Ratio‐dependent significance thresholds in reciprocal ¹⁵N‐labeling experiments as a robust tool in detection of candidate proteins responding to biological treatment

Abstract

Metabolic labeling of plant tissues with (15)N has become widely used in plant proteomics. Here, we describe a robust experimental design and data analysis workflow implementing two parallel biological replicate experiments with reciprocal labeling and series of 1:1 control mixtures. Thereby, we are able to unambiguously distinguish (i) inherent biological variation between cultures and (ii) specific responses to a biological treatment. The data analysis workflow is based on first determining the variation between cultures based on (15)N/(14)N ratios in independent 1:1 mixtures before biological treatment is applied. In a second step, ratio-dependent SD is used to define p-values for significant deviation of protein ratios in the biological experiment from the distribution of protein ratios in the 1:1 mixture. This approach allows defining those proteins showing significant biological response superimposed on the biological variation before treatment. The proposed workflow was applied to a series of experiments, in which changes in composition of detergent resistant membrane domains was analyzed in response to sucrose resupply after carbon starvation. Especially in experiments involving cell culture treatment (starvation) prior to the actual biological stimulus of interest (resupply), a clear distinction between culture to culture variations and biological response is of utmost importance.

Definition of Arabidopsis Sterol-rich Membrane Microdomains by Differential Treatment with Methyl-β-cyclodextrin and Quantitative Proteomics

“…In contrast, among the "non-responsive" proteins we found 71% as annotated membrane proteins, and only a total of 3% has been described as extracellular proteins (Fig. 5B) (31). B, distribution of ratios from two control experiments (yellow), m␤cd-responsive proteins (blue), and other proteins (red).…”

“…ratios in two control experiments showed normal distribution (see Fig. 2B) and were used to define ratio-dependent standard deviations (31). In a second step, the distances to the diagonal in a graphic display of ratios in reciprocal experiments ( Fig.…”

Precision, Proteome Coverage, and Dynamic Range of Arabidopsis Proteome Profiling Using 15N Metabolic Labeling and Label-free Approaches

“…15 N Metabolic Labeling and Label-free Quantitation in Plants tein abundance could still be carried out as described (31). In particular, if a reciprocal experimental design is used (35,36), but also for use as a universal standard (24), significant treatment effects can efficiently be identified also in mixtures deviating from 1:1. The comparatively low efficiency of protein identification and quantitation in 1:1 mixtures observed here, may be less apparent if longer chromatographic gradients are being used leading to overall higher protein identifications (34).…”

Feedback Inhibition of Ammonium Uptake by a Phospho-Dependent Allosteric Mechanism inArabidopsis

“…15 N-labeled seedlings were exposed to 2 mM NH 4 Cl in FN medium for 10 min, seedlings were combined (equal mass) with untreated 14 N-grown seedlings, and plasma membranes were prepared. In a reciprocal experiment, 14 N-grown seedlings were exposed to 2 mM NH 4 Cl in FN medium for 10 min, seedlings were combined (equal mass) with untreated 15 N-grown seedlings, and plasma membranes were prepared (for concept of reciprocal experiments, see Lanquar et al, 2007;Kierszniowska et al, 2009). …”