I would like to extract proteins from mamalian muscle tissues,run 2DE then apply the desired digested spots to mass spectrometry,therefore im looking for a lysis buffer for these kinds of tissue that not contain a detergent which affect the measurement in mass spec.
If you plan to run a 2DE you will have detergents in your gel and thus sample anyway. The detergents will be washed away during the gel extraction process, so you do not need to worry about the detergent aspect, except for that I would avoid Triton-X or NP-40.
About lysis buffer, it depends how you Will perform mass detection.... when mass detection is performed with hyphenated technique as LC-MS, then the lysis buffer Will be washed away with the front of mobile phase phase, while your analyte Will be retained on the column. The front of mobile phaseh shouldn't go into mass spectrometer (mass spectrometers have one extra valve that regulates what goes in mass detector and what doesn't). So, if that is the case just make sure, you Will programme the analyses properly, if not (in the case of direct injection into mass spectrometer), you must use buffers as suggested by Laura Ferrando-Climent.
If you are interested in analyzing proteins, I would recommend you to use a lysis buffer containing detergent. Of all detergents out there, SDS is probably the best for solubilizing the proteins. Since the detergent is not compatible with MS analysis, you can easily remove it bythe filter-aided sample preparation method as described by Wisniewski J.R. et al., Nature Methods, 2009. This is a very nicely designed method that will give you a very pure peptide sample in the end.
Our Lysis buffer for LC-MS/MS contains up to 1% SDS and most methods are working without removing SDS. If the SDS is disturbing your chromatograms you can also remove SDS by precipitating it with Bariumacetate and remove exess Ba with ammonia sulphate.
http://www.sciencedirect.com/science/article/pii/S157002321200102X#
2D electrophoresis requires zwitterionic detergent (CHAPS is common) and chaotropic agents to solublise the proteins from the tissue. Membrane proteins are a problem in 2D electrophoresis, due to solubility problems, and low abundance proteins are not easily visualised in the final 2D gel despite sensitive dyes. An alternative is to use a RIPA buffer which can be found with many variations on Google but you cannot use a charged detergent (neither anionic or cationic) in the first dimension of the electrophoresis. The second dimension requires alkylation, acetamidation and solubilisation in SDS containing buffer performed in the re-equilibration buffer change between dimensions. After the second dimension is completed the gel is visualised (using silver staining is not recommended as it crosslinks proteins and peptides making protein ID very difficult) and the protein spot of interest selected.
You need to be very careful about contaminating the gel during the various procedures that need to be done. Keratin is a very common contaminant from poor care.
The final spot can be digested after destaining and dehydration with acetonitrile so the detergents are also washed out and generally not a problem. The wash steps for the gel plug containing the protein of interest does need to be carried out carefully and extensively otherwise you could end up with detergent in the peptide mix that is being introduced into the mass spectrometer. This can slowly clog up a narrow bore LC column and change retention times with a simultaneous increase in background signal.
Good luck
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques
- Analytical Chemistry
- pH