Abstract

European Journal of Mass Spectrometry
Volume 16 Issue 1, Pages 123–149 (2010)
doi: 10.1255/ejms.1035

Glycoproteome study in myocardial lesions serum by integrated mass spectrometry approach: preliminary insights

Andrea Carpentieri^a, Chiara Giangrande^a, Piero Pucci and Angela Amoresano^*
Dipartimento di Chimica Organica e Biochimica, Università degli Studi Federico II, Complesso Universitario Monte S. Angelo, via Cynthia 4, 80126 Napoli, Italy. E-mail: angamor@unina.it

Bottom up proteomics requires efficient and selective pre-fractionation procedures to simplify the analysis of the enormous number of peptides resulting from the hydrolysis of a cellular extract enabling the detection, identification and the structural characterization of the post-translational modifications. Glycosylation, a well-known post-translational modification, plays a key role in the enormous complexity, and heterogeneity of the human blood serum proteome. Thereby, characterization of glycosylation from serum is a challenging task, even for the existing sophisticated analytical methodologies. Here we report a glycoproteomics study on the identification of even low abundant glycoproteins, including the localization of N-glycosylation sites and the glycan profiling in human sera from healthy and myocarditis affected donors. The strategy is simply based on proteolytic digestion of total serum proteins followed by a single enrichment step of glycopeptides on ConA lectin affinity chromatography. Glycopeptides were then deglycosylated by PNGaseF treatment and nano-liquid chromatography-electrospray ionization tandem mass spectrometry analyses of the free peptides provided the basis for both identification of the individual proteins and elucidation of their modification sites. Moreover, glycan profilings could be obtained by matrix-assisted laser desorption/ionization mass spectrometry analysis of the released oligosaccharides. Our data led to the identification of 68 different glycosylation sites within 49 different proteins. Moreover, the analyses carried out on glycans represent the first picture of a glycosylation pattern in myocardial lesions. As a whole, several differences in the glycosylation patterns from different sera were observed, thus indicating glycan profiling as a possible tool to discriminate among different diseases.

Keywords: human sera, glycoproteomics, mass spectrometry

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