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      Large-scale identification of encystment-related proteins and genes in Pseudourostyla cristata

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          Abstract

          The transformation of a ciliate into cyst is an advance strategy against an adverse situation. However, the molecular mechanism for the encystation of free-living ciliates is poorly understood. A large-scale identification of the encystment-related proteins and genes in ciliate would provide us with deeper insights into the molecular mechanisms for the encystations of ciliate. We identified the encystment-related proteins and genes in Pseudourostyla cristata with shotgun LC-MS/MS and scale qRT-PCR, respectively, in this report. A total of 668 proteins were detected in the resting cysts, 102 of these proteins were high credible proteins, whereas 88 high credible proteins of the 724 total proteins were found in the vegetative cells. Compared with the vegetative cell, 6 specific proteins were found in the resting cyst. However, the majority of high credible proteins in the resting cyst and the vegetative cell were co-expressed. We compared 47 genes of the co-expressed proteins with known functions in both the cyst and the vegetative cell using scale qRT-PCR. Twenty-seven of 47 genes were differentially expressed in the cyst compared with the vegetative cell. In our identifications, many uncharacterized proteins were also found. These results will help reveal the molecular mechanism for the formation of cyst in ciliates.

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          Gene Ontology: tool for the unification of biology

          Genomic sequencing has made it clear that a large fraction of the genes specifying the core biological functions are shared by all eukaryotes. Knowledge of the biological role of such shared proteins in one organism can often be transferred to other organisms. The goal of the Gene Ontology Consortium is to produce a dynamic, controlled vocabulary that can be applied to all eukaryotes even as knowledge of gene and protein roles in cells is accumulating and changing. To this end, three independent ontologies accessible on the World-Wide Web (http://www.geneontology.org) are being constructed: biological process, molecular function and cellular component.
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            Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

            We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.
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              Femtomole sequencing of proteins from polyacrylamide gels by nano-electrospray mass spectrometry.

              Molecular analysis of complex biological structures and processes increasingly requires sensitive methods for protein sequencing. Electrospray mass spectrometry has been applied to the high-sensitivity sequencing of short peptides, but technical difficulties have prevented similar success with gel-isolated proteins. Here we report a simple and robust technique for the sequencing of proteins isolated by polyacrylamide gel electrophoresis, using nano-electrospray tandem mass spectrometry. As little as 5 ng protein starting material on Coomassie- or silver-stained gels can be sequenced. Multiple-sequence stretches of up to 16 amino acids are obtained, which identify the protein unambiguously if already present in databases or provide information to clone the corresponding gene. We have applied this method to the sequencing and cloning of a protein which inhibits the proliferation of capillary endothelial cells in vitro and thus may have potential antiangiogenic effects on solid tumours.
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                Author and article information

                Journal
                Sci Rep
                Sci Rep
                Scientific Reports
                Nature Publishing Group
                2045-2322
                16 June 2015
                2015
                : 5
                : 11360
                Affiliations
                [1 ]School of Life Sciences, East China Normal University , Shanghai 200241, China
                [2 ]Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences , Shanghai 200031, China.
                Author notes
                [*]

                These authors contributed equally to this work.

                Article
                srep11360
                10.1038/srep11360
                4650649
                26079518
                55e0422e-ebb2-4475-b128-57508e6bd0cf
                Copyright © 2015, Macmillan Publishers Limited

                This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

                History
                : 04 February 2015
                : 22 May 2015
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