Thiol-ene-based microfluidic chips for glycopeptide enrichment and online digestion of inflammation-related proteins osteopontin and immunoglobulin G

Proteins, and more specifically glycoproteins, have been widely used as biomarkers, e.g., to monitor disease states. Bottom-up approaches based on mass spectrometry (MS) are techniques commonly utilized in glycoproteomics, involving protein digestion and glycopeptide enrichment. Here, a dual function polymeric thiol-ene-based microfluidic chip (TE microchip) was applied for the analysis of the proteins osteopontin (OPN) and immunoglobulin G (IgG), which have important roles in autoimmune diseases, in inflammatory diseases, and in coronavirus disease 2019 (COVID-19). TE microchips with larger internal surface features immobilized with trypsin were successfully utilized for OPN digestion, providing rapid and efficient digestion with a residence time of a few seconds. Furthermore, TE microchips surface-modified with ascorbic acid linker (TEA microchip) have been successfully utilized for IgG glycopeptide enrichment. To illustrate the use of the chips for more complex samples, they were applied to enrich IgG glycopeptides from human serum samples with antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The dual functional TE microchips could provide high throughput for online protein digestion and glycopeptide enrichment, showing great promise for future extended applications in proteomics and the study of related diseases. Graphical Abstract: [Figure not available: see fulltext.]