(A) iceLogo representations of iP and cP substrate specificity (P4–P4ʹ) at the 480 min assay time point (p≤0.05 for non-grayed residues (Colaert et al., 2009); ‘n’ is norleucine). (B) Quantification …
The following are contained in a supplementary file for the MSP-MS assay: a sample key; the sequences of the peptide library in FASTA format; a full mass spectrometry peptide report; processed cleavage data showing spectral counts for each octapeptide (P4–P4ʹ) and the associated cleavage within the parental peptide; processed cleavage data (indicated as ‘negative’) with cleavages also appearing in the no-enzyme control (NEC) highlighted; all octapeptides (P4–P4ʹ) to be used as the background dataset in iceLogo; ‘positive’ octapeptide (P4–P4ʹ) cleavages with the ‘negative’ cleavage data removed; ‘accumulative’ octapeptide (P4–P4ʹ) cleavages at each assay time point for the proteasome biological replicates; a comparison of cleavages common between biological replicates; and heat maps based on Z-scores showing the specificity at each assay time point.
(A) Progress curves for the iP and cP are shown demonstrating the kinetics of peptide cleavage during the MSP-MS assay time course. (B) Quantification of shared and non-overlapping cleavages is …
These cleavage events are those that tend to occur initially and produce peptides of appropriate size (in m/z). For a given protease, the assay is capable of detecting up to approximately 20% of the …
The heat map from the 480 min assay time point has been repeated from Figure 1 for clarity.
Toes et al used a model protein substrate (enolase-1) for proteasome cleavage, (Toes et al., 2001) whereas Mishto et al used a set of synthetic peptides corresponding to the sequences gp10035-57, …
(A) Example 14-mer peptides from the iP and cP MSP-MS assays associated with Figure 1 illustrating cleavages that were used as a template for rational fluorogenic substrate design. All cleavages …
Mean activity is reported with error bars representing the standard deviation from n = 3 replicates.
CFZ levels were selected based on inhibitor titration against the library to achieve optimal selectivity. Mean activity is reported with error bars representing the standard deviation from n = 3 …
Protocols for synthesis of the Fmoc-ACC fluorophore and its coupling to Rink amide resin have been described. (Maly et al., 2002) (a) 20% 4-methylpiperidine/DMF; (b) Fmoc-protected P1 amino acid (5 …
Mean activity is reported with error bars representing the standard deviation from n = 3 replicates.
(A) Demonstration of EWFW-ACC (10 μM) and iso-VQA-ACC (30 μM) selectivity for the iP and cP, respectively, compared to the universal substrate LLVY-AMC (30 μM) using mixed ratios of purified …
Km = 98.6 μM for the iP and 65.7 μM for the cP with 2.0 fold selectivity at Vmax.
Mean labeled subunit levels are reported with error bars representing the standard deviation from n = 3 replicates.
Mean activity is reported with error bars representing the standard deviation from n = 3 replicates.
(A) Label-free quantitation of relative iP and cP cleavage rates against a library of synthetic peptides derived from sequences flanking sites of MHC I peptide processing (Bassani-Sternberg et al., …
The following are contained in a supplementary file for the MHC cleavage assay: a sample key; the sequences of the peptide library in FASTA format; and a full mass spectrometry peptide report.
(A) Scoring of the MHC I peptidome (Bassani-Sternberg et al., 2015) (n = 22,598) based on the relative favorability for cleavage by the iP or cP. Scores were derived from the P4-P1 cleavage …
(Sutoh et al., 2012) UniProt identification numbers are respectively as follows for β5 and LMP7: P28074 and P28062, Homo sapiens (human); O55234 and P28063, Mus musculus (mouse); P28075 and P28064, R…
(A) Ubiquitin western blot and matched activity assay for the SUP-B15 cell line (with approximately 80:20 LMP7: β5 activity) showing a substantial increase in global ubiquitination levels upon …
Mean ubiquitin levels are reported with normalization to the untreated condition. Statistically significant differences compared to the untreated condition are indicated (*p≤0.05) as determined with …
Mean activity is reported with error bars representing the standard deviation from n = 3 replicates per donor.
Comparison of the reproducibility of the MSP-MS assay for technical triplicate samples.
Percent identity was compared across replicates for the intact library (‘no enzyme control’) and after the addition of the immunoproteasome as an example. The 8 hr time point was chosen to maximize the number of cleavages in the comparison.
Comparison of select ACC substrate Michaelis-Menten parameters.
Comparison of compound IC50 values in the MOLT-4 lysate obtained using the optimal fluorogenic substrates and the ProCISE assay.