Seamlessly Integrated Miniaturized Filter-Aided Sample Preparation Method to Fractionation Techniques for Fast, Loss-Less, and In-Depth Proteomics Analysis of 1 μg of Cell Lysates at Low Cost.

We report an integrated platform that enabled a seamlessly coupling miniaturized filter-aided sample preparation (MICROFASP) method to high-pH reversed phase (RP) or strong cation exchange (SCX) microreactors for low-loss sample preparation and fractionation of 1 μg of cell lysates prior to LC-ESI-MS/MS analysis. Due to the reduced size of the microreactor, only 5 μL of buffer volume is required to generate each fraction, which speeds both elution and lyophilization. The fraction was directly eluted into an autosampler insert vial for LC-MS analysis to reduce sample transfer steps and minimize sample loss as well as contamination. The flow-through sample generated during the loading step was also collected and analyzed. The integrated platform generated 48,890 unique peptides and 4723 protein groups from 1 μg of a K562 cell lysate using MICROFASP and C18 microreactor-based high-pH RP fractionation methods, which are comparable with the state-of-the-art result using in-StageTip sample preparation and nanoflow RPLC-based fractionation methods but with a significant reduction in cost and time. Both pH gradient elution and salt gradient elution approaches provide high reproducibility for the SCX microreactor-based fractionation method. This integrated platform has significant potential in deep proteomics analysis of mass-limited samples with reduced time and equipment requirements.