In the present study we have investigated the human blood plasma mucin-type O-glycoproteome of healthy individuals in an explorative and nontargeted manner
To this end, we have conducted a site-specific large-scale O-glycoproteomic analysis, which combines a broad-specific proteolytic digest, with HILIC enrichment/fractionation and subsequent multistage mass spectrometry measurement (nano-RPLC- ESI-IT- MSn ) with CID and ETD
Centered on the characterization and identification of intact glycopeptides , we could demonstrate the in-depth O-glycoproteomic analysis of a number of important human blood plasma glycoproteins (mainly acute phase proteins ), including alpha-2-HS-glycoprotein , fibrinogen, plasminogen and kininogen-1
Our results are in good agreement with previous findings by other research groups, but also add new aspects to the field, e.g. the identification of a couple of novel O-glycosylation site as well as the benefits and drawbacks of using Proteinase K in large-scale mass spectrometric glycoproteomic studies
Explorative site-specific N- and O-glycoproteomic studies of biofluids, like human blood plasma, human milk, urine or cerebrospinal fluid hold an enormous potential to better understand the implications of protein glycosylation under normal physiological conditions, but also under pathophysiological conditions
By serving as a diagnostic tool, the detection/discovery of relevant glycopeptides (biomarker candidates) can be the basis for targeted quantitative glycoproteomic analyses, which allow for a site-specific monitoring of glycosylation alterations, e.g. during disease progression
Site-specific glycosylation analyses are, moreover, important to produce biopharmaceuticals according to quality by design requirements, in particular if these biopharmaceuticals are produced in heterologous expression systems
In this regard site-specific glycosylation analyses might also enable understanding/controlling important glycan-related features of the final product including its efficacy, half-life, or antigenicity