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Detection of plant protein adulterated in fluid milk using two-dimensional gel electrophoresis combined with mass spectrometry.

ABSTRACT: The illegal or unlabelled addition of plant protein in milk can cause serious anaphylaxis. For sustainable food security, it is therefore important to develop a methodology to detect non-milk protein in milk products. This research aims to differentiate milk adulterated with plant protein using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry. According to the protein spots highlighted on the gel of adulterated milk, ?-conglycinin and glycinin were detected in milk adulterated with soy protein, while legumin, vicilin, and convicilin indicated the addition of pea protein, and ?-amylase and serpin marked wheat protein. These results suggest that a 2-DE-based protein profile is a useful method to identify milk adulterated with soy and pea protein, with a detection limit of 4% plant protein in the total protein.

SUBMITTER: Yang J

PROVIDER: S-EPMC6033788 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Detection of plant protein adulterated in fluid milk using two-dimensional gel electrophoresis combined with mass spectrometry.

Yang Jinhui J Zheng Nan N Yang Yongxin Y Wang Jiaqi J Soyeurt Hélène H

Journal of food science and technology 20180507 7

The illegal or unlabelled addition of plant protein in milk can cause serious anaphylaxis. For sustainable food security, it is therefore important to develop a methodology to detect non-milk protein in milk products. This research aims to differentiate milk adulterated with plant protein using two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry. According to the protein spots highlighted on the gel of adulterated milk, β-conglycinin and glycinin were detected in milk adult ...[more]

PMID: 30042588

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Project description:BACKGROUND:Proteome analysis is frequently applied in identifying the proteins or biomarkers in knee synovial fluids (SF) that are associated with osteoarthritis and other arthritic disorders. The 2-dimensional gel electrophoresis (2-DE) is the technique of choice in these studies. Disease biomarkers usually appear in low concentrations and may be masked by high abundant proteins. Therefore, the main aim of this study was to find the most suitable sample preparation method that can optimize the expression of proteins on 2-DE gels that can be used to develop a reference proteome picture for non-osteoarthritic knee synovial fluid samples. Proteome pictures obtained from osteoarthritic knee synovial fluids can then be compared with the reference proteome pictures obtained in this study to assist us in identifying the disease biomarkers more correctly. RESULTS:The proteomic tool of 2-DE with immobilized pH gradients was applied in this study. A total of 12 2-DE gel images were constructed from SF samples that were free of osteoarthritis. In these samples, 3 were not treated with any sample preparation methods, 3 were treated with acetone, 3 were treated with 2-DE Clean-Up Kit, and 3 were treated with the combination of acetone and 2-D Clean-Up Kit prior to 2-DE analysis. Gel images were analyzed using the PDQuest Basic 8.0.1 Analytical software. Protein spots that were of interest were excised from the gels and sent for identification by mass spectrometry. Total SF total protein concentration was calculated to be 21.98 ± 0.86 mg/mL. The untreated SF samples were detected to have 456 ± 33 protein spots on 2-DE gel images. Acetone treated SF samples were detected to have 320 ± 28 protein spots, 2-D Clean-Up Kit treated SF samples were detected to have 413 ± 31 protein spots, and the combined treatment method of acetone and 2-D Clean-Up Kit was detected to have 278 ± 26 protein spots 2-DE gel images. SF samples treated with 2-D Clean-Up Kit revealed clearer presentation of the isoforms and increased intensities of the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gel images as compared with untreated SF samples and SF samples treated with acetone. CONCLUSIONS:The acetone precipitation method and the combined treatment effect of acetone and 2-DE Clean-Up Kit are not preferred in preparing SF samples for 2-DE analysis as both protein intensities and numbers decrease significantly. On the other hand, 2-D Clean-Up Kit treated SF samples revealed clearer isoforms and higher intensities for the less abundant proteins of haptoglobin, apolipoprotein A-IV, prostaglandin-D synthase, alpha-1B-glycoprotein, and alpha-2-HS-glycoprotein on 2-DE gels. As a result, it is recommended that SF samples should be treated with protein clean up products such as 2-D Clean-Up Kit first before conducting proteomic research in searching for the relevant biomarkers associated with knee osteoarthritis.

Project description:BackgroundCertain wheat gluten proteins form large protein polymers that are extractable in 0.5% SDS only after sonication. Although there is a strong relationship between the amounts of these polymers in the flour and bread-making quality, the protein components of these polymers have not been thoroughly investigated.ResultsFlour proteins from the US bread wheat Butte 86 were extracted in 0.5% SDS using a two-step procedure with and without sonication. Proteins were further separated by size exclusion chromatography (SEC) into monomeric and polymeric fractions and analyzed by quantitative two-dimensional gel electrophoresis (2-DE). When proteins in select 2-DE spots were identified by tandem mass spectrometry (MS/MS), overlapping spots from the different protein fractions often yielded different identifications. Most high-molecular-weight glutenin subunits (HMW-GS) and low-molecular-weight glutenin subunits (LMW-GS) partitioned into the polymer fractions, while most gliadins were found in the monomer fractions. The exceptions were alpha, gamma and omega gliadins containing odd numbers of cysteine residues. These proteins were detected in all fractions, but comprised the largest proportion of the SDS-extractable polymer fraction. Several types of non-gluten proteins also were found in the polymer fractions, including serpins, triticins and globulins. All three types were found in the largest proportions in the SDS-extractable polymer fraction.ConclusionsThis is the first study to report the accumulation of gliadins containing odd numbers of cysteine residues in the SDS-extractable glutenin polymer fraction, supporting the hypothesis that these gliadins serve as chain terminators of the polymer chains. These data make it possible to formulate hypotheses about how protein composition influences polymer size and structure and provide a foundation for future experiments aimed at determining how environment affects glutenin polymer distribution. In addition, the analysis revealed additional layers of complexity to the wheat flour proteome that should be considered when evaluating quantitative 2-DE data.

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Detection of plant protein adulterated in fluid milk using two-dimensional gel electrophoresis combined with mass spectrometry.

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Detection of plant protein adulterated in fluid milk using two-dimensional gel electrophoresis combined with mass spectrometry.

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