期刊
ANALYTICAL BIOCHEMISTRY
卷 408, 期 1, 页码 71-85出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2010.08.010
关键词
Lectin chromatography; Glycopeptide; Plasma; Cancer; Biomarker; Mass spectrometry
资金
- NCRR [S10 RR024615]
- National Cancer Institute [U24 CA126477]
- Buck Institute for Age Research
- NCI Clinical Proteomic Technologies for Cancer initiative
- NATIONAL CANCER INSTITUTE [U24CA126477] Funding Source: NIH RePORTER
- NATIONAL CENTER FOR RESEARCH RESOURCES [S10RR024615] Funding Source: NIH RePORTER
Glycans are cell-type-specific, posttranslational protein modifications that are modulated during developmental and disease processes. As such, glycoproteins are attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow that incorporates lectin affinity chromatography to enrich for proteins that carry specific glycan structures. As increases in sialylation and fucosylation are prominent among cancer-associated modifications, we focused on Sambucus nigra agglutinin (SNA) and Aleuria aurantia lectin (AAL), lectins which bind sialic acid- and fucose-containing structures, respectively. Fucosylated and sialylated glycopeptides from human lactoferrin served as positive controls, and high-mannose structures from yeast invertase served as negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested human plasma from healthy donors. Samples were loaded onto lectin columns, separated by HPLC into flow-through and bound fractions, and treated with peptide: N-glycosidase F to remove N-linked glycans. The deglycosylated peptide fractions were interrogated by ESI HPLC-MS/MS. We identified a total of 122 human plasma glycoproteins containing 247 unique glycosites. Importantly, several of the observed glycoproteins (e.g., cadherin 5 and neutrophil gelatinase-associated lipocalin) typically circulate in plasma at low nanogram per milliliter levels. Together, these results provide mass spectrometry-based evidence of the utility of incorporating lectin-separation platforms into cancer biomarker discovery pipelines. (C) 2010 Elsevier Inc. All rights reserved.
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