Protein Identification by 2D nLC-MS-MS

SDS-PAGE Gel Pixelation - Reverse Phase Chromatography or Strong Cation eXchange - Reverse Phase Chromatography

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. Therefore a number of strategies are used including SDS-PAGE gel pixelation and Strong Cation eXchange (SCX) in order to fractionate the sample prior to 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 prior to Mascot database searching. The resultant Mascot reports are sent by email or provided as hardcopy.

or

Strong Cation eXchange fractionation followed by nanoLC-MS/MS analysis of each SCX fraction: Complex samples are run on SDS-PAGE gels to 1-2 cm then the whole gel section excised, processed, in-gel reductively alkylated and then digested with trypsin. The trypsin digests are then fractionated by SCX prior to 1D nano LC-MS/MS of each SCX fraction. 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.

or

Urea solubilisation with Strong Cation eXchange fractionation followed by 1D nanoLC-MS/MS analysis of each SCX fraction: Complex samples are precipitated with TCA then re-suspended in Urea prior to reductive alkylation and subsequent digestion with trypsin in solution. The trypsin digests are then fractionated by SCX prior to 1D nano LC-MS/MS of each SCX fraction. The resultant ms data from each fraction is merged prior to Mascot database searching for protein identification. The resultant Mascot report can be emailed tothe user or provided as hardcopy.