# Sensitive and Quantitative MS-bases Glycomic Mapping Platform

> **NIH NIH R01** · TEXAS TECH UNIVERSITY · 2020 · $283,987

## Abstract

Glycomics has emerged as an interesting yet challenging area of research in biology. Glycans function in
numerous important biological areas such as, but not limited to: the immune system, cell development, cell
differentiation/adhesion, host-pathogen interactions, protein signaling, and protein stabilization. Abnormal
glycosylation has been associated with several diseases including cancer, cystic fibrosis, and osteoarthritis.
Glycomics/glycoproteomics studies aim to quantify and characterize glycan structures (including linkage and
positional isomers), protein attachment sites, and the protein’s identity. Approximately 50% of mammalian
proteins are glycosylated but their abundance is rather low compared to non-glycosylated proteins. Furthermore,
numerous glycans can occupy the same glycan attachment site on a protein; that is the same protein pool can
have several different types of glycans attached to the same site, each with a potentially different function or a
particular activity. Protein glycans are divided into two classes based on their amino acid attachment sites:
asparagine for N-glycans and threonine, serine, and tyrosine for O-glycans.
 A strategy that has been successfully employed to investigate N-glycans in cells is to release or separate the
glycans from proteins with the enzyme PNGase F, and study the global glycan composition of a sample. A
drawback to this approach is that, so called, native glycans possess low ionization efficiencies which make their
analysis by mass spectrometry quite difficult; however, this sensitivity issue can be overcome by permethylating
glycans. Glycans have many isomers which can make their accurate analysis by LC-MS/MS difficult if the
isomers cannot be resolved. This proposal demonstrates that we are able to separate permethylated glycan
isomerss with a heated PGC column before mass spectrometry analysis (Aim 1), resulting in an extremely
sensitive assay to accurately characterize and quantitate glycan isomers in biological samples. Although the
separation of isomeric glycans has been previously reported, prior studies only resolved native and reducing end
labeled glycan structures. Owing to the fact that permethylated glycans exhibit ionization efficiencies at least two
orders of magnitude higher than the aforementioned structures, the importance of the increase in sensitivity,
for the detection of structures at physiological concentrations, that accompanies isomeric separation of
permethylated glycans (Aim 1) cannot be overstated.
 To overcome the variation in ionization efficiency between LC-MS samples, we have successfully
permethylated glycans with various stable isotope combinations to achieve unprecedented quantitative glycan
comparisons across samples derived from cell culture experiments, biological fluids, and biological tissues.
Through the implementation of our multi-level isotopic labeling strategies (metabolic 15N labeling, 18O reducing
end labeling and multiplex permethylation), the ...

## Key facts

- **NIH application ID:** 10019565
- **Project number:** 5R01GM112490-07
- **Recipient organization:** TEXAS TECH UNIVERSITY
- **Principal Investigator:** Yehia Mechref
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $283,987
- **Award type:** 5
- **Project period:** 2014-09-15 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10019565

## Citation

> US National Institutes of Health, RePORTER application 10019565, Sensitive and Quantitative MS-bases Glycomic Mapping Platform (5R01GM112490-07). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10019565. Licensed CC0.

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