Optimization of Digestion Parameters for Efficient Proteolysis

We assessed the impact of temperature (incubation temperature used 37 ⁰C vs 42 ⁰C) and time on proteolytic efficiency using either manual sample preparation or the i7 automation workstation. In a further optimization step, the impact of incubation length was explored using a 4 hr incubation and the longer incubation (16hr). Yield of peptide and protein identification under different denaturation buffers conditions (TFE 5% vs. 10%) was assessed as well as number of peptides with 0, 1, 2  or 3 miscleavages recovered from digestion when using TFE 5% or TFE 10% denaturation buffers.

Optimization of DIA Acquisition Parameters for Plasma

We used plasma to compare the performance of 8 MS methods covering 4 isolation window widths and 2 resolutions (15 KDa x 77 windows, 18 KDa x 54 windows, 21 KDa x 50 windows, 40 KDa x 20 windows, MS2 resolutions 15 or 30 k).