Overview
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Day/night cycle analysis and first protein map of the cyanobacterium Arthrospira PCC8005 assessed by extensive shotgun and quantitative proteomics approaches Sabine Matallana-Surget, Baptiste Leroy, Jerémy Derock, Ruddy Wattiez * Department of Proteomic and Microbiology, University of Mons,, Mons, Belgium This study assesses progress in cyanobacterial shotgun proteomics, and focuses specially on the proteome of the cyanobacterium Arthrospira PCC8005, a strain of particular interest for the European Space Agency (ESA). This work adds new perspectives in the field of shotgun proteomics by developing a set of methods allowing to get rid of the most abundant proteins (phycocyanin, phycobiliproteins) and access under-represented proteins of a complex sample. Overview IEF (560) IMAC-Cu 2+ (785) SDS (511) 2D-LC (1043) 45 33 1 32 154 261 4 30 143 55 15 123 266 86 56 A total of 1307 proteins was identified, representing 22% coverage of the theoretical proteome. In addition, 450 identified proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Arthrospira PCC8005. The identified proteins participate in the major metabolic and cellular processes of cyanobacterial cells during the exponential growth phase, under continuous light exposure. Introducti on We combined LC-MS/MS approaches with gel-free and gel-based protein/peptides fractionation steps: one-dimensional gel electrophoresis (SDS PAGE and IEF), 2D and 3D-LC MS/MS (IMAC-Copper) on the soluble and/or membrane and secreted fractions. In order to increase the proteome coverage, we also cultivated this cyanobacterium under different light conditions, shifting from continuous light to a 12-hour light/dark cycle and quantitative proteomics was assessed using an ICPL post-digest labeling Methods Shotgun Proteomics - Comparison A protein map of all theoretical proteins in Arthrospira PCC8005 and expressed proteins identified in all the experiments. Arthrospira platensis, better known as “spirulina”, is a motile cyanobacterium, made up of a multicellular coiled filament, found in the ground as well as in water-salt, brackish or lake water. Owing to its exceptional nutritional value, A. platensis was used to face the problem of denutrition in developing countires. It is today used by astronauts on their long-term missions in orbit and is thus a strain of particular interest for the European Space Agency in the MELiSSA project. A. platensis is studied in Mons as a part of the European MEliSSA (Micro-Ecological Life Support System Alternative) project, which aims at developing a closed artificial ecosystem, intended to be used aboard long term manned missions to Mars, in order to recycle wastes and to produce a balanced food. This study presents the first protein of this key cyanobacterium by using a combination of different methods on the soluble and/or membrane secreted proteome. 2 4 6 8 10 12 0 20000 40000 60000 80000 100000 120000 140000 160000 180000 200000 Theoretical P... pI MW When the 4 approaches cited above were compared in term of non-redundant identified proteins, a relative high overlap between the techniques was observed. However, a significant portion of specific proteins for both 2D and 3DLC-MS/MS with immobilized metal affinity chromatography (IMAC- Copper) was observed, with 266 and 123 specific proteins respectively. Venn diagram showing non-redundant proteins identified in the 2D-LC, 3D-LC the IPG-IEF-LC and the SDS-PAGE-LC MS/MS approaches. 109 109 567 Elution fraction (676) Flowthrough fraction (218) A novel aspect of this study is that a 3D LC-MS/MS was used to reduce the complexity of our protein extract and also allowed the examination of the binding behavior to copper of several proteins. We observed a significant difference comparing both flowthrough and elution fractions, with two major bands in the flowhrough, corresponding to phycocyanin proteins. 250 150 100 75 50 37 25 20 15 10 0 0.1 0.2 0.3 0.4 0.5 0.6 Time (min) OD 280nm Shotgun Proteomics 3D-LC-MS/MS Quantitative proteomics 311 proteins were found to be affected by a light-dark cycle. J K L B D V T M N U O C G E F H I P Q R S 0 5 10 15 20 25 30 35 40 Proteins with differential abundance Expressed Proteome COGs Percentage of proteins into COGs categories Percentage of proteins grouped by Clusters of Orthologous Groups (COGs) * * The ratios of differential expression are ranging from 0.2 (ferritin-like protein) to 4.2 (Glycine-rich RNA-binding protein). The study of the light-dark cycle, assessed using an ICPL post-digest method, added 270 newly identified proteins to the total number of proteins characterized by 2D-LC-MS/MS. * * This study has highlighted new characteristics of Arthrospira platensis proteome. The identified expressed proteome was higher than the percentage of proteome coverage found in other cyanobacterial proteome studies. Proteins mostly affected during a light-dark cycle belonged to the translational engineering pathway, energy production and carbohydrate transport/metabolism. The obtained results gave insights into the functional and metabolic processes occurring in an important model organism, and may additionally be significant for the manual annotation of the proteome of Arthrospira PCC8005. Conclusion Fundings for this project was provided by an ESA Grant Aknowledgments Proteins from the phycobilisome (red) (light harvesting antennae) were, as expected, in higher abundance in light condition. However, proteins from the 2 photosystems (blue) were all in higher abundance in the dark condition. This could reflect a increased turnover of this proteins highly challenged in light condition.
description
Day/night cycle analysis and first protein map of the cyanobacterium Arthrospira PCC8005 assessed by extensive shotgun and quantitative proteomics approaches Sabine Matallana- Surget , Baptiste Leroy, Jerémy Derock, Ruddy Wattiez * - PowerPoint PPT Presentation
Transcript of Overview
Day/night cycle analysis and first protein map of the cyanobacterium Arthrospira PCC8005 assessed by extensive shotgun and quantitative
proteomics approachesSabine Matallana-Surget, Baptiste Leroy, Jerémy Derock, Ruddy Wattiez*
Department of Proteomic and Microbiology, University of Mons,, Mons, BelgiumThis study assesses progress in cyanobacterial shotgun proteomics, and focuses specially on the proteome of the cyanobacterium Arthrospira PCC8005, a strain of particular interest for the European Space Agency (ESA). This work adds new perspectives in the field of shotgun proteomics by developing a set of methods allowing to get rid of the most abundant proteins (phycocyanin, phycobiliproteins) and access under-represented proteins of a complex sample.
Overview
IEF (560)
IMAC-Cu2+ (785)
SDS (511)
2D-LC (1043)
45 33 1 32
154 261 4 30
143 55 15 123
266 86 56
A total of 1307 proteins was identified, representing 22% coverage of the theoretical proteome. In addition, 450 identified proteins which were previously either unknown or considered to be hypothetical were shown to be true gene products in Arthrospira PCC8005. The identified proteins participate in the major metabolic and cellular processes of cyanobacterial cells during the exponential growth phase, under continuous light exposure.
Introduction
We combined LC-MS/MS approaches with gel-free and gel-based protein/peptides fractionation steps: one-dimensional gel electrophoresis (SDS PAGE and IEF), 2D and 3D-LC MS/MS (IMAC-Copper) on the soluble and/or membrane and secreted fractions. In order to increase the proteome coverage, we also cultivated this cyanobacterium under different light conditions, shifting from continuous light to a 12-hour light/dark cycle and quantitative proteomics was assessed using an ICPL post-digest labeling
Methods
Shotgun Proteomics - Comparison
A protein map of all theoretical proteins in Arthrospira PCC8005 and expressed proteins identified in all the experiments.
Arthrospira platensis, better known as “spirulina”, is a motile cyanobacterium, made up of a multicellular coiled filament, found in the ground as well as in water-salt, brackish or lake water. Owing to its exceptional nutritional value, A. platensis was used to face the problem of denutrition in developing countires. It is today used by astronauts on their long-term missions in orbit and is thus a strain of particular interest for the European Space Agency in the MELiSSA project.
A. platensis is studied in Mons as a part of the European MEliSSA (Micro-Ecological Life Support System Alternative) project, which aims at developing a closed artificial ecosystem, intended to be used aboard long term manned missions to Mars, in order to recycle wastes and to produce a balanced food. This study presents the first protein of this key cyanobacterium by using a combination of different methods on the soluble and/or membrane secreted proteome.
2 4 6 8 10 120
20000400006000080000
100000120000140000160000180000200000
Theoretical Pro-teome
pI
MW
When the 4 approaches cited above were compared in term of non-redundant identified proteins, a relative high overlap between the techniques was observed.
However, a significant portion of specific proteins for both 2D and 3DLC-MS/MS with immobilized metal affinity chromatography (IMAC-Copper) was observed, with 266 and 123 specific proteins respectively.
Venn diagram showing non-redundant proteins identified in the 2D-LC, 3D-LC the IPG-IEF-LC and the SDS-PAGE-LC MS/MS approaches.
109 109 567
Elution fraction (676)
Flowthrough fraction (218)
A novel aspect of this study is that a 3D LC-MS/MS was used to reduce the complexity of our protein extract and also allowed the examination of the binding behavior to copper of several proteins. We observed a significant difference comparing both flowthrough and elution fractions, with two major bands in the flowhrough, corresponding to phycocyanin proteins.
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Shotgun Proteomics 3D-LC-MS/MS
Quantitative proteomics
311 proteins were found to be affected by a light-dark cycle.
J K L B D V T M N U O C G E F H I P Q R S0
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Proteins with differential abundanceExpressed ProteomeTheoretical Proteome
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Percentage of proteins grouped by Clusters of Orthologous Groups (COGs)*
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The ratios of differential expression are ranging from 0.2 (ferritin-like protein) to 4.2 (Glycine-rich RNA-binding protein).The study of the light-dark cycle, assessed using an ICPL post-digest method, added 270 newly identified proteins to the total number of proteins characterized by 2D-LC-MS/MS.
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This study has highlighted new characteristics of Arthrospira platensis proteome. The identified expressed proteome was higher than the percentage of proteome coverage found in other cyanobacterial proteome studies. Proteins mostly affected during a light-dark cycle belonged to the translational engineering pathway, energy production and carbohydrate transport/metabolism. The obtained results gave insights into the functional and metabolic processes occurring in an important model organism, and may additionally be significant for the manual annotation of the proteome of Arthrospira PCC8005.
Conclusion
Fundings for this project was provided by an ESA GrantAknowledgments
Proteins from the phycobilisome (red) (light harvesting antennae) were, as expected, in higher abundance in light condition. However, proteins from the 2 photosystems (blue) were all in higher abundance in the dark condition. This could reflect a increased turnover of this proteins highly challenged in light condition.
