Dear All,
I digested like 100ug of protein with trypsin which was further purified using S-trap column. My elution was with 50mM TEAB, 0.2% FA and 50% ACN.
Finally, upon speedvac to remove all the volatile solvents; i tried dissolving the peptide sample with 10mM Ammonium formate (pH 10.00) buffer for RP-HPLC but then the peptides showed up precipitates.
Something, which I didn't expect to happen.
Any inputs will be helpful.
Hello Dr. Patra
It is possible that the pH of your buffer might be one of the confounding factors in the precipitation of your digested peptides which would alter the behaviour of your peptides to aggregate and precipitate.
You may want to explore references to solve your problem .
Hope this helps.
Hello Alok Patra
We perform the steps as you describe but dissolve the peptides in a solution of 10 mM ammonium formate (pH 10) containing 2 % acetonitrile.
If we assume that the precipitates are peptides, then the acetonitrile should assist in dissolving them. And 2 % acetonitrile will not affect the peptide binding to your reversed-phase column.
However, if the precipitates are not peptides then your S-trap method may need some optimization. One suggestion would be to split your S-trap eluate into two equal volumes in two different tubes. Dry them both by speedvac. Once dry, add aqueous 0.1 % TFA to one tube and perform a C18 desalting step, elute with acetonitrile, then perform another drying step by speedvac.
You will then have one tube of dried peptides from the S-trap elution only, and one tube of dried peptides from the S-trap elution that has also been C18 desalted. Re-suspend both dried peptide samples in your 10 mM ammonium formate pH 10 and observe if the precipitation still occurs in the additionally desalted sample.
Good luck.
Best,
Peter
hello, did you find out what the problem was?
In my case acetonitrile helps
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