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Development of a rapid and improved two-dimensional electrophoresis method for the separation of protein lysate

Kwan SH, Ismail MN

Biomed Chromatogr, 2019 Dec;33(12):e4686.
PMID: 31452214 DOI: 10.1002/bmc.4686

Researchers frequently use two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) prior to mass spectrometric analysis in a proteomics approach. The i2D-PAGE method, which 'inverts' the dimension of protein separation of the conventional 2D-PAGE, is presented in this publication. Protein lysate of Channa striata, a freshwater snakehead fish, was separated based on its molecular weight in the first dimension and its isoelectric point in the second dimension. The first-dimension separation was conducted on a gel-free separation device, and the protein mixture was fractionated into 12 fractions in chronological order of increasing molecular weight. The second-dimension separation featured isoelectric focusing, which further separated the proteins within the same fraction according to their respective isoelectric point. Advantages of i2D-PAGE include better visualisation of the isolated protein, easy identification on protein isoforms, shorter running time, customisability and reproducibility. Erythropoietin standard was applied to i2D-PAGE to show its effectiveness for separating protein isoforms. Various staining methods such as Coomassie blue staining and silver staining are also applicable to i2D-PAGE. Overall, the i2D-PAGE separation method effectively separates protein lysate and is suitable for application in proteomics research.
Clinical Study on Plant Galactagogue Worldwide in Promoting Women's Lactation: a Scoping Review

Kwan SH, Abdul-Rahman PS

Plant Foods Hum Nutr, 2021 Sep;76(3):257-269.
PMID: 34292494 DOI: 10.1007/s11130-021-00901-y

Human milk is recommended by the World Health Organization (WHO) for the general well-being of infants. However, many mothers face an insufficient milk supply to breastfeed their children. Galactagogue, in particular, plant galactagogue, serves as a method to promote lactation. This in-depth review examines the evidence supporting different plants' galactagogic activity through clinical studies around the globe. A scoping review approach was adopted to establish the research questions, and define the findings, selection and analysis of the study. This scoping review highlights and compiles the clinical research performed globally involving plant galactagogue to better inform the medical practitioners, lactation consultants, nursing mothers, communities and relevant personnel on practicing, guidelines, policymaking and research. In general, a total of 1041 research publications were retrieved from different global bibliographic databases, of which only 13 articles were retained for analysis after applying the exclusion criteria. A total of 14 types of plants have undergone clinical studies in the past decade to verify their galactagogic activity. All but two showed a positive effect on promoting milk production. There were 42 articles categorised as excluded studies. The category includes review articles, surveys, case reports, introductory articles of regional plant galactagogue and preclinical studies, which involves animal testing and the studies exploring other issues related to plant galactagogue. The findings demonstrate that there is a significant research gap on the plant galactagogue using clinical studies. More clinical research is necessary to identify and verify the efficacy of various types of plant galactagogue for the benefit of humankind.
Fulltext Bioactive Proteins in Channa striata Promote Wound Healing through Angiogenesis and Cell Proliferation

Kwan SH, Abdul Aziz NHK, Ismail MN

Protein Pept Lett, 2020;27(1):48-59.
PMID: 31362651 DOI: 10.2174/0929866526666190730121711

BACKGROUND: Channa striata are speculated to contain bioactive proteins with the ability to enhancing wound healing. It is commonly consumed after surgery for a faster recovery of the wound.
OBJECTIVE: To identify the bioactive proteins and evaluate their ability in cell proliferation and angiogenesis promotion.
MATERIAL AND METHODS: Freeze-Dried Water Extracts (FDWE) and Spray-Dried Water Extracts (SDWE) of C. striata were tested with MTT assay using EA.hy926 endothelial cell line and ex-vivo aortic ring assay. Later the proteins were fractionated and analysed using an LC-QTOF mass spectrometer. The data generated were matched with human gene database for protein similarity and pathway identification.
RESULTS: Both samples have shown positive cell proliferation and pro-angiogenic activity. Four essential proteins/genes were identified, which are collagen type XI, actin 1, myosin light chain and myosin heavy chain. The pathways discovered that related to these proteins are integrin pathway, Slit-Robo signalling pathway and immune response C-C Chemokine Receptor-3 signalling pathway in eosinophils, which contribute towards wound healing mechanism.
CONCLUSIONS: The results presented have demonstrated that C. striata FDWE and SDWE protein fractions contain bioactive proteins that are highly similar to human proteins and thus could be involved in the wound healing process via specific biological pathways.
Isolation and identification of O- and N-linked glycoproteins in milk from different mammalian species and their roles in biological pathways which support infant growth

Kwan SH, Wan-Ibrahim WI, Juvarajah T, Fung SY, Abdul-Rahman PS

Electrophoresis, 2021 02;42(3):233-244.
PMID: 33085102 DOI: 10.1002/elps.202000142

Milk serves as the sole nutrition for newborns, as well as a medium for the transfer of immunological components from the mother to the baby. This study reveals different glycoprotein profiles obtained from human, bovine, and caprine milk and their potential roles in supporting infant growth. Proteins from these three milk samples are separated and analyzed using two-dimensional gel electrophoresis (2-DE). Glycosylated proteins from all samples are enriched by affinity chromatography using lectins from the seeds of Artocarpus integer before analysis using LC/MS-QTOF. The glycoproteome profiling demonstrates that glycosylated proteins are higher in caprine milk compared to other samples. Analysis using LC/MS-QTOF identified 42 O-glycosylated and 56 N-glycosylated proteins, respectively. Among those identified, human milk has 17 glycoproteins, which are both O- and N-glycosylated, whereas caprine and bovine have 10 and 1, respectively. Only glycoproteins from human milk have shown positive matching to important human biological pathways, such as vesicle-mediated transport, immune system and hemostasis pathways. Human milk remains unique for human babies with the presence of antibodies in the form of immunoglobulins that are lacking in ruminant milk proteomes.