Mucinomics: The Next Frontier of Mass Spectrometry

Dr. Stacy Malaker from Yale University recently presented advancements in mucin characterization, enrichment techniques, and their translational relevance in disease research on a Nature hosted webinar. Here, we explore the key insights from her presentation.

The complexity of mucin glycosylation

Glycosylation is one of the most diverse post-translational modifications (PTMs), estimated to modify over 50% of the human proteome. There are three primary types of glycosylation:

• N-linked glycosylation: Occurs on asparagine residues, influencing protein folding and cell signaling.
• O-linked glycosylation: Involves serine or threonine residues and is crucial for cell adhesion and matrix interactions.
• O-linked GlcNAcylation: A dynamic intracellular modification similar to phosphorylation, regulating transcription and cell proliferation.

Mucin proteins, characterized by densely glycosylated proline, serine, threonine (PTS) domains, exhibit bottlebrush-like structures that extend from the cell surface or form gel-like secretions. Dysregulation of mucins, particularly MUC1 and MUC16, is implicated in cancer progression, immune evasion, and metastasis. Despite their importance, mucins have remained difficult to analyze due to their complex glycosylation and resistance to standard proteases.

Overcoming analytical challenges with mucinases

Traditional proteomics workflows struggle with mucins due to:

• High heterogeneity: A single glycosylation site can accommodate hundreds of glycan structures.
• Protease resistance: Conventional enzymes like trypsin fail to digest mucin domains.
• Fragmentation issues: Standard collision-induced dissociation (CID) leads to glycan loss, complicating site localization.

Dr. Malaker’s research introduced StcE (pronounced as Sticky), a mucin-selective protease that selectively cleaves glycosylated mucin domains while sparing unmodified peptides. This enzyme has been pivotal in:

• Mass spectrometric sequencing of mucins.
• Cellular assays for mucin function.
• Immunohistochemical staining and mucin enrichment for glycoproteomics studies.

StcE and other mucinases have enabled the first detailed site-specific glycosylation maps of mucins, revealing their structural dynamics and disease relevance.

Deciphering immune-related mucins: the TIM family

The T-cell immunoglobulin and mucin domain-containing (TIM) proteins regulate immune responses, with TIM-3 being a promising checkpoint inhibitor target in cancer therapy. Using mucinases, Dr. Malaker’s team successfully mapped the glycosylation patterns of TIM-1, TIM-3, and TIM-4, revealing:

• TIM-4 adopts a rigid, bottlebrush-like structure, extending beyond the glycocalyx for high-affinity ligand binding.
• TIM-3 exhibits flexible folding, potentially facilitating alternative ligand interactions via galectin-9-mediated clustering.
• Glycosylation regulates immune checkpoint function by influencing receptor-ligand interactions.

These findings underscore the structural and functional roles of mucins in immune regulation and their potential as therapeutic targets.

Advancing mucinomics with enrichment strategies

To enhance mucin analysis, Dr. Malaker’s lab developed an enrichment method using an inactive Sticky mutant conjugated to beads. This strategy:

• Selectively captures mucins from complex biological samples (e.g., serum, cancer cell lysates).
• Enables mass spectrometric analysis of previously undetectable glycoproteins.
• Identifies a broader mucinoma beyond canonical mucins, expanding the scope of mucin research.

This workflow drastically reduces sample preparation time while increasing the detection of mucin-specific glycopeptides, making it a powerful tool for clinical applications.

Improving glycopeptide identification

Existing search algorithms struggle with the complexity of mucin glycoproteomics. The field-wide Human Glycoproteomics Initiative (HGI) aims to evaluate and improve these tools. Key developments include:

• Standardized glycoproteomics reporting.
• Benchmarking of glycopeptide identification software.
• Integration of electron-based fragmentation for precise site localization.

These efforts are critical for establishing robust, reproducible glycoproteomic workflows for biomedical research.

Spatial distribution of mucins in disease

Mass spectrometry imaging (MSI) offers insights into mucin distribution in tissues, but traditional methods are limited to N-glycans. Dr. Malaker’s team pioneered mucinase-assisted MSI to visualize O-glycopeptides in colorectal cancer specimens. Findings included:

• Distinct glycopeptide profiles across tumor regions.
• Localization of tumor-associated carbohydrate antigens (TACAs).
• High-resolution sequencing of MUC2, achieving 90% coverage of its glycosylated domains.

This approach has potential applications in biomarker discovery, disease stratification, and personalized medicine.

Conclusion: Mucinomics represents a rapidly evolving frontier in mass spectrometry, with profound implications for cancer biology, immunology, and clinical diagnostics. The innovations in mucinase-based proteomics, enrichment techniques, and spatial glycoproteomics are paving the way for deeper insights into mucin function and disease mechanisms.

By overcoming long-standing analytical challenges, these advances position mucins as crucial players in biomedical research, opening doors to novel diagnostics and therapeutic strategies. As the field progresses, integrating mucinomics into routine clinical proteomics could revolutionize our understanding of complex diseases and their molecular underpinnings.

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