Use of CID/ETD Mass Spectrometry to Analyze Glycopeptides

Yehia Mechref1

1 Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, Texas
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.11
DOI:  10.1002/0471140864.ps1211s68
Online Posting Date:  April, 2012
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Collision‐induced dissociation (CID) tandem mass spectrometry (MS/MS) does not allow the characterization of glycopeptides because of the fragmentation of glycan structures and limited fragmentation of peptide backbones. Electron transfer dissociation (ETD) MS/MS, on the other hand, offers a complementary approach, prompting only peptide backbone fragmentation while keeping post‐translational modifications intact. Characterization of glycopeptides using both CID and ETD is summarized in this unit. While CID provides information related to the composition of glycan moieties attached to a peptide backbone, ETD permits de novo sequencing of peptides. Radical anion transfer of electrons to the peptide backbone in ETD induces cleavage of the N‐Cα bond. The glycan moiety is retained on the peptide backbone, largely unaffected by the ETD process, thus allowing the identification of the amino acid sequence of a glycopeptide and its glycosylation site. This unit discusses the use of both CID and ETD for better characterization of glycopeptides. Curr. Protoc. Protein Sci. 68:12.11.1‐12.11.11. © 2012 by John Wiley & Sons, Inc.

Keywords: tandem mass spectrometry; ETD; CID; glycoproteins; glycopeptides

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Table of Contents

  • Introduction
  • Basic Protocol 1: Proteolytic Digestion of Glycoproteins
  • Basic Protocol 2: Liquid Chromatography‐Tandem Mass Spectrometry of Glycoprotein Tryptic Digests
  • Limitations of Electron Transfer Dissociation Tandem Mass Spectrometry
  • Concluding Remarks
  • Acknowledgements
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Proteolytic Digestion of Glycoproteins

  • Bovine ribonuclease B or bovine fetuin or horseradish peroxidase
  • Ammonium bicarbonate
  • Dithiothreitol (DTT)
  • Iodoacetamide (IAA)
  • Proteomic‐grade trypsin
  • Formic acid (95% to 97%)
  • 37°, 60°, and 95°C water baths

Basic Protocol 2: Liquid Chromatography‐Tandem Mass Spectrometry of Glycoprotein Tryptic Digests

  • Tryptic digests of glycoproteins (500 fmol to 1 pmol; protocol 1)
  • 90 Å Jupiter C 12 bounded phase (Phenomenex)
  • Acetonitrile (HPLC grade)
  • Formic acid (LC‐MS grade)
  • Water (HPLC grade)
  • Nano liquid chromatograph (e.g., Dionex 3000 Ultimate nano‐LC system; Dionex)
  • Mass spectrometer (e.g., LTQ Orbitrap hybrid mass spectrometer from Thermo Scientific or ultra‐high capacity ion‐trap mass spectrometer, Bruker Daltonics and Agilent Technologies)
  • PepMap300 C18 cartridge (5 µm, 300 Å; Dionex)
  • Pulled‐tip capillary column (150 mm × 75 µm i.d.)
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Literature Cited

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