (EDC; >97.0%; 5.65 M; Sigma‐Aldrich)
  • Formic acid (FA; ACS reagent, >98%; Sigma Aldrich)
  • 10% acetonitrile (ACN; LC‐MS Chromasolv; Sigma Aldrich)
  • Water (HPLC grade; Fisher Scientific)
  • Reaction buffer for neuraminidase (G1; 10×; New England Biolabs)
  • Neuraminidase (α2‐3, α2‐6, α2‐8; desialylation of sialic acid; New England Biolabs)
  • Ammonium bicarbonate (NH 4HCO 3; >99.5%; Sigma Aldrich)
  • Reaction buffer for PNGase F (G7; 10×; New England Biolabs)
  • Ammonium hydroxide (NH 4OH; 28% to 30%; Sigma Aldrich)
  • 0.1% trifluoroacetic acid (TFA) solution: 0.1% (v/v) TFA (LC‐MS Ultra; Sigma Aldrich) in HPLC water
  • DHB‐DMA matrix solution: Dissolve 100 mg of 2,5‐dihydroxybenzoic acid (DHB; Sigma Aldrich) per milliliter into 50% ACN (vol) in the presence of 0.1 mM NaCl and 2% N,N‐dimethylaniline (DMA; >99.5%; Sigma Aldrich)
  • Maltoheptaose (DP7; Oligosaccharide standard for internal standard in MALDI‐MS; Sigma‐Aldrich)
  • Samples used for GIG (for a single GIG experiment, only one sample is used; each sample has been used separately for different purposes)
    • Fetuin from fetal bovine serum (Fetuin; standard glycoprotein for method validation; Sigma Aldrich) (starting from step 9: standard protein sample)
    • Human serum (complex biological sample for glycan extraction; received from Johns Hopkins University) (starting from step 9: complex protein sample)
    • Mucin from porcine stomach (Type III, bound sialic acid 0.5% to 1.5%; glycoproteins for O‐glycan extraction; Sigma Aldrich) (starting from step 9: standard protein sample for O‐glycan)
    • Ribonuclease B from bovine pancreas (RNase B; standard glycoprotein for method development; Sigma Aldrich) (starting from step 9: standard protein sample for N‐glycan)
    • Sialylglycopeptide (α2,6) (SGP; >95%; glycopeptide standard for method development; Fushimi Pharmaceutical; Japan) (starting from step 13: standard peptide for sialic acid derivatization)
  • Eppendorf Research Plus pipets (2.5 μl, 20 μl, 200 μl, and 1 ml; Eppendorf)
  • Thermo Savant SpeedVac SPD121P Centrifugal Evaporator (Thermo Scientific)
  • Allegra 6R centrifuge (Beckman Coulter)
  • Centrifuge 5415 D (Eppendorf AG; used in all steps requiring centrifugation with the exception of step 2)
  • Branson Sonifier 250 (Emerson Industrial Automation)
  • White light
  • Zeba spin desalting column (2 ml; Thermo Scientific)
  • 15‐ml Falcon tubes (BD Biosciences)
  • Corning Costar 96‐well cell culture plate (Corning)
  • DPC MicroMix 5 Shaker (Conquer Scientific)
  • Innova 4000 incubator shaker (37°C; New Brunswick Scientific)
  • μQuant Microplate Spectrophotometer (Biotek Instruments)
  • 1.5‐ and 2‐ml microcentrifuge tubes
  • Lab Dancer Mini Vortexer (VWR International)
  • Snap‐cap spin column (0.5 ml; Thermo Scientific)
  • Barnstead/Thermolyne LabQuake tube shaker (Barnstead International)
  • Boekel 13300 oven (Boekel Scientific)
  • Carbograph extract‐clean columns (Carbograph; 3.0 ml; Grace)
  • Latex dropper bulb (Fisher Scientific), optional
  • μFocus MALDI plate (384 circles; 900 μm diameter; Hudson Surface Technology)
  • MALDI plate holder (For Shimadzu Axima Resonance; Hudson Surface Technology)
  • MALDI‐QIT‐TOF mass spectrometer (Axima Resonance; Shimadzu) equipped with a controllable MSn fragmentation, high‐resolution precursor ion selection
NOTE: DI water is used for general sample preparation.NOTE: HPLC grade water is used for the final step of sample preparation prior to MS detection.
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Literature Cited

Literature Cited
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