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Separation and Identification of Permethylated Glycan Isomers by Reversed Phase NanoLC-NSI-MSⁿ.

ABSTRACT: HPLC 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 multidimensional ion fragmentation (MSⁿ) 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 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 and robust structural characterization and is amenable to autosampling with associated throughput enhancements.

SUBMITTER: Kurz S

PROVIDER: S-EPMC8724860 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The intrinsic nature of glycosylation, namely nontemplate encoded, stepwise elongation and termination with a diverse range of isomeric glyco-epitopes (glycotopes), translates into ambiguity in most cases of mass spectrometry (MS)-based glycomic mapping. It is arguable that whether one needs to delineate every single glycomic entity, which may be counterproductive. Instead, one should focus on identifying as many structural features as possible that would collectively define the glycomic characteristics of a cell or tissue, and how these may change in response to self-programmed development, immuno-activation, and malignant transformation. We have been pursuing this line of analytical strategy that homes in on identifying the terminal sulfo-, sialyl, and/or fucosylated glycotopes by comprehensive nanoLC-MS2-product dependent MS3 analysis of permethylated glycans, in conjunction with development of a data mining computational tool, GlyPick, to enable an automated, high throughput, semi-quantitative glycotope-centric glycomic mapping amenable to even nonexperts. We demonstrate in this work that diagnostic MS2 ions can be relied on to inform the presence of specific glycotopes, whereas their possible isomeric identities can be resolved at MS3 level. Both MS2 and associated MS3 data can be acquired exhaustively and processed automatically by GlyPick. The high acquisition speed, resolution, and mass accuracy afforded by top-notch Orbitrap Fusion MS system now allow a sensible spectral count and/or summed ion intensity-based glycome-wide glycotope quantification. We report here the technical aspects, reproducibility and optimization of such an analytical approach that uses the same acidic reverse phase C18 nanoLC conditions fully compatible with proteomic analysis to allow rapid hassle-free switching. We further show how this workflow is particularly effective when applied to larger, multiply sialylated and fucosylated N-glycans derived from mouse brain. The complexity of their terminal glycotopes including variants of fucosylated and disialylated type 1 and 2 chains would otherwise not be adequately delineated by any conventional LC-MS/MS analysis.

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Separation and Identification of Permethylated Glycan Isomers by Reversed Phase NanoLC-NSI-MSⁿ.

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OmicsDI is part of the ELIXIR infrastructure

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Dataset Information

Separation and Identification of Permethylated Glycan Isomers by Reversed Phase NanoLC-NSI-MSn.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Separation and Identification of Permethylated Glycan Isomers by Reversed Phase NanoLC-NSI-MSⁿ.