We carry out the process of protein isolation from an active substance, post-fermentation medium as well as biological fluids with the use of gel electrophoresis, high performance liquid chromatography (HPLC) and the collector of fractions or the ultra fast liquid chromatography (UFLC), also in tandem with the 4000 QTRAP LC/MS/MS (Applied Biosystems). With the Tempo™ LC MALDI Spotting System (Applied Biosystems) device it is possible to divide and fractionate samples in nano-volumes. The collected fractions placed on a steel plate are to be analyzed on the 4800 Plus MALDI-TOF/TOF mass spectrometer (Applied Biosystems). In addition we perform purification and concentration of samples on RP-18/ RP-4 type of chromatographic bed (e.g. ZipTip® C18, ZipTip® C4, PepClean™ C-18 Spin Column) or with the use of U–Tube™ concentrators and others.
The isolated proteins in the form of a solution or gel stripes stained with Coomassie Blue are further identified based on the peptide maps obtained as a result of proteolysis of the analyzed protein with the use of a dedicated enzyme (e.g. trypsin, Glu-C, Lys-C, Asp-N). Analysis of the resulting MS/MS spectra is carried out on the basis of browsing the database of protein sequences with the use of the MASCOT® system. Prior to digestion the disulphide bridges in proteins usually undergo reduction and alkylation.