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Fulltext Evaluation of sodium deoxycholate as solubilization buffer for oil palm proteomics analysis

Lau BYC, Othman A

PLoS One, 2019;14(8):e0221052.
PMID: 31415606 DOI: 10.1371/journal.pone.0221052

Protein solubility is a critical prerequisite to any proteomics analysis. Combination of urea/thiourea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) have been routinely used to enhance protein solubilization for oil palm proteomics studies in recent years. The goals of these proteomics analysis are essentially to complement the knowledge regarding the regulation networks and mechanisms of the oil palm fatty acid biosynthesis. Through omics integration, the information is able to build a regulatory model to support efforts in improving the economic value and sustainability of palm oil in the global oil and vegetable market. Our study evaluated the utilization of sodium deoxycholate as an alternative solubilization buffer/additive to urea/thiourea and CHAPS. Efficiency of urea/thiourea/CHAPS, urea/CHAPS, urea/sodium deoxycholate and sodium deoxycholate buffers in solubilizing the oil palm (Elaeis guineensis var. Tenera) mesocarp proteins were compared. Based on the protein yields and electrophoretic profile, combination of urea/thiourea/CHAPS were shown to remain a better solubilization buffer and additive, but the differences with sodium deoxycholate buffer was insignificant. A deeper mass spectrometric and statistical analyses on the identified proteins and peptides from all the evaluated solubilization buffers revealed that sodium deoxycholate had increased the number of identified proteins from oil palm mesocarps, enriched their gene ontologies and reduced the number of carbamylated lysine residues by more than 67.0%, compared to urea/thiourea/CHAPS buffer. Although only 62.0% of the total identified proteins were shared between the urea/thiourea/CHAPS and sodium deoxycholate buffers, the importance of the remaining 38.0% proteins depends on the applications. The only observed limitations to the application of sodium deoxycholate in protein solubilization were the interference with protein quantitation and but it could be easily rectified through a 4-fold dilution. All the proteomics data are available via ProteomeXchange with identifier PXD013255. In conclusion, sodium deoxycholate is applicable in the solubilization of proteins extracted from oil palm mesocarps with higher efficiency compared to urea/thiourea/CHAPS buffer. The sodium deoxycholate buffer is more favorable for proteomics analysis due to its proven advantages over urea/thiourea/CHAPS buffer.
Application of Proteomics Technologies in Oil Palm Research

Lau BYC, Othman A, Ramli US

Protein J, 2018 12;37(6):473-499.
PMID: 30367348 DOI: 10.1007/s10930-018-9802-x

Proteomics technologies were first applied in the oil palm research back in 2008. Since proteins are the gene products that are directly correspond to phenotypic traits, proteomic tools hold a strong advantage above other molecular tools to comprehend the biological and molecular mechanisms in the oil palm system. These emerging technologies have been used as non-overlapping tools to link genome-wide transcriptomics and metabolomics-based studies to enhance the oil palm yield and quality through sustainable plant breeding. Many efforts have also been made using the proteomics technologies to address the oil palm's Ganoderma disease; the cause and management. At present, the high-throughput screening technologies are being applied to identify potential biomarkers involved in metabolism and cellular development through determination of protein expression changes that correlate with oil production and disease. This review highlights key elements in proteomics pipeline, challenges and some examples of their implementations in plant studies in the context of oil palm in particular. We foresee that the proteomics technologies will play more significant role to address diverse issues related to the oil palm in the effort to improve the oil crop.
Fulltext Proteomics analysis on lipid metabolism in Elaeis guineensis and Elaeis oleifera

Lau BYC, Amiruddin MD, Othman A

Data Brief, 2020 Aug;31:105714.
PMID: 32462070 DOI: 10.1016/j.dib.2020.105714

Proteome data was obtained from the fruit mesocarps of the two oil palm species, namely, the African Elaeis guineensis (commercial tenera or commonly known as D x P and MPOB-Nigerian tenera) and the South American Elaeis oleifera. Total proteins were extracted from randomly selected fruitlets and subjected to proteomics characterisation by means of liquid chromatography mass spectrometry. Number of proteins identified, the grouping of the biological replicates from five developmental weeks after anthesis, and the localisation of gene corresponded to the detected proteins on the oil palm chromosomes, were presented. A total of 4,116, 4,210 and 4,081 proteins were found in commercial tenera and MPOB Nigerian tenera for Elaeis guineensis; and Elaeis oleifera, respectively. Principal component analysis showed two distinct clusters that corresponded to Elaeis guineensis and Elaeis oleifera. Collectively, genes that corresponded to the identified proteins were found to be located in all 16 oil palm chromosomes. A total of 59 proteins from Elaeis guineensis and Elaeis oleifera were down-regulated for >5-fold change during the peak of lipid biosynthesis compared to the onset. The same comparative analysis revealed that 66 proteins were up-regulated for >5-fold change. About 60.0% of the observed proteins were involved in catalytic activity while 28.5% were associated with redox reaction. Based on same datasets, the tricarboxylic acid cycle and 5-hydroxytryptamine degradation pathways were found to be enriched the most (>36-fold change). These data can be used to support the oil palm gene model validation and lipid metabolism research, particularly in the areas of oil yield and quality. The tabulated protein lists of identified proteins and their expression changes from these varieties were provided as supplementary files. Raw MSF and mzid files for all the oil palm species were deposited in the ProteomeXchange (PXD017436).
Optimization of protein extraction for proteomic analyses of fresh and frozen "Musang King" durian pulps

Tan XY, Misran A, Daim LDJ, Lau BYC

Food Chem, 2021 May 01;343:128471.
PMID: 33143964 DOI: 10.1016/j.foodchem.2020.128471

Four different methods were evaluated to extract proteins from "Musang King" durian pulps and subsequently proteins with different abundance between fresh and long term frozen storage were identified using two-dimensional polyacrylamide gel electrophoresis coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analyses. The acetone-phenol method was found to produce good protein yields and gave the highest gel resolution and reproducibility. Differential protein analyses of the durian pulp revealed that 15 proteins were down-regulated and three other proteins were up-regulated after a year of frozen storage. Isoflavone reductase-like protein, S-adenosyl methionine synthase, and cysteine synthase isoform were up-regulated during frozen storage. The down-regulation of proteins in frozen durian pulps indicated that frozen storage has affected proteins in many ways, especially in their functions related to carbohydrate and energy metabolisms, cellular components, and transport processes. This study will enable future detailed investigations of proteins associated with quality attributes of durians to be studied.
Geographical Diversity of Proteomic Responses to Cold Stress in the Fungal Genus Pseudogymnoascus

Abu Bakar N, Lau BYC, González-Aravena M, Smykla J, Krzewicka B, Karsani SA, et al.

Microb Ecol, 2023 Dec 07;87(1):11.
PMID: 38060022 DOI: 10.1007/s00248-023-02311-w

In understanding stress response mechanisms in fungi, cold stress has received less attention than heat stress. However, cold stress has shown its importance in various research fields. The following study examined the cold stress response of six Pseudogymnoascus spp. isolated from various biogeographical regions through a proteomic approach. In total, 2541 proteins were identified with high confidence. Gene Ontology enrichment analysis showed diversity in the cold stress response pathways for all six Pseudogymnoascus spp. isolates, with metabolic and translation-related processes being prominent in most isolates. 25.6% of the proteins with an increase in relative abundance were increased by more than 3.0-fold. There was no link between the geographical origin of the isolates and the cold stress response of Pseudogymnoascus spp. However, one Antarctic isolate, sp3, showed a distinctive cold stress response profile involving increased flavin/riboflavin biosynthesis and methane metabolism. This Antarctic isolate (sp3) was also the only one that showed decreased phospholipid metabolism in cold stress conditions. This work will improve our understanding of the mechanisms of cold stress response and adaptation in psychrotolerant soil microfungi, with specific attention to the fungal genus Pseudogymnoascus.
Fulltext Transcriptomic and proteomic profiling revealed reprogramming of carbon metabolism in acetate-grown human pathogen Candida glabrata

Chew SY, Brown AJP, Lau BYC, Cheah YK, Ho KL, Sandai D, et al.

J Biomed Sci, 2021 Jan 02;28(1):1.
PMID: 33388061 DOI: 10.1186/s12929-020-00700-8

BACKGROUND: Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection.
METHODS: In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches.
RESULTS: Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages.
CONCLUSION: Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.
Fulltext Computationally Designed Anti-LuxP DNA Aptamer Suppressed Flagellar Assembly- and Quorum Sensing-Related Gene Expression in Vibrio parahaemolyticus

Yusof NAM, Razali SA, Mohd Padzil A, Lau BYC, Baharum SN, Nor Muhammad NA, et al.

Biology (Basel), 2022 Nov 01;11(11).
PMID: 36358301 DOI: 10.3390/biology11111600

(1) Background: Quorum sensing (QS) is the chemical communication between bacteria that sense chemical signals in the bacterial population to control phenotypic changes through the regulation of gene expression. The inhibition of QS has various potential applications, particularly in the prevention of bacterial infection. QS can be inhibited by targeting the LuxP, a periplasmic receptor protein that is involved in the sensing of the QS signaling molecule known as the autoinducer 2 (AI-2). The sensing of AI-2 by LuxP transduces the chemical information through the inner membrane sensor kinase LuxQ protein and activates the QS cascade. (2) Methods: An in silico approach was applied to design DNA aptamers against LuxP in this study. A method combining molecular docking and molecular dynamics simulations was used to select the oligonucleotides that bind to LuxP, which were then further characterized using isothermal titration calorimetry. Subsequently, the bioactivity of the selected aptamer was examined through comparative transcriptome analysis. (3) Results: Two aptamer candidates were identified from the ITC, which have the lowest dissociation constants (Kd) of 0.2 and 0.5 micromolar. The aptamer with the lowest Kd demonstrated QS suppression and down-regulated the flagellar-assembly-related gene expression. (4) Conclusions: This study developed an in silico approach to design an aptamer that possesses anti-QS properties.
Cross-reactivity analysis of milk proteins from different goat breeds with cow's milk allergens using a proteomic approach

Mansor M, Al-Obaidi JR, Ismail IH, Abidin MAZ, Zakaria AF, Lau BYC, et al.

Mol Immunol, 2023 Mar;155:44-57.
PMID: 36696839 DOI: 10.1016/j.molimm.2022.12.016

INTRODUCTION: Goat's milk thought to be a good substitute for cow's milk protein allergic (CMPA) individuals. However, there is growing evidence that their proteins have cross-reactivities with cow's milk allergens. This study aimed to profile and compare milk proteins from different goat breeds that have cross-reactivity to cow's milk allergens.
METHODOLOGY: Proteomics was used to compare protein extracts of skim milk from Saanen, Jamnapari, and Toggenburg. Cow's milk was used as a control. IgE-immunoblotting and mass spectrometry were used to compare and identify proteins that cross-reacted with serum IgE from CMPA patients (n = 10).
RESULTS: The analysis of IgE-reactive proteins revealed that the protein spots identified with high confidence were proteins homologous to common cow's milk allergens such as α-S1-casein (αS1-CN), β-casein (β-CN), κ-casein (κ-CN), and beta-lactoglobulin (β-LG). Jamnapari's milk proteins were found to cross-react with four major milk allergens: α-S1-CN, β-CN, κ-CN, and β-LG. Saanen goat's milk proteins, on the other hand, cross-reacted with two major milk allergens, α-S1-CN and β-LG, whereas Toggenburg goat's milk proteins only react with one of the major milk allergens, κ-CN.
CONCLUSION: These findings may help in the development of hypoallergenic goat milk through cross-breeding strategies of goat breeds with lower allergenic milk protein contents.