Simultaneous and Extensive Site-specific N- and O-Glycosylation Analysis in Protein Mixtures

Abstract

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 non-specific protease digestion, nano-flow 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 nano-flow 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 micro-heterogeneity information was obtained. This approach presents a platform to simultaneously characterize N- and O-glycosites in the same mixture with extensive site heterogeneity.

Sensitive and Structure-Informative <i>N</i>-Glycosylation Analysis by MALDI-MS; Ionization, Fragmentation, and Derivatization

“…[47][48][49][50][51][52] is technique generally yields relatively short glycopeptides (i.e., glycopeptides having short peptide chain), which o en lack basic residues. Glycopeptides lacking basic residues would be detected with signi cantly lower intensities or actually escape detection when the analysis involves the positive-ion mode.…”

MS Platform for Erythropoietin Glycome Characterization

“…37,38 One popular alternative to proteolysis is pronase, one of a large class of nonspecific or 'multi-specific' proteases. [39][40][41][42][43][44] It is much easier to produce 'fingerprint glycopeptide' consisting of a glycan moiety with small peptide tag to identify glycosylation site with glycan heterogeneity.…”

Protein O-glycosylation analysis

“…Lately, the use of unspecifi c proteinases such as pronase E (proteinase mixture from Streptomyces griseus ) or proteinase K (from Tritirachium album ) for glycoprotein cleavage has been shown to be useful for in-depth analysis of the site-specifi c N-and/or O-glycosylation of individual glycoproteins in many cases Wuhrer et al , 2005 ;Clowers et al , 2007 ;Dodds et al , 2009 ;Seipert et al , 2009 ;Christiansen et al , 2010 ;Songsrirote et al , 2010 ;Zauner et al , 2010 ;Froehlich et al , 2011 ;Nwosu et al , 2011 ).…”