No 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.

Before electrophoretic separation is performed, the samples must be dissolved in a lysis buffer (necessary to keep proteins dissolved and unbound during proteomic analyses during for a separation of proteins on polyacrylamide gels). The first step in preparing samples for proteomic analyses is their precipitation using e.g. acetone. The aim of precipitation is to obtain proteins from the sample and to remove the compounds interfering with 2-D electrophoresis. Due to difficulties in dissolving some colostrum and mare's milk samples in buffer lysis electrophoretic separation of this biological material was performed without acetone precipitation of proteins. To assess the effectiveness of the applied method, after two-dimensional separation of proteins (2-DE), the obtained gels were stained and archived. The preparation of mare's colostrum and milk samples for proteomic analyses, consisting of defatting, then precipitation of caseins and separation 2-DE, which was not preceded by precipitation of the samples with acetone, resulted in the loss of many protein spots which made it impossible to identify them later using the mass spectrometer.

Colostrum is an essential feed of foals. It is a source of nutrients and functional proteins significant for foals’ growth and development. In the presented research using two-dimensional electrophoresis coupled via spectrometry mass MALDI-TOF in the mares’ colostrum (whey proteins fraction) were identified 24 proteins representing 15 different gene products. The identified proteins were involved in supporting foals’ immature immune systems and in the transport of various compounds. Further research of mares’ colostrum will allow determining more gene products. An in-depth analysis of mares’ milk will provide information about biochemical processes occurring in the mammary gland of the mare during the lactation period.