| Large-scale glycoprotein profiling is of interest to biomedical researchers because of the diverse physiological roles of these macromolecules and their associated potential as specific molecular diagnostics when the normal expression levels are dysregulated. However, glycoproteomics remains analytically challenging because of measurement-related interference from non-glycoprotein components in the biological samples. To address this challenge, a multimethodological platform has been established that utilizes state-of-the-art liquid chromatography (LC) and mass spectrometry (MS). Glycoproteins were extracted from biological samples with a systematically developed, high-throughput lectin affinity chromatography approach. Furthermore, a novel macroporous chromatography particle was utilized for high-performance affinity chromatography (HPAC). The spherical particle contains an interconnected network of macropores that offers a high accessible surface area for lectin-based capture of glycoproteins. The HPAC columns reproducibly enriched glycoproteins from a mere 1-microliter aliquot of blood serum, as demonstrated by mass-spectrometric measurements downstream. A software program, ProteinQuant, was designed to process the quantitative proteomic data from LC-MS measurements, reducing several hundred hours of manual integration to an automated overnight process. The overall efficacy of the platform was demonstrated in an investigation of fucosylated glycoproteins in a small number of blood serum samples from esophageal cancer patients compared to healthy controls. Finally, the approach was applied to a study of fluids collected from premalignant pancreatic cysts. The cyst fluid glycome was also profiled with a highly sensitive solid-phase permethylation procedure followed by MS. A number of cancer-associated hyperfucosylated glycans were identified during this process, which directed subsequent lectin enrichment and LC-MS measurements of the hyperfucosylated glycoproteins. The combination of glycomic and glycoproteomic profiling analyses of pancreatic cyst fluids, both of which were reported for the first time herein, provided insight into the molecular make-up of this rare and medically important biological material. |
