Hello Meryem Koose,

Trypsin activity is highly dependent on pH. Any change in pH will affect trypsin activity. Trypsin is more active at alkaline pH, typically around 8.0-9.0. At this pH range, trypsin's activity is optimal, and it can effectively catalyze the hydrolysis of peptide bonds. But at lower pH, trypsin gets denatured, leading to a loss of enzymatic activity.

So, in serum proteomics, to stop trypsin activity when you add TFA it helps to lower the pH of the reaction below pH 4, and as a result trypsin loses its activity. The trypsin digestion may also be stopped by formic or acetic acid.

Therefore, maintaining the appropriate pH is crucial for the optimal function of trypsin.

Best.

15% TFA may lead ion suppresion and should be decreased to below 1%. Other than trypsin inactivation, it also represents an ion-pairing agent to improve chromatography. High concentration of ion pair could be detrimental for ESI...As indicated keep the pH around 2 to promote trypsin inactivation and to perform reversed phase chromatography...

İsmail Emir Akyildiz Meryem wrote they add 15% TFA, so that's probably not a final concentration.