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

Research output: Contribution to journalArticle

76 Citations (Scopus)

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 Nglycosylation 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.

Original languageEnglish (US)
Pages (from-to)1291-1302
Number of pages12
JournalAnalytical and Bioanalytical Chemistry
Volume403
Issue number5
DOIs
StatePublished - May 2012

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Glycosylation
Isomers
Polysaccharides
Glycopeptides
Proteins
Glycoproteins
Carbon
Glycomics
Proteolysis
Peptides
Lactoferrin
Microfluidics
Liquid chromatography
Post Translational Protein Processing
Caseins
Chemical analysis
Liquid Chromatography
Proteomics
Mass spectrometry
Mass Spectrometry

Keywords

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

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

Cite this

Hua, S., Nwosu, C. C., Strum, J. S., Seipert, R. R., An, H. J., Zivkovic, A. M., ... Lebrilla, C. B. (2012). Site-specific protein glycosylation analysis with glycan isomer differentiation. Analytical and Bioanalytical Chemistry, 403(5), 1291-1302. https://doi.org/10.1007/s00216-011-5109-x

Site-specific protein glycosylation analysis with glycan isomer differentiation. / Hua, Serenus; Nwosu, Charles C.; Strum, John S.; Seipert, Richard R.; An, Hyun Joo; Zivkovic, Angela M.; German, J. Bruce; Lebrilla, Carlito B.

In: Analytical and Bioanalytical Chemistry, Vol. 403, No. 5, 05.2012, p. 1291-1302.

Research output: Contribution to journalArticle

Hua, S, Nwosu, CC, Strum, JS, Seipert, RR, An, HJ, Zivkovic, AM, German, JB & Lebrilla, CB 2012, 'Site-specific protein glycosylation analysis with glycan isomer differentiation', Analytical and Bioanalytical Chemistry, vol. 403, no. 5, pp. 1291-1302. https://doi.org/10.1007/s00216-011-5109-x
Hua, Serenus ; Nwosu, Charles C. ; Strum, John S. ; Seipert, Richard R. ; An, Hyun Joo ; Zivkovic, Angela M. ; German, J. Bruce ; Lebrilla, Carlito B. / Site-specific protein glycosylation analysis with glycan isomer differentiation. In: Analytical and Bioanalytical Chemistry. 2012 ; Vol. 403, No. 5. pp. 1291-1302.
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