Next generation VAMS®–Trypsin immobilization for instant proteolysis in bottom-up protein determination. | ENEngelskEnglishNext generation VAMS®–Trypsin immobilization for instant proteolysis in bottom-up protein determination.
2022
Reubsaet, Leon | Thiede, Bernd | Halvorsen, Trine Grønhaug
The purpose of this study was to develop a smart proteolysis sampler based on commercially available VAMS® tips employing Volumetric Absorptive Microsampling. In this proof-of-concept paper we show that covalently bound trypsin to VAMS exhibits sufficient activity for proteomic purposes without altering physical conditions. Alkaline pH was needed for the covalent binding of trypsin and at least 50 μg trypsin was required to obtain maximal activity. Prolonged digestion times (up to overnight) of a sample containing 7 model proteins on the VAMS yielded higher peak intensities for signature peptides which are beneficial for quantitative analysis. In-solution digestions of three proteins (carbonic anhydrase 2, catalase and BSA) yielded higher intensities of their signature peptides whereas the other four proteins (cytochrome C, myoglobin, β-lactoglobulin, and phosphorylase B) showed higher intensities for their signature peptides from digestions performed on trypsin modified VAMS. In comparison with conventional in-solution digestions, approximately half of the proteins were identified from diluted serum using trypsin on VAMS. For shotgun serum proteomics, diluted samples collected on trypsin modified VAMS followed by immediate drying yielded similar peptide- (942 vs 875) and protein (96 vs 99) identifications compared to those collected on trypsin modified VAMS followed by overnight digestions. This result shows the potential of trypsin on VAMS digestions.
显示更多 [+] 显示较少 [-]