Systematic evaluation of species-independent serum pre-fractionation strategies revealed cost-effective methods to reduce proteome complexity.

In this study, the efficiency of one commercial (ProteoMiner™ -PM) and five simple and cost-effective laboratory chemicals (Acetone, TCA/acetone, DTT, ACN and DTT-ACN) based serum protein pre-fractionation strategies was compared in pig model by label-free quantitation based mass spectrometric approach to find out the most suitable strategy for reducing the complexity of serum proteome for subsequent proteomic studies. The highest serum protein depletion percentage and highest depletion of albumin, the most abundant serum protein, was observed in DTT-ACN method. The maximum number of serum proteins was identified in ACN followed by DTT-ACN method and importantly, detection of more number of low-abundant proteins (LAPs) could also be achieved by these two methods. Although PM method resulted into lowest dynamic range of protein abundance, quite a less number of proteins were identified by this method. Overall, sequential depletion using DTT-ACN and ACN methods provided advantage of simultaneous detection of more number of proteins along with LAPs with a reasonably high dynamic range of protein abundances over other methods and thus emerged as cheaper and effective alternatives to the commercial methods. Further, these methods are species-independent and hence can be applied in human and in any livestock species to simplify the serum proteome.

Comparative serum proteome analysis reveals potential early pregnancy-specific protein biomarkers in pigs.

In this study, the comparative serum proteome profile of Day 5, 12 and 16 of gestation, representing three early embryonic events, namely formation, elongation and implantation of blastocysts, and non-pregnant control were explored by a label-free quantitation-based mass spectrometric approach to identify early pregnancy biomarkers in pigs. A total of 131 proteins were identified with respect to different groups, out of which 105 were found to be differentially expressed proteins (DEPs). Among the DEPs, 54 and 66 proteins were found to be up and downregulated respectively in early pregnancy groups (fold change >2) and the maximum number of upregulated proteins was observed in the Day 12 pregnancy stage. Functional classification and pathway analysis of the DEPs revealed involvement of most of the proteins in complement and coagulation cascades, metabolic processes and immune and inflammatory responses. Proteins such as glutathione peroxidise (GPX), pregnancy zone protein (PZP), thrombospondin-1 (THBS1), α-1-antitrypsin (AAT) and mannose-binding lectin C (MBLC) were differentially expressed during early pregnancy and actively involved in different pregnancy-related activities. To the best of our knowledge, this is the first report on comparative serum protein profiling of different early pregnancy stages in pigs and our results provide a set of proteins that can be used as potential biomarkers for early pregnancy diagnosis in pigs.

Dynamics of cytokine gene expression in peripheral blood mononuclear cells of indigenous and exotic breeds of pigs in India.

To incorporate immune competence traits in swine breeding programs, association between immune responsiveness and susceptibility to specific infectious diseases must be established. In order to understand the differences in immune competence between indigenous (Zovawk) and exotic (Large White Yorkshire: LWY) pigs reared in India, we carried out a time course expression analysis of immune-regulating key cytokine genes (interleukin (IL)-2, interferon (IFN)-γ, IL-4 and IL-10) in the phytohemagglutinin-P stimulated peripheral blood mononuclear cells (PBMCs). The IL-2 transcript levels in PBMCs increased several thousand-fold when compared to unstimulated cells in both the breeds, albeit the response in that of Zovawk was remarkably higher. Higher and earlier IFN-γ and IL-4 expression levels in Zovawk pigs suggest that both TH 1 and TH 2 immune responsiveness of this indigenous breed affords better preparedness for danger signals. Moreover, the low expression levels of IL-10 depict a regulated adaptive immune responsiveness. Remarkable difference between the two breeds of the pigs is evident showing a clear advantage of the Zovawk over LWY in terms of a shorter lag period of adaptive immune response. These findings provide a lead for understanding the genetic differences with respect to immune competence levels of indigenous pigs compared to exotic counterparts.