The frequencies of site-specific cleavage (SCS) and of cleavages generating the PSP P1 and P1′ positions remarkably differ. The frequencies of site-specific cleavage (SCS) and of cleavages generating the PSP P1 and P1′ positions remarkably differ. The SCS frequency (A) and frequency of cleavages generating the residues at the P1 (B) and P1′ (C) positions (considering all PSPs of the representative substrate gp10035–57) are reported and substantially differ. For example, in the digestions by spleen 20S proteasomes the relative amount of PSPs with T53 at P1′ position is 39.6% (± 15.4). This information is here referred as the frequency of cleavage after the residue W52, which generates the T53 at P1′ position, considering in the computation only the PSPs and not the Σ PCP/PSP. Conversely, the frequency of the cleavage (i.e. SCS) after the residue W52 is 5.7% (± 0.9) if we consider Σ PCP/PSP. The symbol “/” in the x axis refers to all splice-reactants whose N termini have been spliced without previous cleavage, like, for example, [VSRQL] or [VSRQLRT]. This event is, of course, possible only for the P1′ positions. Digestions of 3 nmol of synthetic substrate in 100 μl reactions were carried out by 1.5 μg 20S proteasome purified from human erythrocytes or spleen. The frequencies were computed by SCS algorithm from the QME calculation of the ESI-MS data. Relative frequencies are reported in % and the bars represents S.D. of three experiments measured three times each. Erythrocyte and spleen 20S proteasome data are reported in black or white histograms, respectively. Michele Mishto et al. Mol Cell Proteomics 2012;11:1008-1023 © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.