Solid phase extraction is a common procedure in proteomics to clean-up/desalt samples prior to ESI-LC-MS analysis. I've used it for years in its many forms (ziptips, stagetips, columns, cartridges etc etc etc). Back in time (even before LC-MS), trifluoroacetic acid (TFA) was commonly used to both acidify and 'ion-pair' the process. It was very useful because it can be used as an additive for MALDI, so cleaned up samples could be spotted directly!
I have noticed lately that many protocols call for the use of formic acid in place of TFA in the SPE step for ESI-LC-MS workflows. I tried looking in literature, but can't find any data that can support the use of one or the other. I don't recall formic acid as being an 'ion-pair' reagent, however, as SPE is a 'catch and release' chromatography, I'm not sure how critical the ion-pair reagent is.
So, bottom line, can anybody give me their experience and feedback (and hard data) on using TFA or formic acid in SPE? Is there any support to use one or the other, or just more 'urban myths' in proteomics workflows?
Thanks!
Alex
Hi Alex, hope all's well.
I know exactly what you mean about the FA (formic acid) vs TFA (trifluoroacetic acid) debate for SPE. We use TFA in our LC-MS loading buffer and transfer solution (at
Hi Alex,
I'm with Peter on this one. I tend to use TFA if I'm going on to MALDI, or FA if I'm going on to ESI, but personally I really don't think it makes any practical difference. There may be some cases, as in TiO2 and phosphopeptides, where TFA is advantageous, but for standard C18 clean-up, I don't think it matters.
Thank Peters!
I will do a quick test with both (SPE with TFA or FA at 0.1% in wash and elute buffers) and check whether, after drying and resuspension, I get any difference the the proteins ID. I will add the formylation as a dynamic mod and see if it is significant in our results.
Thanks!
Alex
TFA causes greater ion supression in ESI compared to FA. TFA is a better ion pair for chromatography, but the effects are negligible or not noticeable in C18 nano columns in our experience, especially for sub 2 micron particles. For elution from stage tips or sep-paks normally you don't need to have TFA in your buffers in my experience, but if you speedvac your samples to dryness (which I try to avoid) then it's better to reconstitute in buffers with TFA.
I agree with Tanveer Batth that TFA causes great ion supression in ESI. We avoid to use TFA in every case because trace TFA will distory the sensitivity. I think FA and HAc is good enought to provide H+ for ESI+, with concentration about 0.1% in mobile phase. As far as SPE is concerned, TFA is still not recommended unless MS is not used as the detector.
It is well established that TFA suppresses ionisation in ESI-MS. The question was is there any preference in using TFA or FA when performing SPE. As long as the TFA is removed prior to ESI-MS it should not be a problem. The problems arise when you use TFA as the ion pair in ESI-MS. I would argue you could load a sample that was reconstituted in TFA containing buffers, and run it on a system using FA buffers with little to no effect. Have said that, I would not recommend this course of action.
I use TFA if I'm going on to MALDI, or FA if I'm going on to ESI. I will try to use FA for MALDI next time, because TFA is much more harmful than FA.
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