Abstract
Accumulation of ubiquitin-conjugated proteins in brain tissues has been documented in various neurodegenerative disorders. Identification of such ubiquitin-conjugated proteins is thought to be important to diagnose the pathogenic significance of the protein ubiquitination.
Takada and his coworkers recently reported their original protocol for immunoaffinity separation of the accumulated ubiquitin-conjugated proteins solubilized with SDS. We further assessed the performance of CID-mode MS/MS-ion search on AXIMA-TOF2 for identification of polyubiquitin-conjugated protein separated by 2-DE after immunoaffinity purification.
The target protein-derived peptide fragments were generally very low abundant in the tryptic in-gel digests of polyubiquitinated protein separated by 2-E gel electrophoresis. Therefore, in order to identify the target protein of polyubiquitination, the highest sensitivity of MS/MS-ion search in CID-mode is required for data-dependent analysis of a unique MS signal detected by subtraction of ubiquitin-derived abundant peptides.
Polyubiquitin-conjugated lysozyme was used as an authentic sample to assess the quality of MS/MS signals in this study, and we confirmed that the CID-mode MS/MS-ion search on AXIMA-TOF2 has enough high performance to identify low abundant polyubiquitin-conjugated protein.
