Figure 1.

Experimental setup and systemic proteomics workflow for the comprehensive discovery of bovine milk whey proteome. (A) From 10 individual animals and pooled milk samples, the milk whey proteome is isolated and purified using ultracentrifugation and TCA/acetone precipitation strategy. In-gel fractionation through SDS-PAGE and in-sol digested peptides were subjected to C18 chromatography for long gradient 96 fractionation (see Materials and Methods). (B) C18 separated fractions were concatenated, mixed, and analyzed using a high-resolution qTOF instrument. All the raw data were extracted, and the resulting peptides and proteins were analyzed using the TPP pipeline utilizing Comet, Tandem, and SpectraST search engines, and the obtained results were compiled using iProphet algorithm as described in Materials and Methods section. (C) Brief description of the data analysis; detailed information is provided in the Materials and Methods section. For determination of the high confidence bovine whey proteome, data were filtered using the stringent parameter. Evidence of the proteome at the different levels was determined along with the identification of the different Gene Ontology (GO) classes in it.