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

Abstract

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.

Figure 1.

Workflow for seamlessly integrated MICROFASP method with the fractionation technique for in-depth proteomics analysis of a mass-limited sample.

Figure 2.

(A) Effect of ACN gradients on the number of identifications. (B) Number of peptides identified in each fraction under different gradients. (C) Number of peptides identified in four combined fractions under Gradient-2.

Figure 3.

(A) Numbers of peptides and proteins identified by salt gradient and pH gradient. (B) Numbers of peptides identified in each fraction by salt gradient and pH gradient.

Figure 4.

(A) Label-free quantitation result of the protein expressions identified in duplicate high-pH RP fractionation experiments under Gradient-2. (B) Label-free quantitation result of the protein expressions identified in three fractionation techniques.