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J Proteome Res. 2017 Mar 3;16(3):1249-1260. doi: 10.1021/acs.jproteome.6b00849. Epub 2017 Feb 15.

Parallel Comparison of N-Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC.

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Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine , 3155 Porter Drive MC5483, Palo Alto, California 94304, United States.
ThermoFisher Scientific , 1400 Northpoint Parkway Suite 10, West Palm Beach, Florida 33407, United States.


Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liquid chromatography using strong anion exchange solid-phase extraction (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was observed in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were additionally analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these experiments, 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation analysis in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.


N-linked glycopeptide enrichment; glycoproteomics; mass spectrometry; plasma; protein glycosylation

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