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.