A site-specific map of the human plasma glycome and its age and gender-associated alterations

Alexander A Merleev,Emanual Maverakis,Guillaume Luxardi,Kyoungmi Kim,Carlito B Lebrilla,Yu Wan ,L Renee Ruhaak,Alina I Marusina,Iannis E Adamopoulos,Gege Xu,Qiuting Hong,Dayoung Park,Forum Patel,Michiko Shimoda,Anupum Mitra,Stephanie T Le ,Muchena J Kailemia,Qiongyu Li,Yixuan Xie,Nelvish N Lal

doi:10.1038/s41598-020-73588-x

Abstract

Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR < 0.05) site-specific inter-protein and intra-protein glycan associations, of which the vast majority were previously unknown. Since age and gender are relevant covariants in biomarker research, these variables were also characterized. 13 glycopeptides were found to be associated with gender and 41 to be associated with age. Using just five age-associated glycopeptides, a highly accurate age prediction model was constructed and validated (r2 = 0.62 ± 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.

Highlights

Alterations in the human glycome have been associated with cancer and autoimmunity
Glycan structures are presented as a four digit code where the first numeral represents the total number of mannose and galactose residues combined, the second represents the total number of N-acetylglucosamine residues, the third numeral corresponds to the number of fucose residues, and the final numeral is the number of sialic acid moieties
With knowledge of the retention times and collision induced dissociation (CID) behavior of the most abundant serum glycoforms[17,23] (Table S1), we characterized the relative abundance of 159 glycopeptides

Summary

Introduction

Alterations in the human glycome have been associated with cancer and autoimmunity. constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. On the left of each diagram are the square of the intra-protein Pearson Product Moment Correlation Coefficients (PPMCCs) for connected glycan pair As part of their glycoscience “Roadmap”[2], the National Research Council of the U.S National Academies highlighted the importance of developing a site-specific map of the serum glycome, which would aid in the development of glycans as biomarkers of human diseases. One reason for the excitement around the use of glycans as disease-specific biomarkers is that glycosylation is a process influenced by a variety of factors including: the type of cell and its activation state; environmental factors, such as the presence of available metabolites; the age of the cell, as glycan moieties can be lost over time; and inflammatory mediators, such as cytokines and chemokines All these factors can be altered in the setting of human diseases, making the glycome an expression of the overall health status of an individual. A site-specific glycoprofiling method could theoretically increase the accuracy of a serum protein biomarker by subdividing it into its different glycoforms

Objectives

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Conclusion