The following materials and methods

The following materials and methods section will enable other investigators and laboratories to design similar experimental procedures to study matrix metalloproteinases or any other protease by PICS proteomics. Please refer to our recent dental pulp proteomics and N-terminomics Data in Brief article for more information on TAILS [47].
Experimental design, materials and methods
PICS peptide library preparation. Human whole proteome-derived peptide libraries for MMP specificity profiling were prepared as described in great detail in Nature Protocols [9]: in brief, cell pellets were collected from human lymphoblast cell K562 cultures and lysed in 20mM HEPES (pH 7.5) supplemented with 0.1% (w/v) SDS and protease inhibitors to prevent unwanted proteolysis (1×Roche cOmplete plus 1mM PMSF and 10mM EDTA). Cell debris was removed by centrifugation (26,000g, 1h, 4°C); soluble proteins were denatured using guanidine hydrochloride (4M), and cysteine side-chains were reduced with 20mM DTT (1h, 37°C). Free sulfhydryl groups were protected with 40mM iodoacetamide (3h, 20°C) to avoid peptide crosslinking and reactions were stopped by adding more DTT (5mM, 15min, 20°C). Reaction clean-up was performed using chloroform/methanol precipitation as described elsewhere [48], pellets were air-dried and re-suspended in 100mM HEPES, 5mM CaCl2, pH 7.5, and digested with TPCK-treated trypsin or GluC (Staphylococcus aureus protease V8, Worthington) at a protease to proteome ratio of 1:100 (w/w) overnight at 37°C. Note, another protease often used for PICS library preparation is chymotrypsin. After inactivation of trypsin/GluC with 1mM PMSF (30min, 20°C), undigested protein epinastine hydrochloride were removed by centrifugation (20,000g, 10min, 4°C). Primary amines of peptide N-termini (α-amines) and lysine side chains (ε-amines) were blocked by reductive dimethylation with 30mM formaldehyde (CH2O) and 15mM sodium cyanoborohydride (NaCNBH3, Sterogene) at 20°C for 16h overnight (pH 6–7). To ensure completeness of amine blocking, another 15mM formaldehyde and 15mM sodium cyanoborohydride were added and incubated for additional two hours. Samples were desalted by size exclusion chromatography using Sephadex G-10 columns (10mM potassium phosphate buffer, pH 2.7, 10% (v/v) methanol), and after methanol removal by vacuum concentration (SpeedVac, Thermo), peptides were purified by reversed-phase chromatography on an ÄKTA™ high-performance liquid chromatography system (Äkta Explorer, GE Healthcare) using a RESOURCE RPC column (GE Healthcare); wash buffer contained 0.3% (v/v) formic acid, and samples were eluted in 80% (v/v) acetonitrile, both in HPLC-grade H2O. These PICS peptide libraries were concentrated by rotary evaporation under vacuum, re-suspended in water, and stored in 200–400µg aliquots of 5–15mg/ml at −80°C until use. Peptide concentration was estimated using the bicinchoninic assay (BCA, Pierce). All reagents were purchased from Sigma-Aldrich unless otherwise specified.
PICS cleavage site specificity assay. MMP cleavage assays were performed by incubation of 200–400µg human whole-proteome peptide library with active recombinant MMP at a protease to peptide library ratio of 1:100 (w/w) in 50mM HEPES, 150mM NaCl, 5mM CaCl2 at pH 7.4, overnight, and stopped by heat inactivation at 70°C for 30min. Prime-side cleavage products generated by MMP cleavage were subsequently isolated by positive enrichment using the biotin handle. In short, cleaved peptides with a free primary amine at the N-terminus generated by MMP activity were biotinylated by incubation with 0.5mM Sulfo-NHS-SS-Biotin, an amine-reactive biotin with a redox-sensitive and thus cleavable disulfide linker (Thermo Scientific) for 2h at 20°C. Biotinylated prime-side cleavage products were separated from uncleaved peptides by affinity purification, incubating with 300μl Streptavidin Sepharose slurry (GE Healthcare) for 2h with mild agitation. After extensive washing (50mM HEPES, pH 7.2), biotinylated peptides were eluted with 20mM DTT (2h, 20°C), desalted using reversed-phase solid phase extraction (Sep-Pak C18, Waters) with binding and washing in 0.1% (v/v) formic acid and elution in 80% (v/v) acetonitrile, both in HPLC-grade H2O. Eluates were vacuum dried to near dryness using a SpeedVac concentrator (Thermo), brought to 10μl with 0.1% formic acid, and stored at −80°C until LC–MS/MS analysis.