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


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.


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


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



Bovine lactoferrin


Collision-induced dissociation


Cyanogen bromide


Extracted compound chromatogram












Immunoglobulin G



ManX (where X = 5 to 9)

High mannose glycan of composition GlcNAc2Man X


N-acetylneuraminic acid


Porous graphitized carbon


Quadrupole time-of-flight


Ribonuclease B


Total ion chromatogram



We would like to thank Ning Tang and Keith Waddell (Agilent Technologies Inc.) for instrumentation and technical support. Financial support was provided by the University of California Discovery Grant Program, the California Dairy Research Foundation, the NIEHS Superfund Research Program (P42 ES02710), and the CHARGE Study (P01 ES11269).

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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