Dissolvable Polyacrylamide Gel Electrophoresis‐Enabled High‐Resolution Sample Fractionation for Middle‐Down Proteomics

ABSTRACT Top‐down proteomics (TDP) is a powerful analytical approach for the highly sensitive measurement of intact proteoforms by mass spectrometry. However, its application to high molecular weight proteoforms remains challenging. Middle‐down proteomics (MDP) offers a practical solution but requires pre‐fractionation of the complex peptide mixture generated by limited digestion to successfully achieve trace‐level peptide detection. Here, we present 2D‐GeLC‐FAIMS‐MS, an innovative gel‐based sample pre‐fractionation workflow for in‐depth MDP. This workflow integrates limited Glu‐C digestion with a two‐dimensional sample fractionation strategy that combines a BAC ( N,N′ ‐bis(acryloyl)cystamine)‐cross‐linked dissolvable polyacrylamide gel electrophoresis (BAC‐PAGE) with PEPPI‐MS, a highly efficient passive protein extraction method. Samples are first size‐fractionated by BAC‐PAGE and subsequently subjected to in‐gel Glu‐C digestion. The resulting middle‐down peptides (< 50 kDa) undergo a second fractionation via SDS‐PAGE, followed by peptide recovery using PEPPI‐MS and LC‐FAIMS‐MS analysis. The dissolution properties of BAC gels enable efficient sample transfer between the two PAGE steps with minimal loss, ensuring high‐resolution pre‐fractionation. This novel workflow provides a robust and efficient strategy for the comprehensive characterization of middle‐down peptides, facilitating improved sensitivity and depth in proteome analysis.