Table 2.

Main applied methods in the study of redox proteomics.

Redox Proteomics	Employed Technique	Advantages	Disadvantages
Protein Nitration	Immunochemical methods
	ELISA	High throughput: relatively low cost and sample preparation	Low sensitivity (i.e., accessibility to certain 3-NT) Issues of specificity (i.e., antibodies interactions)
	IHC or ICC	Semiquantitative; it allows a fluorescence intensity comparison 3-NT detection in different cells and tissues	Quantifying results is difficult Cost of the equipment needed
	WB	Primary technique for quantifying 3-NT	Time consuming, relatively expensive, sensitivity issues
	Chromatographic methods
	HPLC-UV	Detection of 3-NT alone or associated with proteins	Low sensitivity and selectivity
	HPLC-fluorescence	Higher sensitivity and selectivity than HPLC-UV	Structural alterations of 3-NT group (derivatization) Production of other fluorescent compounds
	HPLC-ECD	Greater selectivity than UV or fluorescence detection	In general, HPLC disadvantages are the high cost and the regular maintenance
	HPLC-MS	Widely accepted as the gold standard for analyzing biological samples
	GC-MS		Prior derivatization and modifications to functional groups
Protein-Bound HNE	SPH	Carbonyl groups stay intact after hydrolysis	May be coupled with LC-MS/MS
	SDS-PAGE (and WB)	SDS-PAGE is suitable for separating low-molecular-weight molecules	SDS causes a denaturation of proteins WB: time-consuming, expensive, sensitivity issues.
Protein S-glutathionylation	Radio Labeled	Sensitivity and robustness Used in different conditions	Disturbed cell physiology, nonspecific signals Inability to differentiate individual SSG sites
	MS	High sensitivity and specificity	Impurities, difficult mass estimation
	Switch Assay-MS	When the modification is chemically labile
	Detection in situ	May be applied in situ	False positives (oxidative modifications)
	Western Blot	S-glutathionylated proteins are most commonly identified by this method	Time-consuming, expensive, sensitivity issues.
Protein carbonyls	Immunohistochemical detection (ELISA, WB)	Qualitative/quantitative uses, and specificity and sensitivity	Antibodies unspecifically bound to other molecules Impossibility to access the hidden sites of the protein
	DNPH-based photometric assay	The most standardized assay of total protein carbonyls	Limitations inherent to the sample (i.e., heme proteins, DNA) Very limited sensitivity
	Nongel identification	Follows the general approach using MS	Additional purification steps

Abbreviations: DNPH, 2,4-dinitrophenylhydrazine; ELISA, enzyme-linked immunosorbent assay; GC-MS, gas chromatography coupled to mass spectrometry; HPLC-ECD, high-performance liquid chromatography–electrochemical detection; HPLC-MS, high-performance liquid chromatography–mass spectrometry; HPLC-UV, high-performance liquid chromatography–ultraviolet absorption detectors; IHC or ICC, immunohistochemistry or immunocytochemistry; LC-MS/MS, liquid chromatography–mass spectrometry; MS, mass spectrometry; SDS-PAGE, SDS–polyacrylamide gel electrophoresis; SPH, solid-phase hydrazide; SSG, S-glutathionylation; WB, Western blot; 3-NT, 3-nitrotyrosine.