Symposium Sessions
Day 1, June 10（Wed.） 16:00-16:20 Room B (4F 411+412)
- 1B-S2-1600
Next-Generation Strategies Pioneering Post-Translational Modification Proteomics
(1RIKEN IMS, 2Keio Univ., 3Yokohama City Univ., 4Keio Univ., 5Kyoto Univ.)
oKazuya Tsumagari1, Yosuke Isobe1,2,3, Makoto Arita1,2,3,4, Yasushi Ishihama5, Koshi Imami1
We developed efficient enrichment strategies for two classes of PTM peptides: phosphorylation and lipidation, with a particular focus on S-acylation.
Phosphorylation is one of the most frequently observed PTMs and plays critical roles in protein functional regulation, intracellular signal transduction, and so on. We revisited hydroxy acid–modified metal oxide chromatography (HAMMOC) and established a streamlined workflow for phosphopeptide enrichment, termed Rapid HAMMOC. This optimization increased median signal intensity by 7.9-fold for 5 μg of K562 cell digests and enabled the identification of more than 8,000 class I phosphosites from as little as 0.5 μg of HeLa cell digests. Moreover, coupling Rapid HAMMOC with anti-puromycin immunoprecipitation allowed single-day profiling of nascent polypeptides from ultra-low-input samples, yielding 2,310 high-confidence co-translational phosphosites. Lipidation, such as S-palmitoylation on cysteine, alters protein hydrophobicity, regulating protein protein-protein or -membrane interactions. We report a new methodology that combines Phase-transfer surfactant (PTS)-assisted digestion, Liquid–liquid Extraction, and strong cation eXchange chromatography, termed PLEX. Using this approach, we identified more than 4,000 S-palmitoylation sites in mouse brain. We also detected other S-acylations, including stearylation (C18:0).