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protein extraction and peptide quantification from fat breast tissue

fat microBCA

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#1 Vero36

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Posted 04 May 2013 - 05:09 AM

Dear all,

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,

Vero

#2 mdfenko

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Posted 06 May 2013 - 04:26 AM

do you use the same buffer to isolate the proteins from tumors?

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).
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#3 killme00012

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Posted 26 July 2013 - 06:21 PM

yeah, SDS may be a problem due to incomplete digestion, so the inference may be the incompleted digest proteins not fatty, and i suggest that you'd better try out TCA precipitation, it could remove fatty effieciently




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