Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes.
ABSTRACT Proteomics seeks to monitor the flux of protein through cells under variable developmental and environmental influences as programmed by the genome. Consequently, it is necessary to measure changes in protein abundance and turnover rate as faithfully as possible. In the absence of non-invasive technologies, the majority of proteomics approaches involve destructive sampling at various time points to obtain 'snapshots' that periodically report the genomes's product. The work has fallen to separations technologies coupled to mass spectrometry, for high throughput protein identification. Quantitation has become the major challenge facing proteomics as the field matures. Because of the variability of day-to-day measurements of protein quantities by mass spectrometry, a common feature of quantitative proteomics is the use of stable isotope coding to distinguish control and experimental samples in a mixture that can be profiled in a single experiment. To address limitations with separation technologies such as 2D-gel electrophoresis, alternative systems are being introduced including multi-dimensional chromatography. Strategies that accelerate throughput for mass spectrometry are also emerging and the benefits of these 'shotgun' protocols will be considered in the context of the thylakoid membrane and photosynthesis. High resolution Fourier-transform mass spectrometry is bringing increasingly accurate mass measurements to peptides and a variety of gas-phase dissociation mechanisms are permitting 'top-down' sequencing of intact proteins. Finally, a versatile workflow for sub-cellular compartments including membranes is presented that allows for intact protein mass measurements, localization of post-translational modifications and relative quantitation or turnover measurement.
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ABSTRACT: Integral membrane proteins reside within the bilayer membranes that surround cells and organelles, playing critical roles in movement of molecules across them and the transduction of energy and signals. While their extreme amphipathicity presents technical challenges, biological mass spectrometry has been applied to all aspects of membrane protein chemistry and biology, including analysis of primary, secondary, tertiary and quaternary structure, as well as the dynamics that accompany functional cycles and catalysis.Analytical Chemistry 01/2013; · 5.83 Impact Factor
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ABSTRACT: Capsicum (Capsicum annuum L.) is categorised as a non-climacteric fruit that exhibits limited ethylene production during ripening and the molecular mechanisms associated with this process are poorly understood. A proteomic approach was used to identify the differentially expressed proteins during various ripening stages (Green (G), Breaker Red 1 (BR1) and Light Red (LR)) and the genes associated with their synthesis. From 2D gel electrophoresis (2DGE), seven protein spots were identified as selectively present either in G or BR1 and are involved in carbon metabolism, colour and fruit development, protein synthesis and chaperones or biosynthesis of amino acids and polyamines. One candidate of interest, 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) is known to be involved in ethylene biosynthesis and was only present in BR1 and is related to the tomato ACO isoform 4 (LeACO4) and hence named CaACO4. CaACO4 RNA expression as well as total ACO protein expression in multiple stages of ripening (G, Breaker (B), BR1, Breaker Red 2 (BR2), LR and Deep Red (DR)) corresponded to the 2DGE protein spot abundance in breaker stages. Our findings highlight the involvement of the ethylene pathway in non-climacteric fruit ripening.Functional Plant Biology 06/2013; 40(11):1115-1128. · 2.57 Impact Factor
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ABSTRACT: There is a flood of molecular data about many aspects of cellular functioning. This data ranges from sequence and structural data to gene and protein regulation data, including time dependent changes in the concentration. Integration of the different datasets through computational methods is required to extract biological information that is relevant from a systems biology perspective. In this paper we discuss how different computational tools and methods can be made to work together integrating differ-ent types of data, mining these data for biological information, and assisting in pathway reconstruction and biological hy-potheses generation. We review the recent body of literature where such integrative approaches are used and discuss automation of data integration and model building to generate testable biological hypotheses. We analyze issues regarding the design of such automated tools and discuss what limitations and pitfalls can be foreseen for the automation and what solutions can computer science and biologists provide to overcome them.Current Bioinformatics 05/2008; 3:98-129. · 1.73 Impact Factor