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.

Determination of A1 and A2 β-Casein in Milk Using Characteristic Thermolytic Peptides via Liquid Chromatography-Mass Spectrometry

“…To fully dissolve the proteins, a buffer solution is usually prepared with urea, dithiothreitol, or inorganic salts added [ 22 ], which contributes to protein unfolding and thus improves the efficiency of extraction and enzymatic digestion. The sample should be desalted before being subjected to LC-MS analysis, avoiding contamination of the mass spectrometer.…”

Proteomic profile of epidermal mucus from Labeo rohita reveals differentially abundant proteins after Aeromonas hydrophila infection

“…After extraction, protein content was calculated with standard Bovine Serum Albumin (BSA), and a protein curve was made by using a colorimetric Bradford assay. SDS-PAGE was performed and each protein was extracted by the method of [32] with little modifications. The protein samples were put on a 5 % stacking gel at 75V and a 12 % resolving gel at 125V.…”

Advances in separation and identification of biologically important milk proteins and peptides

“…For milk proteome and peptidome characterization, effective sample preparation techniques were developed as well as various proteomic techniques, such as 2DE, electrophoretic, and chromatographic techniques performed in capillaries and followed by MS identification [22][23][24]113]. For example, the qualitative/quantitative efficacy of three protein extraction methods has been evaluated for allergenomic analysis of goat milk [114] using (i) urea/thiourea-based extraction; (ii) methanol/chloroform triphasic extraction, and (iii) sodium sulfite-based extraction. Based on 2DE maps and analysis by MALDI-TOF/TOF MS, the highest protein recovery, number of protein spots, and number of proteins yielded the method with urea/thiourea buffer extraction.…”