The protocols for (brain) tissue digestion I am trying out are: lysis (buffer containing 0.1% SDC or SDS in 100mM TEAB + phosphatase inhibitor which is sort of yellow), reduction with TCEP, alkylation with iodoacetamide, digestion with trypsin, and acidification with trifluoroacetic acid (TFA) to deactivate trypsin. After that I usually dry the sample and do a C18 clean-up.

For some reason my samples become yellow after the addition of TFA (and this was also the case with lysis buffers with SDS or SDC so I don't think it is detergent related). As this happened to a colleague who didn't use the phosphatase inhibitor, I don't think it's related to it either. When I do the C18 clean-up this yellow goes to the eluate (elutes with peptides). Would anyone know why this yellow colour occurs? The most disturbing thing for me is that it does not always happen to every sample, as you can see in the photo 0.1%SDC is clear.brain tissue samples in different lysis buffers

Moreover, for the last digestion, I decided to try out Rapigest (0.1% in 50mM TEAB). When I added the TFA for acidification step, the sample was still clear, but after a while drying it in SpeedVac, when I checked up on it, it had a bloody red pellet-like oddity as you can see in the photo. I left it in the SpeedVac to dry out completely, and when I checked it again, the red colour disappeared and it was clear-whitish again. Would anyone know why this happens and if it interferes with subsequent steps such as C18 clean-up or LC-MS identification? So far, I have asked my colleagues, but they have never seen it before and are calling it magic.

  • $\begingroup$ Is your preparation of TFA fresh and protected from light? $\endgroup$ – CKM Apr 20 '17 at 21:16
  • $\begingroup$ Do you mean is it kept protected from light after it is diluted and added to the sample? Because TFA usually comes in dark bottles, but then when we dilute it to 0.5 or 5% it is kept in light in transparent ones. Once a dilution is prepared, it is used for a long time (couple of months) in our laboratory. Does this usually cause problems? $\endgroup$ – E.T. Apr 20 '17 at 21:30
  • $\begingroup$ After a few months or if the solution begins to take on a yellow hue I'd discard it. TFA on its own is light sensitive, however, and personally I'd try to store it out of direct light. It could also be a reaction breaking down the TFA, but im not sure. In the rapigest tube I'm 100% not sure. I actually saw a protocol somewhere though, if your solution turns yellow you can add some 1% v/v TFA to make it clear again. I'll keep looking into it. $\endgroup$ – CKM Apr 20 '17 at 21:35
  • $\begingroup$ The TFA solution I used was still clear. However, when this happened I made a fresh one, so I will let you know if it happens again. Thank you I will try that next time. 👍 $\endgroup$ – E.T. Apr 20 '17 at 21:48
  • $\begingroup$ @E.T., Rapigest is an acid-cleavavble detergent which precipitates after cleavage, because of which it can be removed before LC-MS analysis. After digestion, TFA would acidify it and you should keep the sample at 37 ºC for 30 min to cleave all Rapigest that then would precipitate and should be removed by centrifugation. $\endgroup$ – ktyagi Sep 14 '17 at 11:54

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