[Comparison of the performance of secretome analysis based on metabolic labeling by three unnatural sugars].

Many secreted proteins, including cytokines, growth factors and hormones, are crucial in processes like intercellular signaling. Dynamic changes in secreted proteins usually reflect the growth and pathological state of the cells. Many drug targets are secretory proteins. The proteins are also important biomarkers. Conditioned cell culture media are important samples for secretory proteomic studies. Biomass spectrometry-based proteomic analysis enables the systematic study of secretory proteins. The main problem in analyzing secretory proteins in conditioned culture media is the low concentration of these proteins and the presence of serum, amino acids, and additives in culture media that may interfere with the protein analysis. Conventional secretory proteome analysis uses serum-free cell culture to reduce sample complexity, and typically involves protein concentration, purification, and desalting using ultrafiltration, dialysis, lyophilization, and trichloroacetic acid (TCA) or acetone precipitation, followed by enzymatic digestion and mass spectrometry analysis. This analytical process does not allow specific enrichment of secreted proteins. Thus, only a few secreted proteins can be identified. In addition, prolonged serum-free incubation of cells also tends to lead to unexpected changes in their activity status. A bioorthogonal-based enrichment approach can effectively avoid this problem. In recent years, unnatural sugars containing bio-orthologous groups, such as azide groups, have been used to metabolically label glycosylated proteins, enabling cellular imaging or selective enrichment of glycoproteins and their use for proteomic analysis. The strategy is a two-step process. First, azide-based sugar analogues are added to the cell culture medium and introduced to glycoproteins via the intracellular glycan biosynthesis pathway. Second, they are specifically covalently labeled with imaging probes or affinity probes via click chemistry. Since secreted proteins are usually glycoproteins, this glycolytic labeling has been used to label and enrich secreted proteins.