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In order to quantify endogenous S-nitrosated cysteines via a mass spectrometry approach, we introduced a mass difference between sample proteins and the control.
350 μg of sample proteins and 150 ng of the provided Tyr 1248 HER2 protein control were used for measurements.
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After adding in sample protein and reaction constant with parameters, we built a simulation-ready model for the network.
The sample was kept at 37 °C, and 1 μg Tris (2-carboxyethyl) phosphine was added per 25 μg sample protein and incubated for 30 min at 60 °C.
30 μL of a solution containing SDS loading buffer and mercaptoethanol were combined with 50 μg of sample protein and lysis buffer up to 100 μL.
A two-third volume of the solution containing 50 μg sample protein and 100 pmol lysozyme was subjected to SDS-PAGE on 12.5% polyacrylamide gel 1-mm-thick.
Then, the sample was digested by the addition of 1 μg trypsin (from a 0.1 μg/μl trypsin stock solution) per 50 μg sample protein and incubated overnight at 37 °C.
DTT (1 mol/L 10 μl) of Caspase-3 detection kit and 50 μl 2 × reaction buffer were added into 10ul supernatant sample protein, and then 10 μl 1 mmol Caspase-3 substrate Ac-DEVD-pNA was added.
The samples (proteins) and control (phosphate buffer (PBS), pH 6.8) were added to the paper disks.
Samples (proteins and peptides) were dissolved in 0.1 % TFA and mixed with the corresponding matrix solution.
The performance of SVM critically depends on the diversity of samples (proteins and peptides) in a training dataset and the appropriate representation of these samples.
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