Computational protocol: A Single Glycan at the 99-Loop of Human Kallikrein-related Peptidase 2 Regulates Activation and Enzymatic Activity*

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Protocol publication

[…] Bz-PFR-pNA, H-PFR-AMC, Chromozym X, and PPACK were obtained from Bachem (Weil am Rhein, Germany), whereas Ac-OFR-AMC was a product of California Peptide (Napa, California). KLK2 activity was determined in 50 mm Tris (pH 7.5), 100 mm NaCl, 10% (v/v) DMSO, 0.1% (w/v) bovine serum albumin (BSA) at 37 °C containing 70–500 nm KLK2, depending on the active fraction of the sample (see below). The maximum substrate concentration ranged from 0.7 to 10 mm, depending on the respective turnover and signal, respectively. The signal of the released pNA was measured photometrically at 405 nm, whereas that of released AMC was fluorimetrically recorded at excitation and emission wavelengths of 380 and 460 nm on an Infinite M200 microplate reader in triplicate or higher multiplicate samples (Tecan, Männedorf, Switzerland). Relative absorbance for the pNA standard (Sigma-Aldrich) and relative fluorescence for the AMC standard (Sigma) were measured at the same conditions and used in the calculations of the rate of product formation. The initial velocity of substrate turnover was fitted to the Michaelis-Menten equation by error-weighted non-linear regression analysis to obtain kcat and Km. Substrate depletion was limited to <10%. The fraction of active KLK2 was determined by active site titration with the irreversible inhibitor PPACK by measuring the activity against H-PFR-AMC. kcat values were calculated by dividing Vmax through the active protease concentrations, which were 15.5% for KLK2e and 45.3% for glyco-KLK2. Data fitting was performed with the ORIGIN software (OriginLab).PICS analysis was done with samples of KLK2e and LEXSY-derived glyco-KLK2 (, ). A variant of the original PICS method was used. Briefly, E. coli was grown in LB medium. Cells were lysed, and lysates were digested by either trypsin or GluC (). The peptide digest was further purified by C18 solid phase extraction (Sep-Pak, Waters) according to the manufacturer's instructions. KLK2e or glyco-KLK2 was incubated with the library at a 1:300 ratio for 3 h at 37 °C in 50 mm Tris (pH 7.5), 100 mm NaCl. The reaction was stopped by 1 μm PMSF. Protease-treated and control samples were isotopically labeled by triplex reductive dimethylation, as described elsewhere (). Liquid chromatography-tandem mass spectrometry was performed on a Q-Exactive plus mass spectrometer coupled to an Easy nanoLC 1000 (both from Thermo Scientific) with a flow rate of 300 nl/min. Buffer A was 0.5% formic acid, and buffer B was 0.5% formic acid in acetonitrile (water and acetonitrile were at least HPLC gradient grade quality). A gradient of increasing organic proportion was used for peptide separation (5–40% acetonitrile in 80 min). The analytical column was an Acclaim PepMap column (2-μm particle size, 100-Å pore size, length 150 mm, inner diameter 50 μm) (Thermo Scientific). The mass spectrometer operated in data-dependent mode with a top 10 method at a mass range of 300–2000. For spectrum to sequence assignment, X! Tandem (version 2013.09.01) was used (). The E. coli proteome database (strain K12, reference proteome) was used as described previously (), consisting of 4304 protein entries and 8608 randomized sequences, derived from the original E. coli proteome entries. The decoy sequences were generated with the software DB toolkit (). X! Tandem parameters included precursor mass error of ± 10 ppm (Q-Exactive), fragment ion mass tolerance of 20 ppm (Q-Exactive), semi-GluC specificity with up to one missed cleavage, and the following static residue modifications: cysteine carboxyamidomethylation (+57.02 Da) and lysine and N-terminal dimethylation (light formaldehyde, 28.03 Da; medium formaldehyde, 32.06 Da; heavy formaldehyde, 36.08 Da). X! Tandem results were further validated by PeptideProphet () at a confidence level of >95%. Peak areas for relative quantitation were calculated by XPRESS (). Semispecific peptides that are increased >8-fold in the KLK2-treated samples were considered as KLK2 cleavage products. The corresponding prime or non-prime sequences were determined bioinformatically through database lookup. Web-PICS was used to generate a heat map style representation of protease specificity (). […]

Pipeline specifications

Software tools X! Tandem, PeptideProphet, XPRESS
Application MS-based untargeted proteomics
Organisms Homo sapiens, Leishmania tarentolae, Escherichia coli
Diseases Prostatic Neoplasms