Assessment of lectin and HILIC based enrichment protocols for characterization of serum glycoproteins by mass spectrometry.
Protein glycosylation is a common post-translational modification that is involved in many biological processes, including cell adhesion, protein-protein and receptor-ligand interactions. The glycoproteome constitutes a source for identification of disease biomarkers since altered protein glycosylation profiles are associated with certain human ailments. Glycoprotein analysis by mass spectrometry of biological samples, such as blood serum, is hampered by sample complexity and the low concentration of the potentially informative glycopeptides and -proteins. We assessed the utility of lectin-based and HILIC-based affinity enrichment techniques, alone or in combination, for preparation of glycoproteins and glycopeptides for subsequent analysis by MALDI and ESI mass spectrometry. The methods were successfully applied to human serum samples and a total of 86 N-glycosylation sites in 45 proteins were identified using a mixture of three immobilized lectins for consecutive glycoprotein enrichment and glycopeptide enrichment. The combination of lectin affinity enrichment of glycoproteins and subsequent HILIC enrichment of tryptic glycopeptides identified 81 N-glycosylation sites in 44 proteins. A total of 63 glycosylation sites in 38 proteins were identified by both methods, demonstrating distinct differences and complementarity. Serial application of custom-made microcolumns of mixed, immobilized lectins proved efficient for recovery and analysis of glycopeptides from serum samples of breast cancer patients and healthy individuals to assess glycosylation site frequencies.
Calvano
Cosima D
CD
Department of Biochemistry & Molecular Biology, University of Southern Denmark, Campusvej 55, Odense M, Denmark. calvano@chimica.uniba.it
Zambonin
Carlo G
CG
Jensen
Ole N
ON
D000595:Amino Acid Sequence; D001943:Breast Neoplasms / Q000175:diagnosis / Q000378:metabolism; D016022:Case-Control Studies; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006020:Glycopeptides / Q000737:chemistry; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006020:Glycopeptides; D037102:Lectins; D008565:Membrane Proteins