Mass spectrometry for high throughput quantitative proteomics in plant research: lessons from thylakoid membranes.
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.
Whitelegge
Julian P
JP
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. jpw@chem.ucla.edu
D013058:Mass Spectrometry / Q000379:methods / Q000639:trends; D010940:Plant Proteins / Q000032:analysis*; D010944:Plants / Q000737:chemistry*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics / Q000379:methods; D020524:Thylakoids / Q000737:chemistry*
D010940:Plant Proteins; D020543:Proteome