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

摘要: Glycans are cell-type-specific, posttranslational protein modifications that modulated during developmental and disease processes. As such, glycoproteins attractive biomarker candidates. Here, we describe a mass spectrometry-based workflow incorporates lectin affinity chromatography to enrich for proteins carry specific glycan structures. increases in sialylation fucosylation prominent among cancer-associated modifications, focused on Sambucus nigra agglutinin (SNA) Aleuria aurantia (AAL), lectins which bind sialic acid- fucose-containing structures, respectively. Fucosylated sialylated glycopeptides from human lactoferrin served as positive controls, high-mannose structures yeast invertase negative controls. The standards were spiked into Multiple Affinity Removal System (MARS) 14-depleted, trypsin-digested plasma healthy donors. Samples loaded onto columns, separated by HPLC flow-through bound fractions, treated with peptide: N-glycosidase F remove N-linked glycans. deglycosylated peptide fractions interrogated ESI HPLC-MS/MS. We identified total of 122 containing 247 unique glycosites. Importantly, several the observed (e.g., cadherin 5 neutrophil gelatinase-associated lipocalin) typically circulate at low nanogram per milliliter levels. Together, these results provide evidence utility incorporating lectin-separation platforms cancer discovery pipelines.