Automated Phosphopeptide Enrichment for Gram-Positive Bacteria

Protein phosphorylation in prokaryotes has gained more attention in recent years as several studies linked it to regulatory and signaling functions, indicating importance similar to protein phosphorylation in eukaryotes. Studies on bacterial phosphorylation have so far been conducted using manual or HPLC-supported phosphopeptide enrichment, whereas automation of phosphopeptide enrichment has been established in eukaryotes, allowing for high-throughput sampling. To facilitate the prospect of studying bacterial phosphorylation on a systems level, we here established an automated Ser/Thr/Tyr phosphopeptide enrichment workflow on the Agilent AssayMap platform. We present optimized buffer conditions for TiO₂ and Fe(III)-NTA-IMAC cartridge-based enrichment and the most advantageous, species-specific loading amounts for Streptococcus pyogenes, Listeria monocytogenes, and Bacillus subtilis. For higher sample amounts (≥250 μg), we observed superior performance of the Fe(III)-NTA cartridges, whereas for lower sample amounts (≤100 μg), TiO₂-based enrichment is equally efficient. Both cartridges largely enriched the same set of phosphopeptides, suggesting no improvement of peptide yield by the complementary use of the two cartridges. Our data represent, to the best of our knowledge, the largest phosphoproteome identified in a single study for each of these bacteria.