UMMS Affiliation

Department of Medicine

Publication Date


Document Type

Article Postprint


Amino Acids, Peptides, and Proteins | Biochemistry | Chemistry | Molecular Biology | Structural Biology


High performance liquid chromatography has been employed for decades to enhance detection sensitivity and quantification of complex analytes within biological mixtures. Among these analytes, glycans released from glycoproteins and glycolipids have been characterized as underivatized or fluorescently tagged derivatives by HPLC coupled to various detection methods. These approaches have proven extremely useful for profiling the structural diversity of glycoprotein and glycolipid glycosylation but require the availability of glycan standards and secondary orthogonal degradation strategies to validate structural assignments. A robust method for HPLC separation of glycans as their permethylated derivatives, coupled with in-line MSn fragmentation to assign structural features independent of standards, would significantly enhance the depth of knowledge obtainable from biological samples. Here, we report an optimized workflow for LC-MS analysis of permethylated glycans that includes sample preparation, mobile phase optimization, and MSn method development to resolve structural isomers on-the-fly. We report baseline separation and MSn fragmentation of isomeric N- and O-glycan structures, aided by supplementing mobile phases with Li+, which simplifies adduct heterogeneity and facilitates cross-ring fragmentation to obtain valuable monosaccharide linkage information. Our workflow has been adapted from standard proteomics-based workflows and, therefore, provides opportunities for laboratories with expertise in proteomics to acquire glycomic data with minimal deviation from existing buffer systems, chromatography media, and instrument configurations. Furthermore, our workflow does not require a mass spectrometer with high-resolution/accurate mass capabilities. The rapidly evolving appreciation of the biological significance of glycans for human health and disease requires the implementation of high-throughput methods to identify and quantify glycans harvested from sample sets of sufficient size to achieve appropriately powered statistical significance. The LC-MSn approach we report generates glycan isomeric separations, robust structural characterization, and is amenable to auto-sampling with associated throughput enhancements.


Glycomics, HPLC, Mass Spectrometry, N-Glycosylation, O-glycosylation, Separation Technologies

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© 2021 THE AUTHORS. Published by Elsevier Inc on behalf of American Society for Biochemistry and Molecular Biology. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Licensed under a Creative Commons license: Attribution 4.0 International (CC BY 4.0)

DOI of Published Version



Kurz S, Sheikh MO, Lu S, Wells L, Tiemeyer M. Separation and identification of permethylated glycan isomers by reversed phase nanoLC-NSI-MSn. Mol Cell Proteomics. 2020 Dec 29:mcp.RA120.002266. doi: 10.1074/mcp.RA120.002266. Epub ahead of print. PMID: 33376194. Link to article on publisher's site

Journal/Book/Conference Title

Molecular and cellular proteomics : MCP

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Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.