Correlation between the amounts of PCPs and PSPs generated from LLO291–317 by 20S s- and i-proteasomes. Correlation between the amounts of PCPs and PSPs generated from LLO291–317 by 20S s- and i-proteasomes. The PCPS activity of 20S proteasomes purified from T2 and T2.27 cell lines was monitored by the kinetics of PSPs and of the correlated splice-reactants during the digestion of the substrate LLO291–317. 4 nmol LLO291–317 were cleaved in 100 μl reactions by 2 μg of 20S proteasomes for 0–4 h at 37 °C. The digestion products were detected by LC-ESI/MS and their absolute amount computed by QME. A, T2.27 20S proteasomes revealed a slightly higher LLO291–317 degradation rate than T2 20S proteasomes. B, T2 and T2.27 20S proteasomes exhibit a similar efficiency for the PSP [AYISSVAY][AYISSVAY] (LLO291–298/291–298) production. C, The production of the PCP [AYISSVAY] (LLO291–298) is only slightly higher when carried out by T2.27 proteasomes compared with the T2 proteasomes. D, T2.27 proteasomes generate more efficiently the PSP [AYISSVAY][AYI] (LLO291–298/291–293) as well as the PCP [AYI] (LLO291–293) than T2 proteasomes (E). F, T2.27 proteasomes produce more efficiently than T2 proteasomes the PSP [AYISSVAY][RQVYLKL] (LLO291–298/300–306) as well as the PCP [RQVYLKL] (LLO300–306) (G). In B-G the pmol of peptides produced per nmol of degraded substrate are reported. Bars represent the S.D. of repeated experiments. Michele Mishto et al. Mol Cell Proteomics 2012;11:1008-1023 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.