Medical College of Wisconsin
CTSICores SearchResearch InformaticsREDCap

A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma. Anal Biochem 2011 Jan 01;408(1):71-85 PMID: 20705048 PMCID: PMC3205961

Abstract

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.

Author List

Drake PM, Schilling B, Niles RK, Braten M, Johansen E, Liu H, Lerch M, Sorensen DJ, Li B, Allen S, Hall SC, Witkowska HE, Regnier FE, Gibson BW, Fisher SJ

Author

Michael Lerch PhD Assistant Professor in the Biophysics department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Chromatography, Affinity
Chromatography, High Pressure Liquid
Databases, Factual
Female
Glycopeptides
Glycoproteins
Humans
Lectins
Male
Neoplasms
Polysaccharides
Protein Binding
Spectrometry, Mass, Electrospray Ionization
Trypsin



View this publication's entry at the Pubmed website PMID: 20705048
jenkins-FCD Prod-153 3ca6710ea990189ceb85f4312e7298b5922ce1a6