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Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC – ESI – MS/MS and LC – MALDI – TOF/TOF

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ABSTRACT

Background: Excretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa.

Methods: E. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC–ESI–MS/MS and LC–MALDI–TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB).

Results: A complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC–ESI–MS/MS and 107 proteins by LC–MALDI–TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC–ESI–MS/MS and LC–MALDI–TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts.

Conclusion: This study showed that complementary use of LC–ESI–MS/MS and LC–MALDI–TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.

Electronic supplementary material: The online version of this article (doi:10.1186/s12014-016-9135-8) contains supplementary material, which is available to authorized users.

No MeSH data available.


SDS-PAGE profile of excretory-secretory (ES) proteins of E. histolytica. ES proteins from E. histolytica separated by 10% SDS-PAGE. Lane 1 protein ladder (BioRad, USA); lane 2, 3, and 4, ES proteins from three independent replicates; lane 5, concentrated RPMI-C-A as control
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Fig1: SDS-PAGE profile of excretory-secretory (ES) proteins of E. histolytica. ES proteins from E. histolytica separated by 10% SDS-PAGE. Lane 1 protein ladder (BioRad, USA); lane 2, 3, and 4, ES proteins from three independent replicates; lane 5, concentrated RPMI-C-A as control

Mentions: In this study, ES proteins and concentrated RPMI-CA medium were resolved in 10% SDS-PAGE, and stained with RAMA solution. Visual observation of distinct protein bands indicated good protein quality while the concentrated protein free RPMI-CA medium (control) showed no protein band (Fig. 1). Furthermore, no protein smearing was observed, and this also suggested that minimal degradation of the proteins had occurred. Thus, the ES proteins were suitable for downstream mass spectrometry analysis.Fig. 1


Proteome analysis of excretory-secretory proteins of Entamoeba histolytica HM1:IMSS via LC – ESI – MS/MS and LC – MALDI – TOF/TOF
SDS-PAGE profile of excretory-secretory (ES) proteins of E. histolytica. ES proteins from E. histolytica separated by 10% SDS-PAGE. Lane 1 protein ladder (BioRad, USA); lane 2, 3, and 4, ES proteins from three independent replicates; lane 5, concentrated RPMI-C-A as control
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5120466&req=5

Fig1: SDS-PAGE profile of excretory-secretory (ES) proteins of E. histolytica. ES proteins from E. histolytica separated by 10% SDS-PAGE. Lane 1 protein ladder (BioRad, USA); lane 2, 3, and 4, ES proteins from three independent replicates; lane 5, concentrated RPMI-C-A as control
Mentions: In this study, ES proteins and concentrated RPMI-CA medium were resolved in 10% SDS-PAGE, and stained with RAMA solution. Visual observation of distinct protein bands indicated good protein quality while the concentrated protein free RPMI-CA medium (control) showed no protein band (Fig. 1). Furthermore, no protein smearing was observed, and this also suggested that minimal degradation of the proteins had occurred. Thus, the ES proteins were suitable for downstream mass spectrometry analysis.Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Excretory-secretory (ES) proteins of E. histolytica are thought to play important roles in the host invasion, metabolism, and defence. Elucidation of the types and functions of E. histolytica ES proteins can further our understanding of the disease pathogenesis. Thus, the aim of this study is to use proteomics approach to better understand the complex ES proteins of the protozoa.

Methods: E. histolytica ES proteins were prepared by culturing the trophozoites in protein-free medium. The ES proteins were identified using two mass spectrometry tools, namely, LC–ESI–MS/MS and LC–MALDI–TOF/TOF. The identified proteins were then classified according to their biological processes, molecular functions, and cellular components using the Panther classification system (PantherDB).

Results: A complementary list of 219 proteins was identified; this comprised 201 proteins detected by LC–ESI–MS/MS and 107 proteins by LC–MALDI–TOF/TOF. Of the 219 proteins, 89 were identified by both mass-spectrometry systems, while 112 and 18 proteins were detected exclusively by LC–ESI–MS/MS and LC–MALDI–TOF/TOF respectively. Biological protein functional analysis using PantherDB showed that 27% of the proteins were involved in metabolic processes. Using molecular functional and cellular component analyses, 35% of the proteins were found to be involved in catalytic activity, and 21% were associated with the cell parts.

Conclusion: This study showed that complementary use of LC–ESI–MS/MS and LC–MALDI–TOF/TOF has improved the identification of ES proteins. The results have increased our understanding of the types of proteins excreted/secreted by the amoeba and provided further evidence of the involvement of ES proteins in intestinal colonisation and evasion of the host immune system, as well as in encystation and excystation of the parasite.

Electronic supplementary material: The online version of this article (doi:10.1186/s12014-016-9135-8) contains supplementary material, which is available to authorized users.

No MeSH data available.