To characterize the structures of N-glycans derived from human serum, we report a strategy that combines microchip electrophoresis, standard addition, enzymatic digestion, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We compared: (i) electrophoretic mobilities of known N-glycans from well-characterized (standard) glycoproteins through standard addition, (ii) the electrophoretic mobilities of N-glycans with their molecular weights determined by MALDI-MS, and (iii) electrophoretic profiles of N-glycans enzymatically treated with fucosidase. The key step to identify the sialylated N-glycans was to quantitatively neutralize the negative charge on both α2,3- and α2,6-linked sialic acids by covalent derivatization with methylamine. Both neutralized and nonsialylated N-glycans from these samples were then reacted with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) to provide a fluorescent label and a triple-negative charge, separated by microchip electrophoresis, and detected by laser-induced fluorescence. The methylamidation step leads to a 24% increase in the peak capacity of the separation and direct correlation of electrophoretic and MALDI-MS results. In total, 37 unique N-glycan structures were assigned to 52 different peaks recorded in the electropherograms of the serum samples. This strategy ensures the needed separation efficiency and detectability, easily resolves linkage and positional glycan isomers, and is highly reproducible.

Colorectal cancer is the fourth most prevalent cancer in the United States, yet there are no reliable non-invasive early screening methods available. Serum-based glycomic profiling has the necessary sensitivity and specificity to distinguish disease states and provide diagnostic potential for this deadly form of cancer. We applied microchip electrophoresis and MALDI-TOF-MS-based glycomic procedures to 20 control serum samples and 42 samples provided by patients diagnosed with colorectal cancer. Within the identified glycans, the position of fucose units was located to quantitate possible changes of fucosyl isomeric species associated with the pathological condition. MALDI-MS data revealed several fucosylated tri- and tetra-antennary glycans which were significantly elevated in their abundance levels in the cancer samples and distinguished the control samples from the colorectal cancer cohort in the comprehensive profiles. When compared to other cancers studied previously, some unique changes appear to be associated with colorectal cancer, being primarily associated with fucosyl isomers. Through MS and microchip electrophoresis-based glycomic methods, several potential biomarkers were identified to aid in the diagnosis and differentiation of colorectal cancer. With its unique capability to resolve isomers, microchip electrophoresis can yield complementary analytical information to MS-based profiling.