PAC/SP3 on-bead carboxyl derivatization allows combined C- and N-terminomics

Abstract 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 labelling by 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 labelling of protein C-termini. Using a prokaryotic model system, Acinetobacter baumannii, we demonstrate that AETMA and ethanolamine labelling each enables the identification of unique protein C-terminal peptides, with AETMA improving the identification of C-terminal peptides lacking 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 viable platform to enable future C-terminomics method development. Competing Interest Statement The authors have declared no competing interest. Data availability All raw files, Fragpipe output files, FASTA files, and the experimental template have been uploaded to PRIDE under accession numbers; PXD068617, PXD068652, PXD068858, PDX068110