Abstract

The Suspension Trapping (S-Trap) method has been a prominent sample preparation technique since its introduction in 2014. Its capacity to induce protein aggregation using organic solvents has significantly improved protein purification and facilitated peptide identification. However, its full potential for automation has been limited by the lack of a suitable liquid han-dling system until recently. In this study, we aimed to enhance the automation of S-Trap sample preparation by optimizing the S-Trap digestion process, incorporating triethylammonium bicarbonate (TEAB) and CaCl 2 . The utilization of TEAB buffer condi-tions in this innovative process led to a noteworthy 12% improvement in protein identification. Additionally, through careful observation of various incubation conditions, we streamlined the entire sample preparation workflow into a concise 4 hours timeline, covering reduction, alkylation, and trypsin incubation stages. This refined and expedited automated S-Trap digestion process not only showcased exceptional time efficiency but also improved trypsin digestion, resulting in increased protein iden- tification.