Biomarker Discovery by Glycopeptide Enrichment and Mass Spectrometry

10th April 2015
Versione stampabile

Venue: Edificio Povo 2, via Sommarive nr. 9, Povo (Tn) - Room B101
 at 2:00 p.m.

  • Marco Gaspari -  Department of Experimental and Clinical Medicine, “Magna Græcia” University of Catanzaro, Catanzaro, Italy

Protein glycosylation is a key post-translational modification present in the majority of cellular proteins expressed on the cell surface and in the extracellular space. Many studies have associated abnormal expression of N-linked glycoproteins to various diseases, such as cancer, suggesting that glycoproteins may be used as biomarkers for cancer diagnosis and/or prognosis. The following strategy can be used to focus mass spectrometry-based proteomics on the detection and quantification of glycoproteins: (i) tryptic digestion of the protein mixture; (ii) enrichment of glycosylated peptides; (iii) peptide deglycosylation by PNGase F; (iv) analysis of the mixture of formerly N-glycosylated peptides by nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). This approach allows the quantification of hundreds of N-linked glycoproteins with very high sensitivity in a single analysis.  The workflow was applied to identify differentially expressed N-linked glycoproteins in colorectal cancer tissue samples compared to healthy colorectal tissue from 19 patients undergoing colorectal cancer surgery. Nine of the identified proteins (LH2, DPEP1, Sel-1L, CD82, PAR-1, LH3, S12A2, LAMP-3, OLM4) were found to be up-regulated in the vast majority of the cohort.  Glycopeptide enrichment and mass spectrometry can be also applied to the analysis of cancer-associated proteins present in serum at concentrations below 10 ng/mL. Results obtained by performing over 100 nanoLC-MS/MS analyses on a prostate cancer sample set will be shown, with particular emphasis on throughput and sensitivity. Proteins like cathepsin-D and LAMP-2 were found to be present at higher levels in patients’ samples compared to controls.