GLYCOPEPTIDE ENRICHMENT USING SOLVENT PRECIPITATION

Glycoproteins have essential roles in biology and medicine. Glycosylation patterns often change during pathophysiological conditions which is also underlined by the fact that most tumor biomarkers are glycoproteins. Biopsies are generally stored in the form of tissue microarrays (TMA) facilitating parallel analysis of healthy, cancerous and metastatic tissues. The aim of our work is to characterize the glycosylation pattern of prostate cancer TMAs following on-surface tryptic digestion. For sensitive detection of minor glycoforms in tissues, especially that using very small sample amounts like that available in TMAs, it is essential to include a glycopeptide enrichment step in the analytical workflow. First we tested a mixed mode graphite and C18 SPE column, but this did not prove efficient in binding glycopeptides. Subsequently we used a solvent (acetone) based precipitation method1, which gave promising results. Following initial trials, we have optimized the method to achieve both high yield and high enrichment for glycopeptides. Modified parameters included the pH, salt concentration, the volume and the type of the precipitating solvent. Results were tested both on a model glycoprotein, alpha-1-acid glycoprotein (AGP) and also on a complex HeLa cell lysate. Glycopeptides and peptides were identified by tandem mass spectrometry in a nano-LC-MS/MS workflow using the Byonic software. Quantitation of the individual glycoforms was performed based on MS1 using our in-house GlycoPattern software. Results of the optimization and glycopeptide enrichment will be presented