protein extraction and peptide quantification from fat breast tissuefat microBCA
Posted 04 May 2013 - 05:09 AM
I am a PhD student and I have a problem : we want to do quantitative proteomic on breast tumor and corresponding normal breast tissue. My problem is the following : normal bresat tissue is really fatty and the lipids interfere with my peptides quantification after trypsin digestion and also with their separation on nano HPLC before mass spectrometry.
I use a 4% SDS, 0,1 M Tris pH 7,6, 0,1 M DTT lysis buffer, then do a buffer exchange into TEAB 0,5M pH 8,5 for trypsin digestion. After desalting, I evaluate the amount of peptides by micro BCA (Pierce) dosage.... The problem is that it overestimates the amount of peptides in normal breast tissue lysates compared to tumor lysates, and I think this is because of lipids that interfere with the mcroBCA dosage.
Would you have any idea to overcome this problem ?
Because to do quantitative proteomics I really need to adjust the amounts of peptides anf for the moment, I have to do a pre-experiment to equilibrate my iTraq labels (which means that I loose material which is precious since it comes from patients, and I loose quite a lot of time).
Is there a trechnique to get rid of the lipids or is there a peptide quantification method that won't be wronged because of fat ?
Thanks a lot,
Posted 06 May 2013 - 04:26 AM
if not then the interference may be due to the sds (sds will remain bound to the proteins unless displaced by a non-ionic surfactant like triton or nonidet or stripped off by alcohol).
genius does what it must
i do what i get paid to do
Posted 26 July 2013 - 06:21 PM