Enrichment method of sulfated glycopeptides by a sulfate emerging and ion exchange chromatography

Sulfated glycoproteins are of growing importance for biomarker discovery, as well as for investigating molecular recognition processes. Mass spectrometry (MS) has become a powerful technique for the characterization of glycans and glycoproteins. However, characterization and detection of sulfated glycopeptides by MS is difficult because of the low abundance and low ionization efficiency of these molecules. To overcome this problem, we developed a novel enrichment procedure for sulfated glycopeptides. The procedure consists of anion exchange chromatography and a sulfate emerging (SE) method which controls the net charge of peptides by utilizing limited proteolyzes and modification with acetohydrazide. Using this procedure, we are able to enrich and characterize the sulfated glycopeptides of bovine luteinizing hormone (bLH). Furthermore, we demonstrate the enrichment and detection of sulfated glycopeptides from a complex mixture comprising human serum spiked with bLH at a concentration of 0.1%.