I plan to use saliva samples for LC-MS peptide analysis and noticed many papers treat them with 0.2% TFA in a 1:1 ratio. Why is TFA used, and does it solubilize / denature proteins?
I know that TFA used with the mobile phase to enhance the ionization, but what about its role in sample preparation?
Trifluoroacetic acid (TFA) is used in LC-MS sample preparation to enhance peptide solubility and denature proteins, facilitating clearer solutions for analysis. Additionally, TFA helps maintain a low pH that improves ionization during the LC-MS process.
Note: If possible, avoid using any TFA in the mobile phase for the LC-MS system. It is too strong of an acid even when diluted. It will strongly bind to the instrument and column resulting in long term contamination of the LC-MS system (Which is very expensive and time consuming to resolve). While it may enhance ionization and provide a low pH, other acids are available (e.g. Formic Acid) which are not as strong and do not bind so strongly to the instrument and column (contaminating the flow path).
As far as I know, TFA improves the retention of some analytes, absorbs moderately at 210 nm, and due to ion-pair interactions the selectivity of the system increases, especially for peptide analysis. If subsequent MS analysis is planned, it is usually advised to replace TFA with formic acid, as TFA reduces the sensitivity of MS (due to ionisation)
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