Optimized mucin-selective enrichment strategy to probe the mucinome

Abstract

Mucin-domain glycoproteins are densely O-glycosylated and play critical roles in a host of healthy and diseasedriven biological functions. Previously, we developed a mucin-selective enrichment strategy by employing a catalytically inactive mucinase (StcE) conjugated to 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 optimized our previous enrichment method to include elution conditions amenable to mucinase digestion and downstream analysis with mass spectrometry. This increased throughput and lowered sample input while maintaining mucin selectivity and enhancing 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 other enrichment techniques tested. This allowed for effective isolation of more mucin-domain glycoproteins, resulting in a high number of O-glycopeptides, thus enhancing our ability to analyze the mucinome.

Analysis of Mucin‐Domain Glycoproteins Using Mass Spectrometry

“…This will assure a depletion of unmodified proteins, e.g., albumin, which will often overtake signal in the mass spectrometer and impede analysis of relevant glycopeptides. To do this, we employ an inactive point mutant of mucinase StcE, which we have demonstrated can selectively enrich mucins from various samples (Mahoney et al, 2023;Malaker et al, 2022;Rangel-Angarita & Malaker, 2021).…”

“…For our purposes, we prefer the use of enzyme SmE, but have found that ImpA provides complementary glycopeptide coverage and can be a useful addition (Chongsaritsinsuk et al, 2023). Further, trypsinizing after mucinase digestion can result in higher coverage of more sparsely glycosylated regions (Mahoney et al, 2023). Finally, N-glycosylation can convolute data analysis and potentially suppress O-glycopeptides desired but can be removed by a universal N-glycosidase PNGase F. Reduction and alkylation 1.…”

“…More recently, the Malaker laboratory introduced the mucinase SmE, which is thus far the most useful enzyme for mass spectrometry analysis of mucin proteins (Chongsaritsinsuk et al, 2023). We also optimized the mucin enrichment procedure to increase throughput and allow for insolution digestion with mucinases (Mahoney et al, 2023). Overall, the last five years has seen considerable improvements in the analysis and enrichment of mucins.…”

“…First, many mucinases have been characterized, which comprise a class of proteases that specifically degrade mucin domains and enable MS analysis of the densely glycosylated peptides (Chongsaritsinsuk et al, 2023;Malaker et al, 2019;Shon et al, 2020Shon et al, , 2021. Additionally, we developed and optimized an enrichment technique to selectively isolate mucin-domain glycoproteins from non-mucin proteins (Mahoney et al, 2023;Malaker et al, 2022). Here, we first describe our protein-level mucin enrichment technique, wherein we conjugate inactive point mutants of mucinases to solid support and use this to selectively isolate mucins from complex samples, such as cell lysate and human serum.…”