Research Abstracts Online
2008 - March 2009
University of Minnesota Twin Cities
College of Food, Agricultural, and Natural Resource Sciences
Department of Horticultural Science
PI: Jerry D. Cohen
Co-PI: William M. Gray
Global Measurement of Turnover of Plant Proteins
The objectives of this project were to develop robust methods for stable isotope labeling of plant proteins for the purpose of determination of absolute rates of turnover (the half-life) for a wide number of different proteins under changing developmental and environmental conditions, as well as for the determination of the effects of specific mutations on rates of protein turnover. The researchers used several different methods for labeling plant proteins, including general methods based on growing plants on 2H2O, in the presence of 13CO2, or on 15nammonium nitrate, as well as methods where a specific stable isotope labeled amino acid, sugar, or other amino acid precursor was used to label plant proteins. Loss of stable isotope from specific proteins after a pre-labeling period was used to determine the suitability of each method for further studies. Changes to normal plant function due to the labeling method and procedures were monitored by measurement of gene expression changes by microarray analyses. The turnover for proteins known to be degraded at different rates and use of mutants altered in their pathways for protein degradation were then used to test the suitability of the procedures developed.
The researchers used the Analyst QS program to process LC-MS-MS data and analyze the mass isotope distribution of labeled and unlabelled peptides for several known target proteins. The information was then used to help design algorithms for the calculation of turnover rates of plant proteins in a high-throughput manner.
Wen-Ping Chen, Research Associate
XiaoYuan Yang, Research Associate