Displaying publications 1 - 20 of 28 in total

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  1. Tan CH, Tan KY, Tan NH
    J Proteomics, 2016 07 20;144:33-8.
    PMID: 27282922 DOI: 10.1016/j.jprot.2016.06.004
    Recent advances in proteomics enable deep profiling of the compositional details of snake venoms for improved understanding on envenomation pathophysiology and immunological neutralization. In this study, the venom of Australian tiger snake (Notechis scutatus) was trypsin-digested in solution and subjected to nano-ESI-LCMS/MS. Applying a relative quantitative proteomic approach, the findings revealed a proteome comprising 42 toxin subtypes clustered into 12 protein families. Phospholipases A2 constitute the most abundant toxins (74.5% of total venom proteins) followed by Kunitz serine protease inhibitors (6.9%), snake venom serine proteases (5.9%), alpha-neurotoxins (5.6%) and several toxins of lower abundance. The proteome correlates with N. scutatus envenoming effects including pre-synaptic and post-synaptic neurotoxicity and consumptive coagulopathy. The venom is highly lethal in mice (intravenous median lethal dose=0.09μg/g). BioCSL Sea Snake Antivenom, raised against the venoms of beaked sea snake (Hydrophis schistosus) and N. scutatus (added for enhanced immunogenicity), neutralized the lethal effect of N. scutatus venom (potency=2.95mg/ml) much more effectively than the targeted H.schistosus venom (potency=0.48mg/ml). The combined venom immunogen may have improved the neutralization against phospholipases A2 which are abundant in both venoms, but not short-neurotoxins which are predominant only in H. schistosus venom.

    SIGNIFICANCE: A shotgun proteomic approach adopted in this study revealed the compositional details of the venom of common tiger snake from Australia, Notechis scutatus. The proteomic findings provided additional information on the relative abundances of toxins and the detection of proteins of minor expression unreported previously. The potent lethal effect of the venom was neutralized by bioCSL Sea Snake Antivenom, an anticipated finding due to the fact that the Sea Snake Antivenom is actually bivalent in nature, being raised against a mix of venoms of the beaked sea snake (Hydrophis schistosus) and N. scutatus. However, it is surprising to note that bioCSL Sea Snake Antivenom neutralized N. scutatus venom much more effectively compared to the targeted sea snake venom by a marked difference in potency of approximately 6-fold. This phenomenon may be explained by the main difference in the proteomes of the two venoms, where H. schistosus venom is dominated by short-neurotoxins in high abundance - this is a poorly immunogenic toxin group that has been increasingly recognized in the venoms of a few cobras. Further investigations should be directed toward strategies to improve the neutralization of short-neurotoxins, in line with the envisioned production of an effective pan-regional elapid antivenom.

  2. Tan CH, Fung SY, Yap MK, Leong PK, Liew JL, Tan NH
    J Proteomics, 2016 Jan 30;132:1-12.
    PMID: 26598790 DOI: 10.1016/j.jprot.2015.11.014
    The venom proteome of the Malayan blue coral snake, Calliophis bivirgata flaviceps from west Malaysia was investigated by 1D-SDS-PAGE and shotgun-LCMS/MS. A total of 23 proteins belonging to 11 protein families were detected from the venom proteome. For the toxin proteins, the venom consists mainly of phospholipase A2 (41.1%), cytotoxin (22.6%), SVMPs (18.7%) and vespryns (14.6%). However, in contrast to the venoms of New World coral snakes and most elapids, there was no post-synaptic α-neurotoxin detected. The proteome also revealed a relatively high level of phosphodiesterase (1.3%), which may be associated with the reported high level of adenosine in the venom. Also detected were 5'-nucleotidase (0.3%), hyaluronidase (0.1%) and cysteine-type endopeptide inhibitor (0.6%). Enzymatic studies confirmed the presence of phospholipase A2, phosphodiesterase, 5'-nucleotidase and acetylcholinesterase activities but not l-amino acid oxidase activity. The venom exhibited moderate cytotoxic activity against CRL-2648 fibroblast cell lines (IC50=62.14±0.87 μg/mL) and myotoxicity in mice, presumably due to the action of its cytotoxin or its synergistic action with phospholipase A2. Interestingly, the venom lethality could be cross-neutralized by a neurotoxic bivalent antivenom from Taiwan. Together, the findings provide insights into the composition and functions of the venom of this exotic oriental elapid snake.
  3. Tan KY, Tan CH, Fung SY, Tan NH
    J Proteomics, 2015 Apr 29;120:105-25.
    PMID: 25748141 DOI: 10.1016/j.jprot.2015.02.012
    Previous studies showed that venoms of the monocled cobra, Naja kaouthia from Thailand and Malaysia are substantially different in their median lethal doses. The intraspecific venom variations of N. kaouthia, however, have not been fully elucidated. Here we investigated the venom proteomes of N. kaouthia from Malaysia (NK-M), Thailand (NK-T) and Vietnam (NK-V) through reverse-phase HPLC, SDS-PAGE and tandem mass spectrometry. The venom proteins comprise 13 toxin families, with three-finger toxins being the most abundant (63-77%) and the most varied (11-18 isoforms) among the three populations. NK-T has the highest content of neurotoxins (50%, predominantly long neurotoxins), followed by NK-V (29%, predominantly weak neurotoxins and some short neurotoxins), while NK-M has the least (18%, some weak neurotoxins but less short and long neurotoxins). On the other hand, cytotoxins constitute the main bulk of toxins in NK-M and NK-V venoms (up to 45% each), but less in NK-T venom (27%). The three venoms show different lethal potencies that generally reflect the proteomic findings. Despite the proteomic variations, the use of Thai monovalent and Neuro polyvalent antivenoms for N. kaouthia envenomation in the three regions is appropriate as the different venoms were neutralized by the antivenoms albeit at different degrees of effectiveness.
  4. Rusmili MR, Yee TT, Mustafa MR, Hodgson WC, Othman I
    J Proteomics, 2014 Oct 14;110:129-44.
    PMID: 25154052 DOI: 10.1016/j.jprot.2014.08.001
    Kraits (Bungarus spp.) are highly venomous elapids that are only found in Asia. In the current study, 103 and 86 different proteins were identified from Bungarus candidus and Bungarus fasciatus venoms, respectively. These proteins were classified into 18 different venom protein families. Both venoms were found to contain a high percentage of three finger toxins, phospholipase A2 enzymes and Kunitz-type inhibitors. Smaller number of high molecular weight enzymes such as L-amino acid oxidase, hyaluronidases, and acetylcholinesterase were also detected in the venoms. We also detected some unique proteins that were not known to be present in these venoms. The presence of a natriuretic peptide, vespryn, and serine protease families was detected in B. candidus venom. We also detected the presence of subunit A and B of β-bungarotoxin and α-bungarotoxin which had not been previously found in B. fasciatus venom. Understanding the proteome composition of Malaysian krait species will provide useful information on unique toxins and proteins which are present in the venoms. This knowledge will assist in the management of krait envenoming. In addition, these proteins may have potential use as research tools or as drug-design templates.
  5. Al-Maleki AR, Mariappan V, Vellasamy KM, Shankar EM, Tay ST, Vadivelu J
    J Proteomics, 2014 Jun 25;106:205-20.
    PMID: 24742602 DOI: 10.1016/j.jprot.2014.04.005
    Colony morphology variation is a characteristic of Burkholderia pseudomallei primary clinical isolates, associated with variations in expression of virulence factors. Here, we performed comparative investigations on adhesion, invasion, plaque-forming abilities and protein profiles of B. pseudomallei wild-type (WT) and a small colony variant (SCV). The percentage of SCV adherence to A549 cells was significantly higher (2.73%) than WT (1.91%). In contrast, WT was significantly more efficient (0.63%) than SCV (0.31%) in invasiveness and in inducing cellular damage. Using 2-DE and MALDI TOF/TOF, 263 and 258 protein spots were detected in WT and SCV, respectively. Comparatively, 49 proteins were differentially expressed in SCV when compared with WT. Of these, 31 proteins were up-regulated, namely, nucleoside diphosphate kinase (Ndk), phosphoglycerate kinase (Pgk), thioredoxin (TrxA), putative ferritin DPS-family DNA-binding protein (DPS) and oxidoreductase (AhpC) that are known to be involved in adhesion, intracellular survival and persistence. However, among the 18 down-regulated proteins, enolase (Eno), elongation factor (EF-Tu) and universal stress-related proteins were associated with invasion and virulence. Differences observed in these protein profiles provide ample clues to their association with the morphotypic and phenotypic characteristics of colony variants, providing additional insights into the potential association of B. pseudomallei colony morphotypes with disease pathogenesis.
  6. Malih I, Ahmad rusmili MR, Tee TY, Saile R, Ghalim N, Othman I
    J Proteomics, 2014 Jan 16;96:240-52.
    PMID: 24269350 DOI: 10.1016/j.jprot.2013.11.012
    The proteome of the venom of Naja haje legionis, the only medically important elapid species in Morocco, has been elucidated by using a combination of proteomic techniques that includes size exclusion chromatography, reverse-phase HPLC, Tricine/SDS-Page, tryptic digestion, Q-TOF tandem mass spectrometry and database search. The sequence analysis of venom fractions revealed a highly complex venom proteome which counts a total of 76 proteins identified from database that can be assigned into 9 proteins families. We report the identification of: cobra venom factor (CVF), l-amino-acid oxidases (LAAO), acetylcholinesterase (AChE), snake venom metalloproteinases (SVMP), cysteine rich secretory proteins (CRISP), venom nerve growth factor (vNGF), phospholipases A2 (PLA2), vespryns, kunitz-type inhibitor, short neurotoxins, long neurotoxins, weak neurotoxins, neurotoxin like proteins, muscarinic toxins, cardiotoxins and cytotoxins. Comparison of these proteins showed high sequence homology with proteins from other African and Asian cobras. Further works are needed to assess the contribution of individual toxins in venom toxicity.
  7. Chang HC, Tsai TS, Tsai IH
    J Proteomics, 2013 Aug 26;89:141-53.
    PMID: 23796489 DOI: 10.1016/j.jprot.2013.06.012
    This study deciphers the geographic variations of king cobra (Ophiophagus hannah) venom using functional proteomics. Pooled samples of king cobra venom (abbreviated as Ohv) were obtained from Indonesia, Malaysia, Thailand, and two provinces of China, namely Guangxi and Hainan. Using two animal models to test and compare the lethal effects, we found that the Chinese Ohvs were more fatal to mice, while the Southeast Asian Ohvs were more fatal to lizards (Eutropis multifasciata). Various phospholipases A2 (PLA2s), three-finger toxins (3FTxs) and Kunitz-type inhibitors were purified from these Ohvs and compared. Besides the two Chinese Ohv PLA2s with known sequences, eight novel PLA2s were identified from the five Ohv samples and their antiplatelet activities were compared. While two 3FTxs (namely oh-55 and oh-27) were common in all the Ohvs, different sets of 3FTx markers were present in the Chinese and Southeast Asian Ohvs. All the Ohvs contain the Kunitz inhibitor, OH-TCI, while only the Chinese Ohvs contain the inhibitor variant, Oh11-1. Relative to the Chinese Ohvs which contained more phospholipases, the Southeast Asian Ohvs had higher metalloproteinase, acetylcholine esterase, and alkaline phosphatase activities.
  8. Tan NH, Fung SY, Tan KY, Yap MK, Gnanathasan CA, Tan CH
    J Proteomics, 2015 Oct 14;128:403-23.
    PMID: 26342672 DOI: 10.1016/j.jprot.2015.08.017
    The venom proteome (venomics) of the Sri Lankan Daboia russelii was elucidated using 1D SDS PAGE nano-ESI-LCMS/MS shotgun proteomics. A total of 41 different venom proteins belonging to 11 different protein families were identified. The four main protein families are phospholipase A2 (PLA2, 35.0%), snaclec (SCL, 22.4%, mainly platelet aggregation inhibitors), snake venom serine proteinase (SVSP, 16.0%, mainly Factor V activating enzyme) and snake venom metalloproteinase (SVMP, 6.9%, mainly heavy chain of Factor X activating enzyme). Other protein families that account for more than 1% of the venom protein include l-amino acid oxidase (LAAO, 5.2%), Kunitz-type serine proteinase inhibitor (KSPI, 4.6%), venom nerve growth factor (VNGF. 3.5%), 5'-nucleotidase (5'NUC, 3.0%), cysteine-rich secretory protein (CRISP, 2.0%) and phosphodiesterase (PDE, 1.3%). The venom proteome is consistent with the enzymatic and toxic activities of the venom, and it correlates with the clinical manifestations of Sri Lankan D. russelii envenomation which include hemorrhage, coagulopathy, renal failure, neuro-myotoxicity and intravascular hemolysis. The venom exhibited remarkable presypnatic neurotoxicity presumably due to the action of basic PLA2 in high abundance (35.0%). Besides, SCLs, Factor X activating enzymes (SVMPs), SVSPs, and LAAOs are potential hemotoxins (50.5%), contributing to coagulopathy and hemorrhagic syndrome in Sri Lankan D. russelii envenomation.
  9. Tan HS, Liddell S, Ong Abdullah M, Wong WC, Chin CF
    J Proteomics, 2016 06 30;143:334-345.
    PMID: 27130535 DOI: 10.1016/j.jprot.2016.04.039
    Oil palm tissue culture is one way to produce superior oil palm planting materials. However, the low rate of embryogenesis is a major hindrance for the adoption of this technology in oil palm tissue culture laboratories. In this study, we use proteomic technologies to compare differential protein profiles in leaves from palms of high and low proliferation rates in tissue culture in order to understand the underlying biological mechanism for the low level of embryogenesis. Two protein extraction methods, namely trichloroacetic acid/acetone precipitation and polyethylene glycol fractionation were used to produce total proteins and fractionated protein extracts respectively, with the aim of improving the resolution of protein species using two-dimensional gel electrophoresis. A total of 40 distinct differential abundant protein spots were selected from leaf samples collected from palms with proven high and low proliferation rates. The variant proteins were subsequently identified using mass spectrometric analysis. Twelve prominent protein spots were then characterised using real-time polymerase chain reaction to compare the mRNA expression and protein abundant profiles. Three proteins, namely triosephosphate isomerase, l-ascorbate peroxidase, and superoxide dismutase were identified to be potential biomarker candidates at both the protein abundant and mRNA expression levels.

    BIOLOGICAL SIGNIFICANCE: In this study, proteomic analysis was used to identify abundant proteins from total protein extracts. PEG fractionation was used to reveal lower abundant proteins from both high and low proliferation embryogenic lines of oil palm samples in tissue culture. A total of 40 protein spots were found to be significant in abundance and the mRNA levels of 12 of these were assessed using real time PCR. Three proteins namely, triosephosphate isomerase, l-ascorbate peroxidase and superoxide dismutase were found to be concordant in their mRNA expression and protein abundance. Triosephosphate isomerase is a key enzyme in glycolysis. Both l-ascorbate peroxidase and superoxide dismutase play a role in anti-oxidative scavenging defense systems. These proteins have potential for use as biomarkers to screen for high and low embryogenic oil palm samples.

  10. Chan SY, Sam IC, Lai JK, Chan YF
    J Proteomics, 2015 Jul 1;125:121-30.
    PMID: 26003530 DOI: 10.1016/j.jprot.2015.05.016
    Hand, foot and mouth disease is mainly caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16), but EV-A71 is also associated with severe neurological complications. Host factors may contribute to the different clinical outcomes of EV-A71 and CV-A16 infections. A neurovirulent EV-A71 strain (EV-A71/UH1) from a fatal case, a non-neurovirulent EV-A71 strain (EV-A71/Sha66) and a CV-A16 strain (CV-A16/22159) from cases of uncomplicated HFMD were used. Replication of the viruses in SK-N-MC (neuronal) and HT-29 (intestinal) cell lines correlated with the severity of clinical disease associated with each virus. EV-A71/UH1 showed the greatest replication in neuronal cells. In HT-29 cells, both EV-A71 strains replicated well, but CV-A16/22159 showed no effective replication. The proteomes of mock and infected SK-N-MC and HT-29 cell lines were compared by 2D-SDS-PAGE. The differentially expressed proteins were identified by MALDI-TOF/TOF analysis. There were 46 and 44 differentially expressed proteins identified from SK-N-MC and HT-29 cells, respectively, categorized under apoptosis, stress, cytoskeletal, energy metabolism proteins and others. Western blot validation showed that EV-A71/UH1 and CV-A16 also differentially induced proteins involved in viral RNA translation and host cell stress responses in neuronal and intestinal cell lines.
  11. Tan CH, Tan KY, Lim SE, Tan NH
    J Proteomics, 2015 Aug 3;126:121-30.
    PMID: 26047715 DOI: 10.1016/j.jprot.2015.05.035
    The venom proteome of Hydrophis schistosus (syn: Enhydrina schistosa) captured in Malaysian waters was investigated using reverse-phase HPLC, SDS-PAGE and high-resolution liquid chromatography-tandem mass spectrometry. The findings revealed a minimalist profile with only 18 venom proteins. These proteins belong to 5 toxin families: three-finger toxin (3FTx), phospholipase A2 (PLA2), cysteine-rich secretory protein (CRISP), snake venom metalloprotease (SVMP) and L-amino acid oxidase (LAAO). The 3FTxs (3 short neurotoxins and 4 long neurotoxins) constitute 70.5% of total venom protein, 55.8% being short neurotoxins and 14.7% long neurotoxins. The PLA2 family consists of four basic (21.4%) and three acidic (6.1%) isoforms. The minor proteins include one CRISP (1.3%), two SVMPs (0.5%) and one LAAO (0.2%). This is the first report of the presence of long neurotoxins, CRISP and LAAO in H. schistosus venom. The neurotoxins and the basic PLA2 are highly lethal in mice with an intravenous median lethal dose of <0.2 μg/g. Cross-neutralization by heterologous elapid antivenoms (Naja kaouthia monovalent antivenom and Neuro polyvalent antivenom) was moderate against the long neurotoxin and basic PLA2, but weak against the short neurotoxin, indicating that the latter is the limiting factor to be overcome for improving the antivenom cross-neutralization efficacy.
  12. Tan CH, Wong KY, Chong HP, Tan NH, Tan KY
    J Proteomics, 2019 08 30;206:103418.
    PMID: 31201947 DOI: 10.1016/j.jprot.2019.103418
    The Philippine cobra, Naja philippinensis, is a WHO Category 1 venomous snake of medical importance responsible for fatal envenomation in the northern Philippines. To elucidate the venom proteome and pathophysiology of envenomation, N. philippinensis venom proteins were decomplexed with reverse-phase high-performance liquid chromatography, and protein fractions were subsequently digested with trypsin, followed by nano-liquid chromatography-tandem mass spectrometry analysis and data mining. Three-finger toxins (3FTX, 66.64% of total venom proteins) and phospholipases A2 (PLA2, 22.88%) constitute the main bulk of venom proteome. Other proteins are present at low abundances (<4% each); these include metalloproteinase, serine protease, cobra venom factor, cysteine-rich secretory protein, vespryn, phosphodiesterase, 5' nucleotidase and nerve growth factor. In the three-finger toxin family, the alpha-neurotoxins comprise solely short neurotoxins (SNTX, 44.55%), supporting that SNTX is the principal toxin responsible for neuromuscular paralysis and lethality reported in clinical envenomation. Cytotoxins (CTX) are the second most abundant 3FTX proteins in the venom (21.31%). The presence of CTX correlates with the venom cytotoxic effect, which is more prominent in murine cells than in human cells. From the practical standpoint, SNTX-driven neuromuscular paralysis is significant in N. philippinensis envenomation. Antivenom production and treatment should be tailored accordingly to ensure effective neutralization of SNTX. BIOLOGICAL SIGNIFICANCE: The venom proteome of Naja philippinensis, the Philippine cobra, is unravelled for the first time. Approximately half the protein bulk of the venom is made up of short neurotoxins (44.55% of the total venom proteins). As the only alpha-neurotoxins present in the venom, short neurotoxins are the causative toxins of the post-synaptic blockade and fast-onset neuromuscular paralysis in N. philippinensis envenomation. A substantial amount of cytotoxins (21.31%) was also detected in N. philippinensis venom, supporting that the venom can be cytotoxic although the effect is much weaker in human cells compared to murine cells. The finding is consistent with the low incidence of local tissue necrosis in N. philippinensis envenomation, although this does not negate the need for monitoring and care of bite wound in the patients.
  13. Guan Q, Kong W, Zhu D, Zhu W, Dufresne C, Tian J, et al.
    J Proteomics, 2021 01 16;231:104019.
    PMID: 33075550 DOI: 10.1016/j.jprot.2020.104019
    Salinity can induce Mesembryanthemum crystallinum to shift its photosynthesis from C3 to crassulacean acid metabolism (CAM), leading to enhanced plant water use efficiency. Studying how M. crystallinum changes its carbon fixation pathways is important for potential translation into crops and enhancing crop resilience. In this study, we examined proteomic changes in guard cells and mesophyll cells in the course of the C3 to CAM transition. We collected enriched guard cells and mesophyll cells during a short period of transition. A total of 1153 proteins were identified and quantified in the two cell-types. During the transition, proteins in the guard cells and mesophyll cells exhibited differential changes. For example, we observed nocturnal carbon fixation in mesophyll cells and proteins involved in cell growth in the two cell-types. Proteins involved in osmotic adjustment, ion transport, energy metabolism and light response may play important roles in the C3 to CAM transition. Real-time PCR experiments were conducted to determine potential correlations between transcript and protein levels. These results have highlighted potential molecular mechanisms underlying the C3 to CAM transition of guard cells and mesophyll cells of the important facultative CAM plant. BIOLOGICAL SIGNIFICANCE: Fresh water resource for agricultural food production is a global challenge. Nature has evolved crassulacean acid metabolism (CAM) plants with enhanced water use efficiency. Using single cell-type proteomics, this study revealed molecular changes taking place in guard cells and mesophyll cells during the shift of ice plant photosynthesis from C3 to CAM. The results have provided important insights into the CAM transition and may facilitate effort toward enhancing crop resilience for global food security.
  14. Kok SY, Namasivayam P, Ee GC, Ong-Abdullah M
    J Proteomics, 2021 02 10;232:104052.
    PMID: 33262095 DOI: 10.1016/j.jprot.2020.104052
    To gain insights on protein changes in fruit setting and growth in oil palm, a comparative proteomic approach was undertaken to study proteome changes during its early development. The variations in the proteome at five early developmental stages were investigated via a gel-based proteomic technique. A total of 129 variant proteins were determined using mass spectrometric analysis, resulting in 80 identifications. The majority of the identified protein species were classified as energy and metabolism, stress response/defence and cell structure during early oil palm development representing potential candidates for the control of final fruit size and composition. Seven prominent protein species were then characterised using real-time polymerase chain reaction to validate the mRNA expression against the protein abundant profiles. Transcript and protein profiles were parallel across the developmental stages, but divergent expression was observed in one protein spot, indicative of possible post-transcriptional events. Our results revealed protein changes in early oil palm fruit development provide valuable information in the understanding of fruit growth and metabolism during early stages that may contribute towards improving agronomic traits. BIOLOGICAL SIGNIFICANCE: Two-dimensional gel electrophoresis coupled with mass spectrometry approach was used in this study to identify differentially expressed proteins during early oil palm fruit development. A total of 80 protein spots with significant change in abundance were successfully identified and selected genes were analysed using real time PCR to validate their expression. The dynamic changes in oil palm fruit proteome during early development were mostly active in primary and energy metabolism, stress responses, cell structure and protein metabolism. This study reveals the physiological processes during early oil palm fruit development and provides a reference proteome for further improvements in fruit quality traits.
  15. Amir SH, Yuswan MH, Aizat WM, Mansor MK, Desa MNM, Yusof YA, et al.
    J Proteomics, 2021 06 15;241:104240.
    PMID: 33894373 DOI: 10.1016/j.jprot.2021.104240
    Mass spectrometry-based proteomics relies on dedicated software for peptide and protein identification. These software include open-source or commercial-based search engines; wherein, they employ different algorithms to establish their scoring and identified proteins. Although previous comparative studies have differentiated the proteomics results from different software, there are still yet studies specifically been conducted to compare and evaluate the search engine in the field of halal analysis. This is important because the halal analysis is often using commercial meat samples that have been subjected to various processing, further complicating its analysis. Thus, this study aimed to assess three open-source search engines (Comet, X! Tandem, and ProteinProspector) and a commercial-based search engine (ProteinPilot™) against 135 raw tandem mass spectrometry data files from 15 types of pork-based food products for halal analysis. Each database search engine contained high false-discovery rate (FDR); however, a post-searching algorithm called PeptideProphet managed to reduce the FDR, except for ProteinProspector and ProteinPilot™. From this study, the combined database search engine (executed by iProphet) reveals a thorough protein list for pork-based food products; wherein the most abundant proteins are myofibrillar proteins. Thus, this proteomics study will aid the identification of potential peptide and protein biomarkers for future precision halal analysis. SIGNIFICANCE: A critical challenge of halal proteomics is the availability of a database to confirm the inferential peptides as well as proteins. Currently, the established database such as UniProtKB is related to animal proteome; however, the halal proteomics is related to the highly processed meat-based food products. This study highlights the use of different database search engines (Comet, X! Tandem, ProteinProspector, and ProteinPilot™) and their respective algorithms to analyse 135 raw tandem mass spectrometry data files from 15 types of pork-based food products. This is the first attempt that has compared different database search engines in the context of halal proteomics to ensure the effectiveness of controlling the FDR. Previous studies were just focused on the advantages of a certain algorithm over another. Moreover, other previous studies also have mainly reported the use of mass spectrometry-based shotgun proteomics for meat authentication (the most similar field to halal analysis), but none of the studies were reported on halal aspects that used samples originated from highly processed food products. Hence, a systematic comparative study is duly needed for a more comprehensive and thorough proteomics analysis for such samples. In this study, our combinatorial approach for halal proteomics results from the different search engines used (Comet, X! Tandem, and ProteinProspector) has successfully generated a comprehensive spectral library for the pork-based meat products. This combined spectral library is freely available at https://data.mendeley.com/datasets/6dmm8659rm/3. Thus far, this is the first and new attempt at establishing a spectral library for halal proteomics. We also believe this study is a pioneer for halal proteomics that aimed at non-conventional and non-model organism proteomics, protein analytics, protein bioinformatics, and potential biomarker discovery.
  16. Tang EL, Tan CH, Fung SY, Tan NH
    J Proteomics, 2016 10 04;148:44-56.
    PMID: 27418434 DOI: 10.1016/j.jprot.2016.07.006
    The venom of Malayan pit viper (Calloselasma rhodostoma) is highly toxic but also valuable in drug discovery. However, a comprehensive proteome of the venom that details its toxin composition and abundance is lacking. This study aimed to unravel the venom complexity through a multi-step venomic approach. At least 96 distinct proteins (29 basic, 67 acidic) in 11 families were identified from the venom. The venom consists of mainly snake venom metalloproteinases (SVMP, 41.17% of total venom proteins), within which the P-I (kistomin, 20.4%) and P-II (rhodostoxin, 19.8%) classes predominate. This is followed by C-type lectins (snaclec, 26.3%), snake venom serine protease (SVSP, 14.9%), L-amino acid oxidase (7.0%), phospholipase A2 (4.4%), cysteine-rich secretory protein (2.5%), and five minor toxins (nerve growth factor, neurotrophin, phospholipase B, 5' nucleotidase and phosphodiesterase, totaling 2.6%) not reported in the proteome hitherto. Importantly, all principal hemotoxins unveiled correlate with the syndrome: SVSP ancrod causes venom-induced consumptive coagulopathy, aggravated by thrombocytopenia caused by snaclec rhodocytin, a platelet aggregation inducer, while P-II rhodostoxin mediates hemorrhage, exacerbated by P-I kistomin and snaclec rhodocetin that inhibit platelet plug formation. These toxins exist in multiple isoforms and/or complex subunits, deserving further characterization for the development of an effective, polyspecific regional antivenom.

    BIOLOGICAL SIGNIFICANCE: Advents in proteomics and bioinformatics have vigorously propelled the scientific discoveries of toxins from various lineages of venomous snakes. The Malayan pit viper, Calloselasma rhodostoma, is a medically important species in Southeast Asia as its bite can cause envenomation, while the venom is also a source of bioactive compounds for drug discovery. Detailed profiling of the venom, however, is inadequate possibly due to the complex nature of the venom and technical limitation in separating the constituents into details. Integrating a multi-step fractionation method, this study successfully revealed a comprehensive and quantitative profile of the composition of the venom of this medically important venomous snake. The relative abundance of the various venom proteins is determined in a global profile, providing useful information for understanding the pathogenic roles of the different toxins in C. rhodostoma envenomation. Notably, the principal hemotoxins were identified in great details, including the variety of toxin subunits and isoforms. The findings indicate that these toxins are the principal targets for effective antivenom neutralization, and should be addressed in the production of a pan-regional polyspecific antivenom. In addition, minor toxin components not reported previously in the venom were also detected in this study, enriching the current toxin database for the venomous snakes.

  17. Tan NH, Wong KY, Tan CH
    J Proteomics, 2017 03 22;157:18-32.
    PMID: 28159706 DOI: 10.1016/j.jprot.2017.01.018
    The venom proteome of Naja sputatrix (Javan spitting cobra) was elucidated through reverse-phase HPLC, nano-ESI-LCMS/MS and data mining. A total of 97 distinct protein forms belonging to 14 families were identified. The most abundant proteins are the three-finger toxins (3FTXs, 64.22%) and phospholipase A2 (PLA2, 31.24%), followed by nerve growth factors (1.82%), snake venom metalloproteinase (1.33%) and several proteins of lower abundance (<1%) including a variety of venom enzymes. At subproteome, the 3FTx is dominated by cytotoxins (48.08%), while short neurotoxins (7.89%) predominate over the long neurotoxins (0.48%) among other neurotoxins of lesser toxicity (muscarinic toxin-like proteins, 5.51% and weak neurotoxins, 2.26%). The major SNTX, CTX and PLA2 toxins were isolated with intravenous median lethal doses determined as 0.13, 1.06 and 0.50μg/g in mice, respectively. SABU, the Indonesia manufactured homologous tri-specific antivenom could neutralize the CTX and PLA2 fraction with moderate potency (potency=0.14-0.16mg toxin per ml antivenom). The SNTX, however, was very poorly neutralized with a potency level of 0.034mg/ml, indicating SNTX as the main limiting factor in antivenom neutralization. The finding helps elucidate the inferior efficacy of SABU reported in neutralizing N. sputatrix venom, and supports the call for antivenom improvement.

    BIOLOGICAL SIGNIFICANCE: The Javan spitting cobra, Naja sputatrix is by itself a unique species and should not be confused as the equatorial and the Indochinese spitting cobras. The distinction among the spitting cobras was however unclear prior to the revision of cobra systematics in the mid-90's, and results of some earlier studies are now questionable as to which species was implicated back then. The current study successfully profiled the venom proteome of authenticated N. sputatrix, and showed that the venom is made up of approximately 64% three-finger toxins (including neurotoxins and cytotoxins) and 31% phospholipases A2 by total venom proteins. The findings verified that the paralyzing components in the venom i.e. neurotoxins are predominantly the short-chain subtype (SNTX) far exceeding the long-chain subtype (LNTX) which is more abundant in the venoms of monocled cobra and Indian common cobra. The neurotoxicity of N. sputatrix venom is hence almost exclusively SNTX-driven, and effective neutralization of the SNTX is the key to early reversal of paralysis. Unfortunately, as shown through a toxin-specific assay, the immunological neutralization of the SNTX using the Indonesian antivenom (SABU) was extremely weak, implying that SABU has limited therapeutic efficacy in treating N. sputatrix envenomation clinically. From the practical standpoint, actions need to be taken at all levels from laboratory to production and policy making to ensure that the shortcoming is overcome.

  18. Akpunarlieva S, Weidt S, Lamasudin D, Naula C, Henderson D, Barrett M, et al.
    J Proteomics, 2017 02 23;155:85-98.
    PMID: 28040509 DOI: 10.1016/j.jprot.2016.12.009
    Leishmania parasites multiply and develop in the gut of a sand fly vector in order to be transmitted to a vertebrate host. During this process they encounter and exploit various nutrients, including sugars, and amino and fatty acids. We have previously generated a mutant Leishmania line that is deficient in glucose transport and which displays some biologically important phenotypic changes such as reduced growth in axenic culture, reduced biosynthesis of hexose-containing virulence factors, increased sensitivity to oxidative stress, and dramatically reduced parasite burden in both insect vector and macrophage host cells. Here we report the generation and integration of proteomic and metabolomic approaches to identify molecular changes that may explain these phenotypes. Our data suggest changes in pathways of glycoconjugate production and redox homeostasis, which likely represent adaptations to the loss of sugar uptake capacity and explain the reduced virulence of this mutant in sand flies and mammals. Our data contribute to understanding the mechanisms of metabolic adaptation in Leishmania and illustrate the power of integrated proteomic and metabolomic approaches to relate biochemistry to phenotype.

    BIOLOGICAL SIGNIFICANCE: This paper reports the application of comparative proteomic and metabolomic approaches to reveal the molecular basis for important phenotypic changes Leishmania parasites that are deficient in glucose uptake. Leishmania cause a very significant disease burden across the world and there are few effective drugs available for control. This work shows that proteomics and metabolomics can produce complementary data that advance understanding of parasite metabolism and highlight potential new targets for chemotherapy.

  19. Faisal T, Tan KY, Sim SM, Quraishi N, Tan NH, Tan CH
    J Proteomics, 2018 07 15;183:1-13.
    PMID: 29729992 DOI: 10.1016/j.jprot.2018.05.003
    The venom proteome of wild Pakistani Russell's viper (Daboia russelii) was investigated through nano-ESI-LCMS/MS of the reverse-phase HPLC fractions. A total of 54 venom proteins were identified and clustered into 11 protein families. Phospholipase A2 (PLA2, 63.8%) and Kunitz-type serine protease inhibitor (KSPI, 16.0%) were most abundant, followed by snake venom serine protease (SVSP, 5.5%, mainly Factor V activating enzyme), vascular endothelial growth factor (VEGF, 4.3%), snake venom metalloproteinase (SVMP, 2.5%, mainly Factor X activating enzyme) and phosphodiesterase (PDE, 2.5%). Other minor proteins include cysteine-rich secretory protein (CRiSP), snake venom C-type lectin/lectin-like protein (snaclec), nerve growth factor, L-amino acid oxidase and 5'-nucleotidase. PLA2, KSPI, SVSP, snaclec and SVMP are hemotoxic proteins in the venom. The study indicated substantial venom variation in D. russelii venoms of different locales, including 3 Pakistani specimens kept in the USA. The venom exhibited potent procoagulant activity on human plasma (minimum clotting dose = 14.5 ng/ml) and high lethality (rodent LD50 = 0.19 μg/g) but lacked hemorrhagic effect locally. The Indian VINS Polyvalent Antivenom bound the venom immunologically in a concentration-dependent manner. It moderately neutralized the venom procoagulant and lethal effects (normalized potency against lethality = 2.7 mg venom neutralized per g antivenom).

    BIOLOGICAL SIGNIFICANCE: Comprehensive venom proteomes of D. russelii from different locales will facilitate better understanding of the geographical variability of the venom in both qualitative and quantitative terms. This is essential to provide scientific basis for the interpretation of differences in the clinical presentation of Russell's viper envenomation. The study revealed a unique venom proteome of the Pakistani D. russelii from the wild (Indus Delta), in which PLA2 predominated (~60% of total venom proteins). The finding unveiled remarkable differences in the venom compositions between the wild (present study) and the captive specimens reported previously. The integration of toxicity tests enabled the correlation of the venom proteome with the envenoming pathophysiology, where the venom showed potent lethality mediated through coagulopathic activity. The Indian VINS Polyvalent Antivenom (VPAV) showed binding activity toward the venom protein antigens; however the immunorecognition of small proteins and PLA2-dominating fractions was low to moderate. Consistently, the antivenom neutralized the toxicity of the wild Pakistani Russell's viper venom at moderate efficacies. Our results suggest that it may be possible to enhance the Indian antivenom potency against the Pakistani viper venom by the inclusion of venoms from a wider geographical range including that from Pakistan into the immunogen formulation.

  20. Tan BC, Lim YS, Lau SE
    J Proteomics, 2017 10 03;169:176-188.
    PMID: 28546092 DOI: 10.1016/j.jprot.2017.05.018
    Proteomics is a rapidly growing area of biological research that is positively affecting plant science. Recent advances in proteomic technology, such as mass spectrometry, can now identify a broad range of proteins and monitor their modulation during plant growth and development, as well as during responses to abiotic and biotic stresses. In this review, we highlight recent proteomic studies of commercial crops and discuss the advances in understanding of the proteomes of these crops. We anticipate that proteomic-based research will continue to expand and contribute to crop improvement.

    SIGNIFICANCE: Plant proteomics study is a rapidly growing area of biological research that is positively impacting plant science. With the recent advances in new technologies, proteomics not only allows us to comprehensively analyses crop proteins, but also help us to understand the functions of the genes. In this review, we highlighted recent proteomic studies in commercial crops and updated the advances in our understanding of the proteomes of these crops. We believe that proteomic-based research will continue to grow and contribute to the improvement of crops.

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