Profiling of 54 plasma glycoproteins by label-free targeted LC-MS/MS

Dahabiyeh, Lina A.; Tooth, David; Barrett, David A.

Abstract

Glycoproteins play a central role in diverse biological processes and are linked with many serious human diseases. In this paper we present a simple, reproducible and cost-effective analytical workflow that enables the reliable quantification of clinically relevant human plasma glycoproteins using label free microflow LC-MS/MS analysis. Plasma N-glycoproteins were selectively extracted via ConA Sepharose lectin affinity chromatography then separated into two fractions using reversed-phase solid phase extraction. LC-MS/MS analysis of the tryptic digest of both fractions identified 90 proteins from which 54 clinically relevant glycoproteins were selected for protein profiling. Careful assessment of the chosen peptides and transitions in terms of reproducibility, selectivity, signal intensity and peak shape was carried out. Measurement of the analytical precision of the method revealed the majority of glycoproteins showed a coefficient of variation (CV) ≤15% (median CV 11.8%, range 3.6–33%). The method was successfully applied to compare glycoproteins in plasma and serum and to detect changes in glycoprotein profile in perturbed plasma pre-treated with ammonium sulphate. Our results show that label-free methodology can be a cost-effective affordable alternative to stable isotope standard workflow when applied for relative protein quantification, especially when targeting a large number of proteins in bioanalytical measurements.

Citation

Dahabiyeh, L. A., Tooth, D., & Barrett, D. A. (2019). Profiling of 54 plasma glycoproteins by label-free targeted LC-MS/MS. Analytical Biochemistry, 567, 72-81. https://doi.org/10.1016/j.ab.2018.12.011

• Journal Article Type: Article
• Acceptance Date: Dec 15, 2018
• Online Publication Date: Dec 13, 2018
• Publication Date: Feb 15, 2019
• Deposit Date: May 1, 2019
• Publicly Available Date: Dec 14, 2019
• Journal: Analytical Biochemistry
• Print ISSN: 0003-2697
• Electronic ISSN: 1096-0309
• Publisher: Elsevier
• Peer Reviewed: Peer Reviewed
• Volume: 567
• Pages: 72-81
• DOI: https://doi.org/10.1016/j.ab.2018.12.011
• Keywords: Glycoprotein; Label-freeTargeted LC-MS/MS; ConA; RP-SPE; Stable isotope standard
• Public URL: https://nottingham-repository.worktribe.com/output/1861888
• Publisher URL: https://www.sciencedirect.com/science/article/pii/S0003269718310327?via%3Dihub
• Additional Information: This article is maintained by: Elsevier; Article Title: Profiling of 54 plasma glycoproteins by label-free targeted LC-MS/MS; Journal Title: Analytical Biochemistry; CrossRef DOI link to publisher maintained version: https://doi.org/10.1016/j.ab.2018.12.011; Content Type: article; Copyright: © 2019 Elsevier Inc. All rights reserved.