Proteins with high turnover rate in barley leaves estimated by proteome analysis combined with in planta isotope labelling.

Plant physiology

PubMedID: 25082890

Nelson CJ, Alexova R, Jacoby RP, Millar AH. Proteins with high turnover rate in barley leaves estimated by proteome analysis combined with in planta isotope labelling. Plant Physiol. 2014;.
Protein turnover is a key component in cellular homeostasis, however there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15N labelling of barley (Hordeum vulgare) plants and GC-MS analysis of free amino acids and LC-MS analysis of proteins to track enrichment of 15N into the amino acid pools in barley leaves, and then into tryptic peptides derived from newly synthesised proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14N labelled proteins, we calculate the turnover rates of 508 different proteins in barley and show they vary by >100 fold. There was approximately a nine hour lag from label application until 15N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of MS spectra filtering steps, demonstrates that 15N labelling can be used for large-scale LC-MS studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turnover significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turnover as rapidly as the well-known D1 subunit of PSII. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis.