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Particle-based N-linked glycan analysis of selected proteins from biological samples using nonglycosylated binders
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
Stockholm University, Faculty of Science, Department of Environmental Science and Analytical Chemistry.
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Number of Authors: 6
2017 (English)In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 132, 125-132 p.Article in journal (Refereed) Published
Abstract [en]

Glycosylation is one of the most common and important post-translational modifications, influencing both the chemical and the biological properties of proteins. Studying the glycosylation of the entire protein population of a sample can be challenging because variations in the concentrations of certain proteins can enhance or obscure changes in glycosylation. Furthermore, alterations in the glycosylation pattern of individual proteins, exhibiting larger variability in disease states, have been suggested as biomarkers for different types of cancer, as well as inflammatory and neurodegenerative diseases. In this paper, we present a rapid and efficient method for glycosylation analysis of individual proteins focusing on changes in the degree of fucosylation or other alterations to the core structure of the glycans, such as the presence of bisecting N-acetylglucosamines and a modified degree of branching. Streptavidin-coated magnetic beads are used in combination with genetically engineered immunoaffinity binders, called VHH antibody fragments. A major advantage of the VHHs is that they are nonglycosylated; thus, enzymatic release of glycans from the targeted protein can be performed directly on the beads. After deglycosylation, the glycans are analyzed by MALDI-TOF-MS. The developed method was evaluated concerning its specificity, and thereafter implemented for studying the glycosylation pattern of two different proteins, alpha-1-antitrypsin and transferrin, in human serum and cerebrospinal fluid. To our knowledge, this is the first example of a protein array-type experiment that employs bead-based immunoaffinity purification in combination with mass spectrometry analysis for fast and efficient glycan analysis of individual proteins in biological fluid.

Place, publisher, year, edition, pages
2017. Vol. 132, 125-132 p.
Keyword [en]
Glycosylation pattern, Biomarkers, Magnetic beads, VHH antibody fragments, Cerebrospinal fluid, MALDI-MS
National Category
Chemical Sciences Medicinal Chemistry
Identifiers
URN: urn:nbn:se:su:diva-137554DOI: 10.1016/j.jpba.2016.09.029ISI: 000389015700017PubMedID: 27718394OAI: oai:DiVA.org:su-137554DiVA: diva2:1064774
Available from: 2017-01-13 Created: 2017-01-09 Last updated: 2017-01-13Bibliographically approved

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Karlsson, IsabellaNdreu, LorenaQuaranta, AlessandroThorsén, Gunnar
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