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

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.