I'm currently analysing proteins from S.cerevisiae, and while analysing it by ESI and MALDI the CHAPS peak keeps appearing. I already cleaned the samples with Chlorophorm/MetOH protocol. Is there any efficient method to clean my sample?
I would suggest a buffer exchange via size exclusion to a more volatile buffer.
Without more info on your peptides or your goals I can only recommend general methods. IF the protein sample is trypsin digested peptides then I would put the entire sample through a C18 column or ZipTip. Protea Biosciences makes a cheaper alternative to Millipore Ziptips that is quite good. You can elute in an acetonitrile gradient and go directly to MS. I do this to MALDI routinely but ESI is also possible. With the C18 column you may be able to skip your chloroform wash step. https://proteabio.com
Thank you for the reply, my samples are trypsin digested and resuspended in % Formic acid. I'm just trying to get a profile of proteins excreted to the medium, the misture should be too complex, 10-15 proteins according to the 2D
So you need to isolate proteins from the medium then separate and then tryptic digestion? I did something similar. I have attached the manuscript. I grew the bugs in filtered media to limit the background molecular weights and then isolated the proteins that were produced. I determined what was important based on human immune reactivity. Could you pick from your 2-D gels? You could also capture the proteins before trypsinization with a sepak column and a syringe barrel. Then wash and elute to a buffer compatible with whatever downstream column you use to separate.
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I usually avoid CHAPS (I use C7BzO at 1%) but got some samples prepared with 4% CHAPS. Not surprisingly, after shotgun digest and SPE clean up with OASIS HLB, there was still loads of CHAPS which caused awesome ion supression in nanoESI. We got around it by loading 10ug into 1D SDS-PAGE and running the gel until the proteins were
if it is possible you can use strong cation exchange chromatographic at acid pH. The chaps molecule is neutral (it has a fixed positive charge and a sulpho group.
Yes, use ZIP-TIPs. They are made by Millipore. http://www.millipore.com/catalogue/module/c5737
CHAPS (and other detergent) cannot be completely removed by precipitation or by C18 solid phase extraction. As long as the CHAPS peak (should be m/z 615 and 1229) is under control, it won't hurt your experiment.
If you want complete detergent removal (SDS, triton, CHAPS, etc), you have to go for SCX SPE or use those commercial detergent removal kits. For SCX, you can wash with 1% TFA, then elute with 5% ammonium hydroxide solution. Avoid using salt buffer solution so you don't have to use C18 desalt afterward.
Dilute the sample up to 0.1% CHAPS (MCI) and then filter it with a MW filter 10KDa 4 rounds. You will see no CHAPS in your sample. C18 doesn´t work . CHAPS has a big carbon structure that binds to C18.
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