trouble shooting for low labeling efficiency in TMT experiment

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cavaliersun2003
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trouble shooting for low labeling efficiency in TMT experiment

Postby cavaliersun2003 » Mon May 11, 2015 9:06 pm

Hi,
Recently I came across this problem and couldn't find a possible explanation... hopefully somebody here can give some hints of what might went wrong.
we enriched proteins from pig serum samples using CPLL method (elute with 4%SDS+100mM DTT boiling) and use standard FASP protocol to digest protein, and then collect the peptides to do the TMT labeling as per manual. However, my search results suggest most hits were unlabeled. We test the samples before and after labelling and concluded that the peptide abundance and digest efficiency are normal, and I'm pretty sure we avoided primary amines throughout the whole procedure. We use TEAB buffer in the whole protocol.
We also tested this protocol with a recent introduced DiART label which is very similar to TMT and iTRAQ, and disappointingly the labeling efficiency is also very low. So at least I believe there might be something wrong with the sample.
By the way, we used to perform this protocol by dimethylation and the labeling efficiency was good. We switched to TMT since now we have 10 samples to be compared.
Is there any other secrets that I didn't know about this isobaric labeling?
Thank you very much!

SunSun

Infinity
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Postby Infinity » Tue May 12, 2015 7:09 am

I was wondering if you desalted your samples after FASP?

cavaliersun2003
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Postby cavaliersun2003 » Tue May 12, 2015 4:43 pm

Hi, Infinity,
we use 200ul 50mM TEAB, then 200ul 500mM NaCl to elute the peptide from FASP, dry the sample and resuspend to 100ul, so yes, we do have salt before labeling. But there was no warning in Thermo's protocol that salt might be the reason of poor labeling. Did you have suggestion on how much salt the TMT labels can handle?
thank you!

Infinity
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Postby Infinity » Fri May 15, 2015 6:15 am

It is always advisable to desalt your sampler prior to any labeling. I would definitely recommend to desalt peptides after FASP, dry on speedvac and then resuspend in the labeling buffer (TEAB). We routinely use HLB cartridges from Waters for desalting (but my guess is that any C18 cartridge will work just fine). Just in case I would also inject small part of the sample just after the desalting step to have a QC prior to labeling.

cavaliersun2003
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Postby cavaliersun2003 » Sat May 16, 2015 6:32 am

Thank you, I will try to desalt the sample and see if the problem can be solved.

Infinity wrote:It is always advisable to desalt your sampler prior to any labeling. I would definitely recommend to desalt peptides after FASP, dry on speedvac and then resuspend in the labeling buffer (TEAB). We routinely use HLB cartridges from Waters for desalting (but my guess is that any C18 cartridge will work just fine). Just in case I would also inject small part of the sample just after the desalting step to have a QC prior to labeling.


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