Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots

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.