Does anyone know why TFA (ion pairing agent in HPLC) is better for TiO2 and Phosphopeptides?
I am doing SILAC experiment on a kinase module of mediator, and I would like to get some inputs regarding to the steps usually carried out, especially in Tandem M/S.
Thank you
Here is a good paper on the subject Article Enrichment techniques employed in phosphoproteomics
Also try reading some of the work of the labs of Boris Macek in Tubingen, they specialize in phosphoproteomics.Article Parallel reaction monitoring on a Q Exactive mass spectromet...
https://uni-tuebingen.de/fakultaeten/mathematisch-naturwissenschaftliche-fakultaet/fachbereiche/interfakultaere-institute-und-zentren/ifiz/proteome-center-tuebingen/home/
TFA was used decades ago (the dark ages in HPLC) in the mobile phase as there was no other means of separating these molecules. Today is different (thank God) since many new generation columns (higher carbon load) which are more hydrophobic can do the same separation (or even better). TFA will foul the MS detector and is really HARD to clean out. In addition HPLC columns exposed to TFA have to be dedicated as thier separatory power has been changed.
The issue is ionization. TFA has a very low pKa compared to the ionizable residues of peptides, particularly the ionizaiton of phosphate-modifications of phosphopeptides. You can get efficient ionization of any peptide as long as the proton source has a lower pKa than that of the peptide. I've successfully used phenol as the low pH modifier for simple peptides, but not any donor with a higher pKa, without losing ionization efficiency. The pKa of acetic acid and formate are above 3. TCA and TFA are 1 or below.
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