Optimization of Protein Extraction Method for 2DE Proteomics of Goat's Milk

Abstract

Two-dimensional electrophoretic (2DE)-based proteomics remains a powerful tool for allergenomic analysis of goat's milk but requires effective extraction of proteins to accurately profile the overall causative allergens. However, there are several current issues with goat's milk allergenomic analysis, and among these are the absence of established standardized extraction method for goat's milk proteomes and the complexity of goat's milk matrix that may hamper the efficacy of protein extraction. This study aimed to evaluate the efficacies of three different protein extraction methods, qualitatively and quantitatively, for the 2DE-proteomics, using milk from two commercial dairy goats in Malaysia, Saanen, and Jamnapari. Goat's milk samples from both breeds were extracted by using three different methods: a milk dilution in urea/thiourea based buffer (Method A), a triphasic separation protocol in methanol/chloroform solution (Method B), and a dilution in sulfite-based buffer (Method C). The efficacies of the extraction methods were assessed further by performing the protein concentration assay and 1D and 2D SDS-PAGE profiling, as well as identifying proteins by MALDI-TOF/TOF MS/MS. The results showed that method A recovered the highest amount of proteins (72.68% for Saanen and 71.25% for Jamnapari) and produced the highest number of protein spots (199 ± 16.1 and 267 ± 10.6 total spots for Saanen and Jamnapari, respectively) with superior gel resolution and minimal streaking. Six milk protein spots from both breeds were identified based on the positive peptide mass fingerprinting matches with ruminant milk proteins from public databases, using the Mascot software. These results attest to the fitness of the optimized protein extraction protocol, method A, for 2DE proteomic and future allergenomic analysis of the goat's milk.

Keywords: 2DE; gel electrophoresis; goat’s milk; protein extraction; proteomics.

Figure 1

The protein recovery rate of each extraction method is presented as a percentage of extracted protein concentration calculated against skim milk protein concentration. Error bars are from Bradford assay technical replicates and presented as SD (n = 3).

Figure 2

SDS-PAGE profiles of goat’s milk from Saanen (S) and Jamnapari (J) extracted using three different extraction methods: (A) urea/thiourea-based extraction, (B) methanol/chloroform-based triphasic separation, and (C) sodium sulfite-based extraction.

Figure 3

Two-dimensional electrophoretic (2DE) profiles of three different total protein extraction methods: (A) urea/thiourea-based, (B) methanol/chloroform triphasic separation, and (C) sodium sulfite-based extraction methods, of milk from (S) Saanen and (J) Jamnapari goats. Protein extracts (400 µg) were run on 13 cm pH 4–7 IPG strip and resolved on 12% bis-acrylamide gel.

Figure 4

Spots chosen for protein identification by MALDI-TOF/TOF MS/MS for (S) Saanen and (J) Jamnapari: (A) serum albumin region [36,41]; (B) casein region [37,38]; (C) beta-lactoglobulin region [26,39], and (D) S100 calcium-binding protein region [40].

Figure 5

(A) Images of protein spots from 2DE gels of Saanen and Jamnapari goat’s milk extracted using method A that were chosen for identification by MALDI-TOF/TOF MS/MS. (B) Five spots from each breed were selected based on milk protein regions that were present in both breeds, with no significant differences in their levels, and two spots based on the significant differences in their abundance levels when analyzed by Progenesis SameSpot software. * Denotes protein spots with p-values < 0.05.

Figure 5

(A) Images of protein spots from 2DE gels of Saanen and Jamnapari goat’s milk extracted using method A that were chosen for identification by MALDI-TOF/TOF MS/MS. (B) Five spots from each breed were selected based on milk protein regions that were present in both breeds, with no significant differences in their levels, and two spots based on the significant differences in their abundance levels when analyzed by Progenesis SameSpot software. * Denotes protein spots with p-values < 0.05.

Figure 6

A workflow overview of the three extraction methods used in this study on milk proteins from Saanen and Jamnapari goats; n indicates the number of technical replicates used for each stage of the study.