Preparing mare milk sample for proteomic analysis using two-dimensional electrophoresis

Abstract

There is no universal method to prepare physiological fluids for 2-DE proteomic analysis. Furthermore, interspecies differences in milk composition require the formulation of a species-specific sample preparation procedure. The study was carried out on mare's milk which was prepared for 2-DE in four different methods: the first sample (M1) was defatted, sample M2 was defatted and after casein precipitation, sample M3 was sample M2 after reduction of high molecular proteins and sample M4 was desalted sample M3. The milk samples prepared in different methods were separated by 1-DE and 2-DE. The obtained gels were analysed qualitatively and quantitatively. Furthermore, selected protein spots were identified by MALDI-TOF MS. Analysis of 1-DE and 2-DE gel images indicated that the optimal procedure for preparing mare milk samples for 2-DE and identification of proteins by MS is a method based on defatting and precipitation of caseins. The preparation of mare's milk samples by defatting and then precipitation of caseins is enough to obtain an optimal 2-DE separation for identification of proteins of this body fluid. The method of caseins precipitation should be improved in order to reduce the proportion of these high-abundance protein in milk samples which could increase in the identification of low-abundance proteins in mare's milk.