For very complex samples (100s-1000s of proteins) multi-dimensional approaches have to be used in order to reduce sample complexity prior to mass spectrometry analysis. A number of strategies are used to reduce the sample complexity and include gel pixelation and High pH Reverse Phase Chromatorgaphy (High pH RPC). The subsequent sample fractions are then analysed by standard 1D nanoLC-MS/MS analysis. This process reduces sample complexity so giving the ms system time to analyse the sample in more depth.
Sample Process:
SDS-PAGE Gel Pixelation (e.g. 10 gel sections) followed by nanoLC-MS/MS analysis of each section: Complex samples run on 1D gel lanes are cut into 10 sections and each section processed in a laminar flow cabinet, using our optimised in-gel digestion protocol, in order to minimise dust contamination (i.e. keratin). The digests of each gel section are then run on our 1D nano LC-MS/MS system. The resultant ms data from each gel section is merged prior to Mascot database searching for protein identification. The resultant Mascot report can be emailed to the user or provided as hardcopy.
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FASP or S-TRAP processing followed by fractionation by High pH Reverse Phase Chromatography (High pH RPC) prior to analysis by 1D nanoLC-MS/MS. Complex samples are processed by Filter Aided Sample Preparation (FASP, M Mann) or S-TRAP (Protifi), reductively alkylated and then digested with trypsin. The trypsin digests are then fractionated by High pH RPC prior to 1D nano LC-MS/MS. The resultant ms data from each fraction is merged prior to Mascot database searching for protein identification. The resultant Mascot report can be emailed to the user or provided as hardcopy.
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Urea solubilisation followed by fractionation by High pH RPC prior to 1D nanoLC-MS/MS analysis: Complex samples are precipitated with TCA then re-suspended in 8M Urea prior to reductive alkylation, dilution and subsequent digestion with trypsin. The trypsin digests are then fractionated by High pH RPC prior to 1D nano LC-MS/MS. The resultant ms data from each fraction is merged prior to Mascot database searching for protein identification. The resultant Mascot report can be emailed to the user or provided as hardcopy.