Analytical and Bioanalytical Chemistry

, Volume 403, Issue 5, pp 1291–1302 | Cite as

Site-specific protein glycosylation analysis with glycan isomer differentiation

  • Serenus Hua
  • Charles C. Nwosu
  • John S. Strum
  • Richard R. Seipert
  • Hyun Joo An
  • Angela M. Zivkovic
  • J. Bruce German
  • Carlito B. Lebrilla
Original Paper

Abstract

Glycosylation is one of the most common yet diverse post-translational modifications. Information on glycan heterogeneity and glycosite occupancy is increasingly recognized as crucial to understanding glycoprotein structure and function. Yet, no approach currently exists with which to holistically consider both the proteomic and glycomic aspects of a system. Here, we developed a novel method of comprehensive glycosite profiling using nanoflow liquid chromatography/mass spectrometry (nano-LC/MS) that shows glycan isomer-specific differentiation on specific sites. Glycoproteins were digested by controlled non-specific proteolysis in order to produce informative glycopeptides. High-resolution, isomer-sensitive chromatographic separation of the glycopeptides was achieved using microfluidic chip-based capillaries packed with graphitized carbon. Integrated LC/MS/MS not only confirmed glycopeptide composition but also differentiated glycan and peptide isomers and yielded structural information on both the glycan and peptide moieties. Our analysis identified at least 13 distinct glycans (including isomers) corresponding to five compositions at the single N-glycosylation site on bovine ribonuclease B, 59 distinct glycans at five N-glycosylation sites on bovine lactoferrin, 13 distinct glycans at one N-glycosylation site on four subclasses of human immunoglobulin G, and 20 distinct glycans at five O-glycosylation sites on bovine κ-casein. Porous graphitized carbon provided effective separation of glycopeptide isomers. The integration of nano-LC with MS and MS/MS of non-specifically cleaved glycopeptides allows quantitative, isomer-sensitive, and site-specific glycoprotein analysis.

Figure

Overlaid chromatograms and associated structural assignments of glycopeptides from bovine κ-casein. Color denotes the site(s) of glycosylation from which the glycopeptide originated

Keywords

Site-specific glycosylation Glycopeptide Non-specific protease Isomer Quantitation LC/MS 

Abbreviations

bLF

Bovine lactoferrin

CID

Collision-induced dissociation

CNBr

Cyanogen bromide

ECC

Extracted compound chromatogram

Fuc

Fucose

Gal

Galactose

Glc

Glucose

Hex

Hexose

HexNAc

N-acetylhexosamine

IgG

Immunoglobulin G

Man

Mannose

ManX (where X = 5 to 9)

High mannose glycan of composition GlcNAc2ManX

NeuAc

N-acetylneuraminic acid

PGC

Porous graphitized carbon

Q-TOF

Quadrupole time-of-flight

RNAse B

Ribonuclease B

TIC

Total ion chromatogram

Supplementary material

216_2011_5109_MOESM1_ESM.pdf (145 kb)
ESM 1(PDF 144 kb)

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Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Serenus Hua
    • 1
  • Charles C. Nwosu
    • 1
  • John S. Strum
    • 1
  • Richard R. Seipert
    • 1
  • Hyun Joo An
    • 2
  • Angela M. Zivkovic
    • 3
    • 4
  • J. Bruce German
    • 3
    • 4
  • Carlito B. Lebrilla
    • 1
    • 4
    • 5
  1. 1.Department of ChemistryUniversity of CaliforniaDavisUSA
  2. 2.Graduate School of Analytical Science and TechnologyChungnam National UniversityDaejeonSouth Korea
  3. 3.Department of Food Science and TechnologyUniversity of CaliforniaDavisUSA
  4. 4.Foods for Health InstituteUniversity of CaliforniaDavisUSA
  5. 5.Department of Biochemistry and Molecular MedicineUniversity of CaliforniaDavisUSA

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