Cardiovascular redox and ox stress proteomics

Abstract

Significance: Oxidative post-translational modifications (OPTMs) have been demonstrated as contributing to cardiovascular physiology and pathophysiology. These modifications have been identified using antibodies as well as advanced proteomic methods, and the functional importance of each is beginning to be understood using transgenic and gene deletion animal models. Given that OPTMs are involved in cardiovascular pathology, the use of these modifications as biomarkers and predictors of disease has significant therapeutic potential. Adequate understanding of the chemistry of the OPTMs is necessary to determine what may occur in vivo and which modifications would best serve as biomarkers.

Recent advances: By using mass spectrometry, advanced labeling techniques, and antibody identification, OPTMs have become accessible to a larger proportion of the scientific community. Advancements in instrumentation, database search algorithms, and processing speed have allowed MS to fully expand on the proteome of OPTMs. In addition, the role of enzymatically reversible OPTMs has been further clarified in preclinical models.

Critical issues: The identification of OPTMs suffers from limitations in analytic detection based on the methodology, instrumentation, sample complexity, and bioinformatics. Currently, each type of OPTM requires a specific strategy for identification, and generalized approaches result in an incomplete assessment.

Future directions: Novel types of highly sensitive MS instrumentation that allow for improved separation and detection of modified proteins and peptides have been crucial in the discovery of OPTMs and biomarkers. To further advance the identification of relevant OPTMs in advanced search algorithms, standardized methods for sample processing and depository of MS data will be required.

FIG. 1.

Interaction of proteome with reactive nitrogen oxygen species (RNOS). In addition to traditional post-translational protein modifications (phosphorylation, acetylation, ubiquitination, etc.), cellular proteins can be modified by RNOS, adding another layer of protein regulation to the proteome classified as oxidative post-translational modifications (OPTMs). OPTMs are reversible or irreversible—reversible oxidation is generally involved in the regulation of cell signaling or the protection of proteins from irreversible oxidation, whereas irreversible oxidation is mostly associated with pathophysiology and protein degradation. Both reversible and irreversible modified proteins may be potential biomarkers of oxidant-mediated pathologies.

FIG. 2.

Oxidative cleavage of proteins–peptide bond cleavage.

FIG. 3.

Enhanced cysteine (Cys) reactivity in the presence of catalytic diad/triad.

FIG. 4.

Overview comparison of isotope-coded affinity tags (ICAT) strategies for oxICAT with standard ICAT. The first section in the figure shows components of the ICAT reagent consisting of a biotin-based affinity tag for purification, a linker group that can be cleaved by an acid after labeling, an isotopic tag which varies because it has heavy nine ¹³C atoms instead of light ¹²C atoms, and an iodoacetamide-based group which binds the reduced Cys in proteins. The second section shows a general strategy used for ICAT; all reversible thiols are reduced and labeled with ICAT. The third section shows the OxICAT (a modified ICAT method) that is used to study oxidized thiols; free thiols are blocked with a blocking reagent such as iodoacetomide, and, reversibly, oxidized thiols are labeled with ICAT. The fourth section depicts an overview of the ICAT strategy; samples are first labeled with heavy or light isotope reagent individually, then mixed, and, finally, subjected to tryptic digestion after affinity purification. Finally, both quantification and identification are achieved by mass spectrometry from MS and MS/MS data, respectively.

FIG. 5.

OPTMs by alpha, beta unsaturated aldehydes. The family of alpha, beta unsaturated aldehydes possess electrophilic and aldehyde groups that allow for two types of reactions: (i) Schiff base formation occurs with primary amine groups, primarily on lysine (Lys) residues but can modify arginine as well. (ii) Michael addition primarily occurs through interactions of the electrophilic group with the sulfhydryl group of Cys. Michael addition can also modify the imidazole group of histidine residues, the ɛ-amino group of Lys, and, to a lesser extent, the arginine side chains. The electrophilic and aldehydic moieties of this species allow for dual reactions to occur, thereby forming inter- and intramolecular crosslinks between protein chains under favorable conditions.

FIG. 6.

Workflow for biomarker discovery.