Relative and accurate measurement of protein abundance using 15N stable isotope labeling in Arabidopsis (SILIA)

In the quantitative proteomic studies, numerous in vitro and in vivo peptide labeling strategies have been successfully applied to measure differentially regulated protein and peptide abundance. These approaches have been proven to be versatile and repeatable in biological discoveries. N-15 metabolic labeling is one of these widely adopted and economical methods. However, due to the differential incorporation rates of N-15 or N-14. the labeling results produce imperfectly matched isotopic envelopes between the heavy and light nitrogen-labeled peptides. In the present study, we have modified the solid Arabidopsis growth medium to standardize the N-15 supply, which led to a uniform incorporation of N-15 into the whole plant protein complement. The incorporation rate (97.43 +/- 0.11%) of N-15 into N-15-coded peptides was determined by correlating the intensities of peptide ions with the labeling efficiencies according to Gaussian distribution. The resulting actual incorporation rate (97.94%) and natural abundance of N-15/N-14-coded peptides are used to re-calculate the intensities of isotopic envelopes of differentially labeled peptides, respectively. A modified N-15/N-14 stable isotope labeling strategy, SILIA, is assessed and the results demonstrate that this approach is able to differentiate the fold change in protein abundance down to 10%. The machine dynamic range limitation and purification step will make the precursor ion ratio deriving from the actual ratio fold change. It is suggested that the differentially mixed N-15-coded and N-14-coded plant protein samples that are used to establish the protein abundance standard curve should be prepared following a similar protein isolation protocol used to isolate the proteins to be quantitated. Crown Copyright (C) 2011 Published by Elsevier Ltd. All rights reserved.