Abstract
The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a (15)N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (K(S)) and degradation (K(D)) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify K(S) and K(D) for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. K(S) and K(D) correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive (15)N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.
Highlights
Genome wide analysis of Arabidopsis mRNA turnover rates has confirmed that knowledge of transcript decay rates can provide insights into diverse biological processes [6]
The advent of mass spectrometry as a key tool in proteomics has provided a means to use enrichment of the natural abundance of stable isotopes to provide mass rather than radio decay signals to track the synthesis of new proteins
Stable isotope labeling using individual amino acids (SILAC)1 has proven highly successful in mammalian cell culture systems [14]
Summary
Genome wide analysis of Arabidopsis mRNA turnover rates has confirmed that knowledge of transcript decay rates can provide insights into diverse biological processes [6]. The advent of mass spectrometry as a key tool in proteomics has provided a means to use enrichment of the natural abundance of stable isotopes to provide mass rather than radio decay signals to track the synthesis of new proteins. The ratio between light and heavy isotopes and the degrees of enrichment provided by mass spectrometry provides a powerful means to measure synthesis and degradation rates of individual proteins [5, 13]. A variety of factors including differences in the labeling of amino acids, changes in incorporation rates over time and the range of turnover rates provide a heterogeneous response and complicate data interpretation. We have sought to overcome these obstacles and provide a data processing approach to measure kinetics of changes in 15N incorporation of peptides from in-gel digestions of separated protein spots
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