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

Abstract 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.