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

Anal Biochem. 2011 Jan 1;408(1):71-85. doi: 10.1016/j.ab.2010.08.010. Epub 2010 Aug 10.


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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Biomarkers, Tumor / blood
  • Biomarkers, Tumor / chemistry*
  • Chromatography, Affinity / methods
  • Chromatography, High Pressure Liquid / methods*
  • Databases, Factual
  • Female
  • Glycopeptides / chemistry
  • Glycoproteins / blood
  • Glycoproteins / chemistry*
  • Glycoproteins / metabolism
  • Humans
  • Lectins / chemistry*
  • Male
  • Neoplasms / diagnosis
  • Polysaccharides / chemistry*
  • Polysaccharides / isolation & purification
  • Protein Binding
  • Spectrometry, Mass, Electrospray Ionization / methods*
  • Trypsin / metabolism


  • Biomarkers, Tumor
  • Glycopeptides
  • Glycoproteins
  • Lectins
  • Polysaccharides
  • lectin, Aleuria aurantia
  • Trypsin