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

Muzammeer Mansor,Atiqah Farah Zakaria,Jameel R Al-Obaidi,Nuzul Noorahya Jambari,Intan Hakimah Ismail,Nurain Nadiah Jaafar,Jinap Selamat,Mohd Azri Zainal Abidin,Son Radu

doi:10.3390/molecules25112625

Muzammeer Mansor, Atiqah Farah Zakaria + Show 7 more

Open Access

https://doi.org/10.3390/molecules25112625

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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.

Highlights

Cow’s milk protein allergy (CMPA) is prevalent among young children and has been reported to affect around 2.5% of children during their early years [1,2]
Protein extraction efficiency and reproducibility were evaluated based on the amount of extracted protein from 0.5 mL of skim goat’s milk samples
Protein concentration of each extract was tabulated in. This is followed by method B, which is a methanol/chloroform-based protein triphasic separation method, and method C, a modified sodium-sulfite-based method that consists of Tris/HCl, Tween 20, sodium dodecyl sulphate (SDS), and sodium sulfite

Summary

Introduction

Cow’s milk protein allergy (CMPA) is prevalent among young children and has been reported to affect around 2.5% of children during their early years [1,2]. Goat’s milk has been proposed as a hypoallergenic cow’s milk substitute for CMPA patients. Despite their close milk protein homology due to sharing the same phylogenetic origin [4], about 40% of cow’s milk protein allergic patients were estimated to have tolerance toward goat’s milk proteins [5]. Most of the previous studies performed on evaluating the goat’s milk protein cross-reactivities with cow’s milk allergens [9,10] employed 1D SDS-PAGE and IgE-immunoblotting to suggest presumptive allergenic proteins, yet there has been no study focusing on profiling the complete allergenic proteins (allergenome) of milk from different goat breeds and their cross-reactivities with cow’s milk allergens

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