A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma

• Published in: Analytical biochemistry Vol. 408; no. 1; pp. 71 - 85
• Main Authors: Drake, Penelope M., Schilling, Birgit, Niles, Richard K., Braten, Miles, Johansen, Eric, Liu, Haichuan, Lerch, Michael, Sorensen, Dylan J., Li, Bensheng, Allen, Simon, Hall, Steven C., Witkowska, H. Ewa, Regnier, Fred E., Gibson, Bradford W., Fisher, Susan J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 2011
• Subjects: affinity chromatography; agglutinins; Aleuria; beta-fructofuranosidase; Biomarker; biomarkers; Biomarkers, Tumor - blood; Biomarkers, Tumor - chemistry; blood plasma; cadherins; Cancer; Chromatography, Affinity - methods; Chromatography, High Pressure Liquid - methods; Databases, Factual; Female; fucose; Glycopeptide; glycopeptides; Glycopeptides - chemistry; glycoproteins; Glycoproteins - blood; Glycoproteins - chemistry; Glycoproteins - metabolism; glycosidic linkages; high performance liquid chromatography; Humans; lactoferrin; Lectin chromatography; lectins; Lectins - chemistry; Male; Mass spectrometry; neoplasm proteins; Neoplasms - diagnosis; neutrophils; Plasma; polysaccharides; Polysaccharides - chemistry; Polysaccharides - isolation & purification; Protein Binding; Sambucus nigra; sialic acids; Spectrometry, Mass, Electrospray Ionization - methods; trypsin; Trypsin - metabolism; yeasts; Plasma; Biomarker; Glycopeptide; Mass spectrometry; Lectin chromatography; Cancer
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2010.08.010

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.