Toward Tubulin Proteomics: Analysis of Human Microtubule-Associated Proteins Using Offline High-pH RP-HPLC Coupled with Low-pH LC-MS/MS

摘要

Offline high-pH RP-HPLC coupled with traditional online low-pH RP-HPLC and MS/MS analysis identified 621 microtubule-associated proteins from human Jurkat T cells. High-pH RP-HPLC separates peptides based on their hydrophobicity and acidity: 1. Peptides with similar GRAVY scores elute according to increasing isoelectric points. 2. Peptides with similar pI values elute according to increasing GRAVY scores. Acidic C-terminal tubulin peptides may elute in a predictable manner using high-pH mobile phases, which would facilitate tubulin isotype characterization. (There are 9 alpha-tubulin isotypes and 7 beta-tubulin isotypes). Future work: Investigate changes in MAPs composition in response to MTA drug treatment in Jurkat T cells; Identify each tubulin isotype based on its unique C-terminal peptide; Characterize tubulin isotype post-translational modifications.