We are attempting non-quantitative shotgun proteomics of unicellular algal culture cell lysates using LC-ESI-MS/MS Orbitrap, but are encountering the issue of only detecting a few dozen very high-abundance, mostly chloroplast-associated proteins (light harvesting proteins, rubisco, etc.). Other than separating the chloroplast from the sample (not possible in these samples), how can we deplete these high-abundance proteins so we can detect lower-abundance proteins in our sample? Is fractionation by SDS-PAGE acceptable?
We often use SDS-PAGE for complexity reduction, followed by in-gel digest. For plant material we would definitely cut the rubisco band (small & large SU) separate and run it at the very end of the MS sequence. The other parts of the gel also can be cut in more bands. We do not recommend to cut equal slices but cut rather larger slices at the bottom of the gel and small ones at the top of the gel (according to its complexity). This will allow you to easily identify a few thousand of proteins.
Another option which we use is a HILIC fractionation followed by a FASP digest. For Arabidopsis tissue (green) we routinely achieve more than 4K proteins.
Hello
I often use 1D gels followed by ingel digests as suggested by Jonas Grossman. Depanding of your cells, you can also deplete for rubisco using some columns with anti rubisco antibody, this has been published, and it is commercialised not sure by whom, maybe biorad.
IT is expensive but apparently you can use a column 100x.
I haven't yet used FASEP for in gel digest but apparently it can help.
Regards.
Laurence
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