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.