I made 1D-western blotting of different crude antigens of Schistosoma mansoni. Now I need to perform the MS. I have read about the subject and I would like to know if I have to do the 2D-gel because my sample is small.
As far as I know, 1-D gels can be used for proteomics analysis, all you have to do is carefully divide the gel into about 10-15 lane cuts and do in-gel digestion and Mass Spec measurement. However you should bear in mind that a 2-D gel has a better resolution because several proteins with similar or relatively close molecular weights will be superimposed and hence 2-D gel will separate such by isoelectric point/ immobilized pH gradient (IPG). If you have your way I will either suggest an automated 2-D spot cut or do in-solution digest or FASP.
What mass spectrometer are you using? Nowadays the resolution of mass spectrometers is such that there is virtually no need for the extra resolution of 2D gels. Proteins superimposed in the same band won't be a problem with MS/MS. The only reason you'd need a 2D gel is for comparative proteomics with 2D DiGE (if SILAC isn't an option) or for very specific cases when you are looking for a particular protein. That is not to say there can't be issues, but usually you can only notice that when looking for a single protein (when you are looking at the entire proteome, it's hard to tell if anything is missing given the amount of data generated). With that said, a 1D gel shouldn't be a limitation compared to 2D.
Gel-based (either 1- 0r 2- D) proteomics is often fraught with a lot of upstream and downstream issues. Many researchers have gradually phased out gel-based to embrace gel-free systems. In my experience, there are too many potential sources of variance in gel-based systems and as such its use especially for global proteomics must be considered with a lot of caution.
i read a paper in where they used 1D for myelin proteomics. the reason was that at high pI, 2D resolution is not good. but they cut about hundred pieces of a line for MS sampling. so everything is possible.
You should have no problem identifying proteins from 1D bands. There is possibility that you may get good identification score for more than one protein from a single band. Two- dimensional (2-DE) is better for resolution but 1D should give you sufficient information.
Regarding the issue Henry mentioned (electrophoretic vs chromatographic sample preparation), I think those two papers are a good starting point to understand the pros and cons of each. The first paper is about intact proteins preparation (top down mass spec) and the second for digested protein (bottom up) which is more common:
http://www.ncbi.nlm.nih.gov/pubmed/21689823
http://www.ncbi.nlm.nih.gov/pubmed/19816929
Vanessa, some things to keep in mind here:
- an SDS-PAGE band from a biological sample in my experience never contains less than a few dozen proteins, no matter how accurately you cut. You will therefore need LC/MS/MS with upfront separation to identify more than the topmost 1-3 proteins in the band. Peptide Mass Fingerprinting or infusion experiments are probably not going to cut it
- if you start from a Western: that is a highly selective detection technique, and also very sensitive. Consequently, the protein causing the Western stain is not necessarily the most abundant in an SDS-PAGE band - often quite the opposite. You should expect quite a number of other proteins to score higher in your MS experiment. Again this strongly points to using a good LC/MS/MS setup to obtain analytical depth
- how sure are you of the specificity of the antibody used for the Western?
If your sample is limited, SDS-PAGE followed by LC/MS/MS is still the best option in my opinion. Yes you could move to gel-free setups as suggested by others, however you would give away the correlation with the Western.
- Badges
- Science method