PAC/SP3 On-Bead Carboxyl Derivatization Allows Combined C- and N-Terminomics.

On-bead single-pot solid-phase enhanced sample preparation, SP3, also known as protein aggregation capture (PAC), is a robust, high-throughput, and widely utilized approach for proteomic sample preparation. Recent studies have highlighted PAC/SP3 as an ideal platform for chemoproteomics, allowing chemical labeling while minimizing sample loss and improving recovery of derivatized peptides. In this work, we establish an on-bead PAC/SP3 protein-level amine and carboxyl derivatization approach to facilitate C-terminal focused proteomics. We demonstrate that on-bead protein derivatization of carboxyl groups can be achieved using ethanolamine, (2-aminoethyl)trimethylammonium (AETMA), and (carboxymethyl)trimethylammonium (Girard's reagent T, GT) via EDC/HOBt coupling, enabling the labeling of protein C-termini. Using a prokaryotic model, Acinetobacter baumannii, we demonstrate that AETMA and ethanolamine labeling both enable identification of discrete protein C-terminal peptides, with AETMA enabling the detection of unique C-terminal peptides that lack basic residues. Finally, we apply this approach to interrogate both N- and C-termini in response to etoposide-induced apoptosis within Jurkat cells, demonstrating that combined N- and C-terminomics is achievable using on-bead derivatization, yet provides modest coverage of the C-terminome in its current form. Overall, this work establishes bead-based carboxyl group derivatization as a platform to enable future C-terminomics method development.