Smart proteolysis samplers for pre‐lab bottom‐up protein analysis – Performance of on‐paper digestion compared to conventional digestion

Here the relation between digestion of proteins by trypsin covalently bound to paper and trypsin in-solution is investigated. The trypsin acting on paper is covalently bound. A trypsin concentration of 0.5% (w/v) results in the highest digestion activity of all concentrations tested. Additionally, it can be seen that trypsin on-paper has retained approx. 50% of its activity. Unlike trypsin in-solution, the stability of the smart proteolysis samplers was regarded to be stable for at least four months when kept refrigerated. Autolysis was very small for covalently bound trypsin: less than 2% compared to in-solution trypsin. Proteomic analysis of diluted human serum showed more protein identifications (214) in-solution digestions than on-paper digestions (76). Also, higher coverage for the in-solution digestion was obtained. Those proteins identified after on-paper digestion with no or few disulfide bonds seem to have more similar sequence coverages compared to those identified after in-solution digestion. Smart samplers allow the determination of at least 70-75 proteins without performing the overnight digestion. All in all, trypsin covalently bound to paper shows to retain high proteolytic activity and is a stable alternative for conventional digestions. In this way, smart proteolytic samplers show their feasibility in pre-lab sample preparation.