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Methods Mol Biol. 2017;1550:99-113. doi: 10.1007/978-1-4939-6747-6_9.

Multi-Lectin Affinity Chromatography for Separation, Identification, and Quantitation of Intact Protein Glycoforms in Complex Biological Mixtures.

Author information

1
Department of Radiology, Canary Center at Stanford for Cancer Early Detection, Stanford University School of Medicine, 3155 Porter Drive, MC 5483, Palo Alto, CA, 94304, USA.

Abstract

Protein glycosylation is considered to be one of the most abundant post-translational modifications and is recognized for playing key roles in cellular functions. Aberrant N-linked glycosylation has been associated with several human diseases and has prompted the development and constant improvement of analytical tools to separate, characterize, and quantify glycoproteins in complex mixtures extracted from various biological samples (such as blood and tissue). Lectins, or carbohydrate-binding proteins, have been used as valuable tools for enriching for glycoproteins and selecting for specific types of glycosylation. Herein a method using multidimensional intact protein fractionation and LC-MS/MS analysis is described. Immunodepletion is used to remove highly abundant proteins from human plasma, followed by glycoform separation using multi-lectin affinity chromatography, in which specific lectins are chosen to capture and elute specific types of glycosylation. Reversed-phase chromatography prior to digestion is used for further fractionation, allowing for an increased number of protein identifications of moderate- to low-abundant proteins detectable in plasma. This method also incorporates isotopic labeling during alkylation for relative quantitation between two samples (such as a case and control). A bottom-up, tandem mass spectrometry-based proteomics approach is used for protein identification and quantitation, and allows for screening glycoform-specific changes across hundreds of plasma proteins.

KEYWORDS:

Glycoproteomics; Multi-lectin affinity chromatography; Plasma proteomics; Protein glycosylation

PMID:
28188526
DOI:
10.1007/978-1-4939-6747-6_9
[Indexed for MEDLINE]

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