Discovery of sialyl Lewis A and Lewis X modified protein cancer biomarkers using high density antibody arrays.

We report on a high-dimensional method to globally profile glycoproteins that are modified with sialyl Lewis A or Lewis X glycans. Specifically, glycoproteins in serum or plasma are fractionated on a high-density antibody microarray (i.e., each are localized to their specific antibody spot) and are specifically detected via fluorescently labeled anti-sialyl Lewis A or anti-Lewis X antibodies with quantification in a microarray scanner. Non-glycosylated proteins or glycoproteins with other glycan motifs do not interfere with this assay. The whole process is very rapid and applicable for high-throughput screening without the need for purification of glycoproteins from the samples. Using these methods, sialyl Lewis A or Lewis X moieties were found to be expressed on many previously unreported secreted or membrane associated proteins. Furthermore, the combination of sialyl Lewis A or Lewis X content with protein level increased the ability of certain glycoproteins to distinguish 30 patients with stage III and IV colon cancer from 60 control samples. Thus, this highly sensitive method is capable of discovering novel specific glycan modifications on proteins, many of which will likely be useful for disease detection and monitoring. BIOLOGICAL SIGNIFICANCE: In this paper, we show that we can detect cancer-specific glycan modifications on thousands of proteins using a high-density antibody array paired with a glycan specific antibody to probe the bound glycoproteins. To our knowledge, our array is by far the largest and densest that has ever been used for global profiling of specific glycan modification on proteins. Analysis of colon cancer patient plasma for sialyl Lewis A and Lewis X modifications revealed previously unknown protein carriers of these modifications and significant increases in these specific glycans on some proteins in people with cancer versus healthy controls, suggesting this method could be used to discover novel biomarkers.