Use of glycan targeting antibodies to identify cancer-associated glycoproteins in plasma of breast cancer patients

Immunoaffinity chromatography (IAC) was used to isolate and identify potential cancer biomarker glycoproteins by targeting disease-associated glycans. Glycoproteins were selected from plasma of disease-free and breast cancer patients with an anti-Lewis x (Le(x)) IAC column. After extensive washing of the IAC column to remove abundant proteins, the selected proteins were eluted with an acidic mobile phase and identified in two ways. The protocol used in route A involved the steps of tryptic digestion, reversed-phase chromatographic fractionation of the digest, and identification of peptides in collected RPC fractions by MALDI-MS/MS. Route B differed in that IAC-selected proteins were further fractionated by reversed-phase chromatography before proteolysis of individual chromatographic fractions and identification by MALDI-MS/MS. Route A was the more efficacious of the two protocols in total number of proteins identified. Of the 26 proteins identified, 9 were found to be potential breast cancer marker candidates based on their elevation in breast cancer patients. The potential of these candidates as cancer markers remains to be validated in much larger, more diverse populations of breast cancer patients.