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

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

Authors

Lina A. Dahabiyeh

David Tooth

David A. Barrett



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.

Journal Article Type Article
Publication Date Feb 15, 2019
Journal Analytical Biochemistry
Print ISSN 0003-2697
Electronic ISSN 1096-0309
Publisher Elsevier
Peer Reviewed Peer Reviewed
Volume 567
Pages 72-81
APA6 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
DOI https://doi.org/10.1016/j.ab.2018.12.011
Keywords Glycoprotein; Label-freeTargeted LC-MS/MS; ConA; RP-SPE; Stable isotope standard
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.

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