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Targeted, Site-specific quantitation of N- and O-glycopeptides using 18O–labeling and product ion based mass spectrometry

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Abstract

The site-specific quantitation of N- and O-glycosylation is vital to understanding the function(s) of different glycans expressed at a given site of a protein under physiological and disease conditions. Most commonly used precursor ion intensity based quantification method is less accurate and other labeled methods are expensive and require enrichment of glycopeptides. Here, we used glycopeptide product (y and Y0) ions and 18O–labeling of C-terminal carboxyl group as a strategy to obtain quantitative information about fold-change and relative abundance of most of the glycoforms attached to the glycopeptides. As a proof of concept, the accuracy and robustness of this targeted, relative quantification LC-MS method was demonstrated using Rituximab. Furthermore, the N-glycopeptide quantification results were compared with a biosimilar of Rituximab and validated with quantitative data obtained from 2-AB-UHPLC-FL method. We further demonstrated the intensity fold-change and relative abundance of 46 unique N- and O-glycopeptides and aglycopeptides from innovator and biosimilar samples of Etanercept using both the normal-MS and product ion based quantitation. The results showed a very similar site-specific expression of N- and O-glycopeptides between the samples but with subtle differences. Interestingly, we have also been able to quantify macro-heterogeneity of all N- and O-glycopetides of Etanercept. In addition to applications in biotherapeutics, the developed method can also be used for site-specific quantitation of N- and O-glycopeptides and aglycopeptides of glycoproteins with known glycosylation pattern.

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Abbreviations

2-AB:

2-amino benzamide

AGP:

aglycopeptide or unoccupied glycopeptide

Asp-N:

endoprotinase AspN;

CE:

collision energy

CID:

collision induced dissociation

Cmc:

carboxymethylation

DDA:

data-dependent acquisition

DIA:

data-independent acquisition

ECD:

electron capture dissociation

ER:

endoplasmic reticulum

ETD:

electron transfer dissociation

Fuc:

fucose

GalNAc:

N-acetylgalactosamine

GlcNAc:

N-acetylglucosamine

GP:

glycopeptide(s)

HCD:

higher-energy collision dissociation

HILIC:

hydrophilic interaction liquid chromatography

IR:

insulin resistance

iTRAQ:

isobaric tag for relative and absolute quantitation

LC:

liquid chromatography

Man:

mannose

MRM:

multiple reaction monitoring

MRM-HR:

multiple reaction monitoring – high resolution

NeuAc:

N-acetylneuraminic acid

PGC:

porous graphitized carbon

PRM:

parallel reaction monitoring

RP:

reversed phase

RT:

retention time

SILAC:

stable isotope labeling with amino acids in cell culture

SPE:

solid phase extraction

XIC:

extracted ion chromatogram

UHPLC-FL:

Ultra-high performance liquid chromatography with fluorescence detector.

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Acknowledgments

We thank Department of Biotechnology, Govt. India, Ramaswamy, S. and Saiyed, T. of C-CAMP for their support. This work was supported through internal technology development funding to mass spectrometry facility of C-CAMP. We would like to thank AB Sciex, Pvt. Ltd. for supporting with mass spectrometer and accessories through collaboration. We thank the reviewers for their suggestion to improve readability of the manuscript.

Author contributions

PB conceived the project, designed experiments, analyzed data and written the paper. SJ and AR performed the experiments, analyzed the data and contributed to writing the paper.

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Authors and Affiliations

  1. Glycomics and Glycoproteomics & Biologics Characterization Facility, Centre for Cellular and Molecular Platforms, National Centre for Biological Sciences (NCBS) - TIFR, GKVK Post, Bellary Road, Bangalore, Karnataka, 560065, India

    Jandhyam Srikanth, Rathinasamy Agalyadevi & Ponnusamy Babu

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  1. Jandhyam Srikanth
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  2. Rathinasamy Agalyadevi
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Correspondence to Ponnusamy Babu.

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The online version of this article contains supplementary material, which is available to authorized users. The supplementary information file (pdf) includes detailed experimental section, supplementary Tables S1 to S3, Figure legends, and supplementary Figs. S1 to S22.

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Srikanth, J., Agalyadevi, R. & Babu, P. Targeted, Site-specific quantitation of N- and O-glycopeptides using 18O–labeling and product ion based mass spectrometry. Glycoconj J 34, 95–105 (2017). https://doi.org/10.1007/s10719-016-9733-8

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  • DOI: https://doi.org/10.1007/s10719-016-9733-8

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