My sample consists of a mixture of proteins. After size exclusion chromatography, the fraction samples were subjected to ESI MS. The SDS-PAGE gel showed the sIze exclusion fraction has many proteins so, my query is whether we can analyze three or more proteins in ESI MS?
If you fractionate the samples by SDS-PAGE, SEC or SCX, you can analyse hundreds of proteins by ESI-MS
the smaller the fractions in volumes, the better the number of hits or coverage as well as scores
Combining SEC eluents and MS detection with ESI is one of the top-down protein analysis workflows. Fractionation will improve the identification score, whether it is top-down or bottom-up. You can analyze many proteins simultaneously by using these techniques but I would suggest in-gel digestion and bottom-up approach since you have indicated SEC was not satisfying based on fractionation performance. Excising the bands from PAGE and extraction of the protein followed by protease digestion will both give fractionation efficiency and bottom-up peptide level identification ability. Intact level LC separation and CID/HCD/ETD fragmentation of POI that is extracted from the gel is also possible but I personally prefer to continue from the tryptic digest. It should be determined depending on your purpose (identification, quantitation, PTM characterisation, quality attributes etc.)
Thanks,
Emir
Absolutely! As other posters suggested, tryptic or other digestions can be performed to prepare smaller, surrogate peptides which ionize more efficiently. If interested in low intensity compounds (regardless of whether you perform tryptic digestion or not), you may wish to add a supercharging agent such as dimethyl sulfoxide (DMSO) or m-nitrobenzyl alcohol (m-NBA) to mobile phase solutions to coalesce charge states and increase intensity.
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