Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures

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

Research output: Contribution to journalArticlepeer-review

105 Scopus citations


Extensive site-specific glycosylation analysis of individual glycoproteins is difficult due to the nature and complexity of glycosylation in proteins. In protein mixtures, these analyses are even more difficult. We present an approach combining nonspecific protease digestion, nanoflow liquid chromatography, and tandem mass spectrometry (MS/MS) aimed at comprehensive site-specific glycosylation analysis in protein mixtures. The strategy described herein involves the analysis of a complex mixture of glycopeptides generated from immobilized-Pronase digestion of a cocktail of glycoproteins consisting of bovine lactoferrin, kappa casein, and bovine fetuin using nanoflow liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (nano-LC-Q-TOF MS). The resulting glycopeptides were chromatographically separated on a micro fluidic chip packed with porous graphitized carbon and analyzed via MS and MS/MS analyses. In all, 233 glycopeptides (identified based on composition and including isomers) corresponding to 18 glycosites were observed and determined in a single mixture. The glycopeptides were a mixture of N-linked glycopeptides (containing high mannose, complex and hybrid glycans) and O-linked glycopeptides (mostly sialylated). Results from this study were comprehensive as detailed glycan microheterogeneity information was obtained. This approach presents a platform to simultaneously characterize N- and O-glycosites in the same mixture with extensive site heterogeneity.

Original languageEnglish (US)
Pages (from-to)2612-2624
Number of pages13
JournalJournal of Proteome Research
Issue number5
StatePublished - May 6 2011


  • glycopeptides
  • immobilized-Pronase
  • N-glycosites
  • nano-LC-Q-TOF MS
  • O-glycosites
  • protein mixtures
  • site-specific glycosylation
  • tandem MS

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)


Dive into the research topics of 'Simultaneous and extensive site-specific N- and O-glycosylation analysis in protein mixtures'. Together they form a unique fingerprint.

Cite this