The Pasarow Mass Spectrometry Laboratory, Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine and College of Letters and Sciences, University of California, Los Angeles, CA, USA.
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.
"Of these, in vivo 15 N-metabolic labeling holds great potential for quantitative proteomics in animal and plant models. The first metabolic labeling was conducted in 15 N-enriched media to grow yeast Saccharomyces cerevisiae (Oda et al., 1999), and the method was extended to prokaryotic photosynthetic Cyanobacterium (Whitelegge, 2004) and multicellular non-plant organisms, such as Drosophila melanogaster, Caenorhabditis elegans and mammalian cells (for review, see Gouw et al., 2010). In multicellular terrestrial plants, the first 15 N stable isotope metabolic labeling was performed using hydroponic setup for proteomic analysis in potato plant (Ippel et al., 2004), PTM proteomics in Arabidopsis (Benschop et al., 2007) and using 15 N-enriched soil for quantitative proteomics in adult tomato plants (Schaff et al., 2008). "
"Due to the high complexity and the dynamic nature of the cereals proteome, it is generally accepted that a single analytical approach for protein separation will not supply comprehensive exploration of the entire proteome. Therefore, it is often needed to use multiple technologies to improve proteome resolution and coverage, and to provide complementary results (Whitelegge, 2004). Currently there are two complementary proteomic approaches: the so-called ''gel-based'' approach, and the ''gel-free'' approach. "
[Show abstract][Hide abstract] ABSTRACT: In the last decade, the improved performance and versatility of the mass spectrometers together with the increasing availability of gene and genomic sequence database, led the mass spectrometry to become an indispensable tool for either protein and proteome analyses in cereals. Mass spectrometric works on prolamins have rapidly evolved from the determination of the molecular masses of proteins to the proteomic approaches aimed to a large-scale protein identification and study of functional and regulatory aspects of proteins. Mass spectrometry coupled with electrophoresis, chromatographic methods, and bioinformatics tools is currently making significant contributions to a better knowledge of the composition and structure of the cereal proteins and their structure-function relationships. Results obtained using mass spectrometry, including characterization of prolamins, investigation of the gluten toxicity for coeliac patients, identification of proteins responsible of cereal allergies, determination of the protein pattern and its modification under environmental or stress effects, investigation of genetically modified varieties by proteomic approaches, are summarized here, to illustrate current trends, analytical troubles and challenges, and suggest possible future perspectives.
Mass Spectrometry Reviews 07/2012; 31(4):448-65. DOI:10.1002/mas.20347 · 7.71 Impact Factor
"An additional consideration is whether all plant tissues can be labeled to a similarly high efficiency with this method. However, recent studies indicate that only partial metabolic labeling in plants may prove effective for comparative studies (Whitelegge et al., 2004; Huttlin et al., 2007). "
[Show abstract][Hide abstract] ABSTRACT: The ability to compare dynamic changes in the proteome is an exciting new addition to the research programs of many plant biologists. With alternative transcription/translation and the potential addition of over 200 different posttranslational modifications to proteins, the complexity of the proteome is likely to exceed the complexity of the transcriptome by one to two orders of magnitude, making the proteome as vast and complex as it is dynamic. A variety of options for performing quantitative proteomic comparisons in plants is available and currently in use by a number of laboratories. As we hope we have emphasized, presently no single method is more highly preferred over another. However, neither will any single method provide a complete overview of all the changes in a proteome. This admission is something that should simply be accepted rather than serve as a deterrent from initiating proteomic studies. Any quantitative proteomic method can yield new insights into the biological system, regardless of whether some information has been missed. With some of these quantitative methods beginning to reach technical maturity, we look forward to comparative proteomic studies moving out of the realm of technical experts and spreading throughout the community of biological researchers.
The Plant Cell 12/2007; 19(11):3339-46. DOI:10.1105/tpc.107.053991 · 9.34 Impact Factor
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