When biological stresses induce the overproduction of reactive oxygen within the cell, redox-sensitive proteins may undergo oxidative modifications. The result may be oxidative damage to cellular components, including lipids, proteins and DNA. The modifications may also play a role in regulating signal transduction pathways and gene expression.1 For many plants, including Arabidopsis species, the thiol group of cysteine residues is especially prone to stress-triggered oxidation and reduction.2,3
Researchers have relied upon fluorescent labeling and gel electrophoresis for the identification of most redox-sensitive proteins; however, low resolution and inadequate sensitivity render this approach ineffective for the identification of low-abundance proteins.4.5 Other approaches, including tandem mass tagging (TMT) and isotope coded affinity tagging (ICAT), couple isotope labeling with mass spectrometry (MS). The expense associated with these methods, however, limits their applicability for whole-proteome analysis.
In this study, Liu et al. (2013) describe OxiTRAQ, a high-throughput quantitative approach in which biotin-tagged, iTRAQ-labeled peptides are analyzed with parallel higher energy collisional dissociation–collision-induced dissociation (HCD-CID) on an LTQ-Orbitrap mass spectrometer (Thermo Scientific).6 Using this technology, the researchers isolated 195 cysteine-containing peptides from 179 oxidatively modified Arabidopsis proteins, including 151 redox-sensitive proteins that had not been previously identified using other methods.
To perform OxiTRAQ, Liu et al. used hydrogen peroxide to induce oxidative stress in Arabidopsis cells prior to extracting proteins in the presence of free thiol-blocking N-ethylmaleimide (NEM). They then reduced reversibly oxidized thiols (including disulfide bonds, sulfenic acid, and S-nitrosylated thiols) with dithiothreitol (DTT) before tagging with biotin-HPDP. After trypsin digestion and affinity purification, they cleaved the disulfide bonds between the tags and the cysteine residues and eluted the peptides for identification by MS. Following HCD-CID, the researchers analyzed the mass spectral data with Proteome Discoverer Software (Thermo Scientific) and verified their results using multiple reaction monitoring (MRM).
In two identical MS runs, the researchers identified 1,327 and 1,226 cysteine-containing peptides with a false discovery rate of less than 1% for both proteins and peptides. They isolated 1,098 cysteine-containing peptides derived from 820 distinct proteins for further analysis. Of these, 195 peptides met the researchers’ criteria for identification as redox-sensitive. These included 164 cysteines from 150 proteins that became more oxidized in the treated sample and 31 cysteines from 29 proteins that became more reduced in the treated sample. The researchers note that their previous study using gel-based analysis identified only 20 redox-sensitive proteins,7 indicating that the OxiTRAQ approach is more sensitive and capable of identifying low-abundant transcription factors.
The research team observed that redox-sensitive proteins were less likely to be extracellular and that 31% were located in the chloroplasts and 26% were located in the mitochondria (as compared to 28% and 16%, respectively, for total proteins). They also noted that the identified redox-sensitive proteins operate in various systems, including transcription regulation, protein synthesis and metabolism.
Liu et al. assert that the OxiTRAQ approach may be used to isolate oxidatively modified proteins in plants that have undergone physiological stress, such as infection. However, stress treatment that alters the abundance of any given redox-sensitive protein may complicate results. The researchers indicate that tissue samples undergoing extended stress treatment may require parallel proteomic evaluation to accurately determine protein abundance. Overall, they offer OxiTRAQ as a sensitive, cost-effective proteomic solution for the quantitation of redox-sensitive proteins across various plant species.
1. Apel, K., and Hirt, H. (2004) “Reactive oxygen species: Metabolism, oxidative stress, and signal transduction,” Annual Review of Plant Biology, 55 (pp. 373–99).
2. Møller, I.M., et al. (2007) “Oxidative modifications to cellular components in plants,” Annual Review of Plant Biology, 58 (pp. 459–81).
3. Foyer, C.H., and Noctor, G. (2005) “Redox homeostasis and antioxidant signaling: A metabolic interface between stress perception and physiological responses,” Plant Cell, 17 (pp. 1866–75).
4. Gygi, S.P., et al. (2000) “Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology,” Proceedings of the National Academy of Sciences, 97 (pp. 9390–5).
5. Ong, S.E., and Pandey, A. (2001) “An evaluation of the use of two-dimensional gel electrophoresis in proteomics,” Biomolecular Engineering, 18 (pp. 195–205).
6. Liu, P., et al. (2013) “Identification of redox-sensitive cysteines in the Arabidopsis proteome using OxiTRAQ, a quantitative redox proteomics method,” Proteomics, doi: 10.1002/pmic.201300307.
7. Wang, H., et al. (2012) “Proteomic analysis of early-responsive redox-sensitive proteins in Arabidopsis,” Journal of Proteome Research, 11 (pp. 412–24).
Post Author: Melissa J. Mayer. Melissa is a freelance writer who specializes in science journalism. She possesses passion for and experience in the fields of proteomics, cellular/molecular biology, microbiology, biochemistry, and immunology. Melissa is also bilingual (Spanish) and holds a teaching certificate with a biology endorsement.