Mass Spectrometry-Compatible Elution Technique Enables an Improved Mucin-Selective Enrichment Strategy to Probe the...

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and disease-driven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to a solid support. While this method was effective, it suffered from low throughput and high sample requirements. Further, the elution step required boiling in SDS, thus necessitating an in-gel digest with trypsin. Here, we introduce innovative elution conditions amenable to mucinase digestion and downstream analysis using mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing the glycopeptide signal. We then benchmarked this technique against different O-glycan binding moieties for their ability to enrich mucins from various cell lines and human serum. Overall, the new method outperformed our previous procedure and all of the other enrichment techniques tested. This allowed for the effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.