Deep middle-down proteomics using dissolvable BAC cross-linked Polyacrylamide gel electrophoresis

In the analysis of proteoforms by mass spectrometry (MS)-based proteomics, it is difficult to glean comprehensive information about proteoforms using the bottom-up approach because short peptide fragments, obtained by enzymatic digestion such as trypsin, are used for analysis. As an alternative, the top-down approach, in which proteoforms are introduced into a mass spectrometer in intact form allowing analysis of their intact chemical structures, has become the mainstream. However, most of the proteoforms that can be measured in current top-down proteomics are 50 kDa or smaller, and the analysis of higher molecular weight proteoforms has remained a challenge. As a middle-ground approach, middle-down proteomics, which analyzes middle-down peptides obtained by limited proteoform digestion, is attracting attention as a practical solution for obtaining proteoform information from a wide range of molecular weight regions.

We have developed a multidimensional separation workflow, GeLC-FAIMS-MS, which combines high-resolution intact proteoform fractionation by SDS-PAGE with an LC-FAIMS-MS system, and have succeeded in achieving in-depth top-down proteomics from small sample amounts. GeLC-FAIMS-MS has also demonstrated its superior performance in Glu-C-based middle-down proteomics, dramatically increasing the number of detectable middle-down peptides and enabling broader sequence coverage. In this presentation, we report on 2D-GeLC-FAIMS-MS, a novel workflow that combines GeLC-FAIMS-MS with pre-fractionation of middle-down peptides using dissolvable BAC cross-linked polyacrylamide gel electrophoresis (BAC-PAGE) to achieve even deeper middle-down analysis.