Biology subjects

Kelly, M. I.

Publications and source records attributed to Kelly, M. I..

3 recordsLinked to original sources

bioRxiv2026

Contextualised real-time mass spectrometry improves glycosylation detection and characterisation

Glycosylation is a structurally diverse, non-template-driven modification whose analysis by liquid chromatography-mass spectrometry is constrained by discovery-mode acquisition rules developed for proteomics. Data-dependent acquisition filters, such as intensity-based precursor selection and charge-state exclusion, map poorly onto glycan analysis, which span wide ranges of charge state and abundance independent of their biological importance. Here we present glycosylation real-time mass spectrometry (GlycoRTMS), an instrument-API method that annotates observed precursor masses with glycan compositions in real time and uses this context to guide fragmentation. Composition-aware precursor prioritisation sampled deeper into the precursor space, expanding MS2 coverage of a hyaluronic acid hydrolysate from four to eight oligosaccharide subunits. Charge-state-specific collision energy equations tailored to oligosaccharides produced complete fragment ladders where fixed normalised collision energy did not. MS3 triggering gated by both diagnostic ions and glycan composition matching enabled efficient, chromatography-compatible characterisation of O-acetylated sialic acids and identified product ions specific to O-acetylation. Together, these strategies improve both the depth and quality of glycan detection and characterisation within a single injection.

biochemistry↗

bioRxiv2026

GlyComboCLI enables command line-based FAIR workflows for glycan composition assignment in mass spectrometry data

Glycans are integral biomolecules whose presence cannot be predicted from genomic data alone, necessitating experimental characterisation through approaches including mass spectrometry. Assignment of glycan compositions to observed mass to charge ratios is computationally challenging due to the potential monosaccharide diversity and existing tools lack the required flexibility for integration into automated bioinformatic workflows. Here, we present GlyComboCLI, an open-source command-line application for the assignment of glycan compositions to mass spectrometry data which expands upon the previous GUI application, GlyCombo. GlyComboCLI accepts mass lists and vendor-neutral mzML files, supports diverse monosaccharides, derivatisation states, reducing-end modifications and adducts, and is validated through automated tests spanning supported search parameters, input formats, and instrument vendors. Outputs are compatible with downstream tools including Skyline and GlycoWorkBench while GlyTouCan accessions support persistent identification of registered base compositions. Deployment as a standalone executable, a Docker container, and a Galaxy tool, supports FAIR and reproducible workflows. Applied to published mouse and human glycomics datasets, GlyComboCLI reproduced major qualitative glycomic motifs and relative abundance trends, while Galaxy workflow reproduced expected effects of sialidase treatment. These results demonstrate a flexible, scalable, and reproducible approach for glycan composition assignment within automated glycomics workflows.

bioinformatics↗

bioRxiv2025

Anion supercharging enables structural assignment of oxidatively released O-glycans

O-glycan analysis faces analytical challenges due to poor fragmentation characteristics of oxidatively released O-glycan acids, which can preserve labile O-acetyl modifications but suffer from charge fixation at the reducing end. This study introduces a supercharging approach using hexafluoroisopropanol (HFIP) and butylamine mobile phase additives for enhanced mass spectrometric analysis of bleach-released O-glycan acids. HFIP/butylamine increased average charge states by up to 50% compared to traditional mobile phases, dramatically improving MS2 fragmentation quality and enabling discrimination between isomeric compositions and structures. Enhanced fragmentation enabled confident identification of sialic acid variants and acetylation patterns, overcoming the analytical bottleneck of poor O-glycan acid fragmentation. Computational optimization reduced database search times by up to ninefold while maintaining sensitivity. Application to multi-species gastric mucins revealed distinct glycosylation profiles. Porcine mucin showed predominant serine-linked neutral structures, while bovine and ovine mucins exhibited primarily threonine-linked sialylated glycans. A minor fraction of porcine and bovine mucins contained acetylated serine-linked sialylated glycans. This methodology provides a comprehensive framework for O-glycan characterization while preserving biological modifications.

biochemistry↗