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
Date
08/14/2010Pubmed ID
20705048Pubmed Central ID
PMC3205961DOI
10.1016/j.ab.2010.08.010Scopus ID
2-s2.0-77958450102 (requires institutional sign-in at Scopus site) 57 CitationsAbstract
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 SJAuthor
Michael Lerch PhD Associate Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Biomarkers, TumorChromatography, Affinity
Chromatography, High Pressure Liquid
Databases, Factual
Female
Glycopeptides
Glycoproteins
Humans
Lectins
Male
Neoplasms
Polysaccharides
Protein Binding
Spectrometry, Mass, Electrospray Ionization
Trypsin
