title: A lectin affinity workflow targeting glycosite-specific, cancer-related carbohydrate structures in trypsin-digested human plasma.
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.
authors: Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, 94143, USA.
authors: Penelope M
authors: PM
authors: Drake
authors: Birgit
authors: B
authors: Schilling
authors: Richard K
authors: RK
authors: Niles
authors: Miles
authors: M
authors: Braten
authors: Eric
authors: E
authors: Johansen
authors: Haichuan
authors: H
authors: Liu
authors: Michael
authors: M
authors: Lerch
authors: Dylan J
authors: DJ
authors: Sorensen
authors: Bensheng
authors: B
authors: Li
authors: Simon
authors: S
authors: Allen
authors: Steven C
authors: SC
authors: Hall
authors: H Ewa
authors: HE
authors: Witkowska
authors: Fred E
authors: FE
authors: Regnier
authors: Bradford W
authors: BW
authors: Gibson
authors: Susan J
authors: SJ
authors: Fisher
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood / Q000737:chemistry*; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016208:Databases, Factual; D005260:Female; D006020:Glycopeptides / Q000737:chemistry; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000737:chemistry*; D008297:Male; D009369:Neoplasms / Q000175:diagnosis; D011134:Polysaccharides / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
chemical_list: D014408:Biomarkers, Tumor; D006020:Glycopeptides; D006023:Glycoproteins; D037102:Lectins; D011134:Polysaccharides; C064549:lectin, Aleuria aurantia; D014357:Trypsin