¹⁵N‐Metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells

Abstract

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope 15N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that 15N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of 14N versus 15N MS signals reflects the relative abundance of 14N and 15N proteins in the sample analyzed. We describe the use of 15N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that 15N-metabolic labeling is a useful technique for comparative membrane proteome studies.

Quantitative detection of changes in the leaf‐mesophyll tonoplast proteome in dependency of a cadmium exposure of barley (Hordeum vulgare L.) plants

“…Lanquar et al [25] undertook an approach to identify changes in the abundance of plasma membrane proteins in response to Cd 2+ treatment by in vivo labelling Arabidopsis suspension cells with 15 N. They identified 25 proteins, 10 corresponding to plasma membrane transporters.…”

Semidominant Mutations in Reduced Epidermal Fluorescence 4 Reduce Phenylpropanoid Content in Arabidopsis

“…Unfortunately, neither REF4 nor RFR1 has been identified in any of the proteomic studies performed to date (e.g., Alexandersson et al 2004;Marmagne et al 2004;Dunkley et al 2006;Morel et al 2006;Hartman et al 2007;Heazlewood et al 2007;Lanquar et al 2007;Mitra et al 2007). The Aramemnon database for plant membrane spanning proteins (Schwacke et al 2003) annotates REF4 and its homologs as having between 10 to 12 transmembrane regions (TMRs), although for any individual protein, less than half of these score above a cutoff score of 0.5.…”

Stable isotopic labeling in proteomics

“…Since then, other labs have described similar labeling methods on various small organisms and cell lines (e.g., Deinococcus radiodurans and mouse B16 melanoma cells [3], Arabidopsis thaliana cells [4], and Rhodopseudomonas palustris [5], see also Section 2.2). This type of metabolic labeling in cell lines or small organisms poses high demands on software algorithms for automated quantification.…”