TFA alternatives, peptide purification

[JimH]

I am presently working with 0.1%TFA buffers for purification of peptides, and following up with repeated lyophilizations in HCl to drive off the undesirable TFA. Ideally I'd like to eliminate the TFA altogether to avoid this issue, as well as speed up the process. I've read small bits on using a 5mM HCl mobile phase, but not a lot of info as to compatibility with the instruments or selectivity. Basically looking for others' thoughts on this, as well as other potential options. The desired end result would be minimum salt content, "cell friendly" non-TFA (acetate, cl, etc.)

I'm generally using C18 columns through ELSD & electrospray, and the sequences I'm working with average ~20 residues and generally have 3-4 of both positive and negatively charged aminos, so options are fairly wide open (although I'd prefer to stay away from higher pH buffers for the occasional cys/met-containing sequences).

[Kostas Petritis]

HCl is very corrosive and not recommended for use with LC. Is there any reason you can not use formic acid instead of TFA?

[JimH]

Formic acid is not off the table, I just haven't had any experience with it but will look into it. The desired end result is a peptide with minimal salt content compatible with cell-based assays. Currently we are converting from TFA to Cl salt, and switching to 5mM HCl would have kept the end product consistent. Unfortunately if it is detrimental to the system that is not a desirable option either.

[Kostas Petritis]

Formic or acetic acid will work just fine, although your peptides peak widths will be a little bit wider and probably you will experience somewhat smaller retention...

[Uwe Neue]

HCl should not be used with a stainless steel system. However, it has been used successfully and without problems with so-called metal-free systems.

As mentioned already above, formic acid is a good alternative. The quality of the separation that you get with formic depends on the packing. Not all packings are created equal. Some work superbly with formic acids, others don't.

[peptidemetdev]

Is HCl really all that damaging as dilute as it would be?

I currently use a 0.1M sodium perchlorate mobile phase for some analytical work on peptides with many basic residues, and I usually end up adjusting the pH with 1N HCl to a final concentration of around 1mN. Also, I'm constantly reading that 5mN HCl is recommended as an alternative to TFA for protein/peptide analysis/purification, and I've never seen any warnings about stainless steel (although I have seen warnings about not adding any HCl to the acetonitrile for your B mobile phase).

To the original poster:

Acetic or Formic acid are both pretty common. Adding TEA to the mobile phase can help tighten up the peaks a bit by deactivating silanol interactions. Would it be economical for your application to consider a multi-step purification? You could find an analytical method that works really well for your peptide without too much concern about counterion or salt contaminants, and then take the result from that and either put it through an exchange column, or just reinject it on another RP column equilibrated with an Acetic Acid mobile phase. The result would be the same either way, your peptide would stick to the column and all of the salt contaminants would elute in the garbage peak. Then you'd just kick the peptide off the column and lyo down to the acetate salt.

Your yeild will decrease because you won't be able to recover EVERYTHING from the second step, but it's a very convenient way to do things.

[Kostas Petritis]

Uwe,

As you mentioned that some columns work well and some not with formic acid... What makes a column to work well with formic acid? Are there some specific characteristics in terms of chemistry that will make a C18 column to work better than others? Can you recommend some commercial columns that will work very well for formic acid and peptide separations (if they can reach peak widths that can be achieved by TFA that would be perfect).

Thanks!

[Uwe Neue]

The packings that work well for peptide separations using formic acid as the mobile phase additive are Atlantis dC18, Atlantis T3, and XBridge C18 or XBridge300 C18. I personally have not found a generic reason why some packings work better than others. One can always speculate about silanols etc., and there is definitely an element of this, but it does not appear to be that straightforward.

[Victor]

I do not doubt that some columns will differ from others on the question of TFA vs formic acid due to silanol effects. The pH of commonly used concentrations of TFA is significantly lower than that of commonly used formic acid solutions, as TFA is a stronger acid. Thus, I guess on columns with acidic silanols, the lower pH of the TFA mobile phases is beneficial to the analysis of basic peptides that can undergo silanol interactions.

However, I believe that overloading of peptides (especially basic peptides) is a far more important contributor to the wider peaks seen with formic or acetic acids than with TFA when modern RP columns are used. This is due to the low ionic strength of formic acid solutions that is compounded by the presence of acetonitrile, which makes formic acid an even weaker acid. The ionic strength of TFA solutions is much higher, and its ionisation is much less affected by the presence of acetonitrile. In consequence, you will see much better peak shapes with TFA for such peptides even on the most inert silica based columns that have few if any ionised silanols in acid solutions. Overloading is reduced in solutions of higher ionic strength. You can even see the same effect on purely polymeric columns (polystyrene/divinylbenzene) that obviously have no silanols at all.

In consequence Kostas, I do not believe that you will find any column that will work as well with formic acid as with TFA. You can throw in some salt into your formic acid solutions to increase the ionic strength, but as a mass spectroscopist, this would be heresy to you. Of course, you can dilute your samples, and as you inject less and less, the peak shapes in formic acid will approach those obtained in TFA...but again this is hardly a general answer, as some of your peaks will disappear into the background noise.......

[Kostas Petritis]

Assuming that Jim's question was answered I will highjack the threat.

I have a personal interest on the subject as I will be doing a TFA vs. formic acid study for tryptic peptides. The field here is proteomics and mass spectrometry and the criteria of sucess might be different than just best peak shapes or resolution (in proteomics you analyze simultaneously thousands of peptides). We are currently using the Phenomenex Jupiter 300 A column (a choice made 7-8 years ago) which works great with TFA. We have used the same column with formic acid with relatively good outcome but I am currently searching around for a packing that will work the best with formic acid and tryptic peptides...

Victor, from what you are saying, if you decrease the quantity of TFA accordingly in order to match the ionic strength of formic acid (i.e. 0.1 % of TFA for 1% formic acid - these are arbitory numbers as I have to make the necessary calculations) you will achieve similar performance for TFA and formic acid. Would you agree?

There have been made several studies on the subject but few of them in the scope of proteomics where again the criteria of sucess are different... Other things to take under consideration of course is ion-supression etc...

[peptidemetdev]

I had another thought:

How high is too high? Have you considered trying ammonium bicarb? At lower concentrations (5-10mM), it supposedly works just fine with electrospray LC-MS, and you could probably go as low as pH 5-5.5 and still get good buffering. I've used it as an analytical mobile phase with a 5mM concentration and no other additives, and saw great peak shapes. The peptides I was analyzing were fairly short and only had a few ionizable residues, though, so I can't say for sure that you will see the same results. But if you are desperate, and the residual ammonia won't be a problem, maybe it's worth a shot?

[Victor]

Kostas,

Another point is that TFA can produce ion pairs with peptides whereas formic acid has only extremely small such effects. When such neutral ion pairs are held on the surface, the effects of overloading are likely to be reduced. So I do not think it is just a matter of matching the ionic strength of formic acid and TFA. Another consideration is that it may be inadequate to use the aqueous pKa of these acids, due to the effects of the ACN.

You need to look carefully through the literature before you start your TFA vs formic acid comparison, as you could be in danger of re-inventing the wheel. As you say there are a number of papers on this issue but many of them got it wrong in my opinion, from a chromatographic point of view.

Clearly many proteomics people will be more interested in the better MS detection with formic acid than the better chromatographic performance with TFA.

[HW Mueller]

On HCl and stainless steel: There have been extensive discussions on this.

Victor, when you talk about ionic strength are you thinking of I (1/2 the Sum of all mx^2), or are you referring to the principle of chaotropy, etc.? At 5mM it probably does not matter, as all ions have a rather chaotropic effect there, but still it would be interesting to know from an academic view.

Kostas, I wondered before how things would compare if the different type of modifications (TFA, Formic, acetic) would all have been optimized. So, I have a feeling that if you optimize regarding pH, ion influence, org. modifyer, flow rate, maybe even temp. the results might look quite different from present "common knowledge". One can imagine, though, that setting up good comparisons will be difficult. For instance, I can only think of keeping the ion effects constant by using a salt above 100 mM in all experiments . Obviously, it looks like you have to make some tough choices to keep this manageable. I am sure you will come up with something useful!

[Kostas Petritis]

Peptidemetdev,

There is a good reason that nobody in proteomics use ammonium bicarb with the exception of speciality applications (maybe) due to the decreased amount of peptide ID's achieved with that buffer when compared to solely acidic mobile phases.

Victor,

We have preliminary results and we have already some novel results and conclusions. Especially when it comes to what you are refering better MS detection with formic acid than TFA that depends on some other factors too. These experiments were small scale and we need to demonstrate them to a more systematic way. At the same time I am trying to show some more things along the way to make the paper more useful.

Hans,

As I said the study will be looked from a practical and proteomic point of view. There is no way I am going to add any salt because your number of peptide ID's will go down significantly. Also I do not have unlimited time or resources to do this but I will try to address the main ones. I guess the whole subject might be a good one for a PhD thesis...

PS:There are some approaches which might be useful to be investigated more in terms of using TFA and trying to enchance sensitivity in mass spectrometry (by adding the modifier in the mobile phase instead of sheath liquid...).

Title: Signal enhancement for gradient reverse-phase high-performance liquid chromatography electrospray ionization mass spectrometry analysis with trifluoroacetic and other strong acid modifiers by postcolumn addition of propionic acid and isopropanol
Author(s): Kuhlmann FE, Apffel A, Fischer SM, et al.
Source: JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Volume: 6 Issue: 12 Pages: 1221-1225

Title: ENHANCED SENSITIVITY FOR PEPTIDE-MAPPING WITH ELECTROSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY IN THE PRESENCE OF SIGNAL SUPPRESSION DUE TO TRIFLUOROACETIC ACID-CONTAINING MOBILE PHASES
Author(s): APFFEL A, FISCHER S, GOLDBERG G, et al.
Source: JOURNAL OF CHROMATOGRAPHY A Volume: 712 Issue: 1 Pages: 177-190

[HW Mueller]

The diminishing of peptide ID, is this a MS phenomenon or a matter of chrom. separation?