Computational protocol: The Autolysis of Human HtrA1 Is Governed by the RedoxState of Its N-Terminal Domain

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Protocol publication

[…] HtrA1 (0.5 μg, 20 μg/mL) was incubated at 37 °C in the presence or absence of 5 mM DTT for 3 or 20 h. The reaction was quenched by the addition of formic acid to a final concentration of 1%. The samples, incubated for 20 h, were pretreated with 10 mM fresh DTT for 15 min to ensure complete reduction of the disulfides before the formic acid quenching step. Then they were micropurified using C18 stage tips (Thermo Fisher Scientific), dried down in a SpeedVac, and dissolved in 0.1% formic acid for liquid chromatography–tandem mass spectrometry analysis. The samples were run three consecutive times on an AB Sciex TripleTOF 5600 instrument. Data analysis was conducted using the Mascot search engine with a peptide fragment mass tolerance of 0.2 Da. Only peptides with a significance score of <0.01 and an ion score cutoff of >30 were considered reliable hits and included in the final list of identified HtrA1 N-terminal peptides. For an autolytic cut site to be valid, the given P1 residue had to be identified in at least two of the three replicates. MS Data Miner was used for the extraction of relevant data from the Mascot search file. […]

Pipeline specifications

Software tools Mascot Server, MS Data Miner
Application MS-based untargeted proteomics
Organisms Homo sapiens
Chemicals Cysteine, Disulfides