Concluding Remarks

Oxidative/nitrative-induced tyrosine nitration in a protein is an important modification and is associated with multiple pathophysiological processes [1-3, 26, 27, 75]. In addition, studies found a denitrase in mammalian cells and tissues. This enzyme implies that the reciprocal processes of nitration and denitration might modulate biological events and regulate cell signaling events [17, 18]. Nitration dynamically alters protein function [117], including activation or inactivation [118-120]. MS is the key technique used to identify nitroproteins and nitration sites and to understand the biological roles of tyrosine nitration [121-123]. However, characterization of endogenous nitro- proteins and nitration sites is a very challenging issue because of extreme low abundance of nitration in biological samples and various MS behaviors among MALDI UV-laser-, ESI-, CID-, ECD-, ETD-, and MAD-MS. It is necessary to preferentially enrich endogenous nitroproteins and nitropeptides before MS analysis. The immunoaffinity enrichment, biotin affinity enrichment, and COFRADIC are currently developed enrichment strategies for the analysis of endogenous nitroproteins, and nitration sites have been found in different pathophysiological status. TMT- or iTRAQ-based quantitative nitroproteom- ics are promising methods to quantify the key nitroproteins and nitropeptides in a disease. Furthermore, protein domain/motif analysis, systems pathway analysis, and structural biological analysis of nitroproteins [38, 124] are needed to completely clarify the biological functions of tyrosine nitration.

However, it is very important to realize clearly that no high-sensitivity, high- reproducibility, and highly reliable methods currently exist for the analysis of the extreme low-abundance endogenous tyrosine nitration in a proteome [38, 125]. Many different approaches remain under development. Currently, antinitrotyrosine antibody-based immunoaffinity methods, for example, 2D western blotting and NTAC, have all been used to identify endogenous tyrosine nitration sites; however, an overwhelming amount of nonnitrated tryptic peptides negatively impacts the identification of tryptic nitropeptides in these studies. Therefore, it would be more effective to develop immunoaffinity enrichment of tryptic nitropeptides, but not nitroproteins, before MS analysis. Until now, most chemical derivation-based target enrichment methods have succeeded in in vitro experiments but not in in vivo endogenous tyrosine nitration site analysis. The COFRADIC-based methods succeeded in the identification of endogenous nitropeptides in a serum proteome; however, its throughput and sensitivity were very low, and it has not been used extensively in endogenous tissue nitroproteomes. Therefore, a better method is needed to analyze endogenous tyrosine nitration sites.

The following aspects are worth considering alone or in combination including (i) derivation of a nitro to amino group to stabilize MS behaviors,

  • (ii) developing specific amino group tags to enrich nitrotyrosine peptides,
  • (iii) enriching nitrotyrosine or aminotyrosine peptides but not proteins for sensitivity, (iv) choosing the appropriate ion source and collision model to fragment nitropeptide or aminopeptides, (v) developing super high-sensitivity mass spectrometers, (vi) improving liquid chromatography isolation, and (vii) developing reliable software for data analysis. We recommend the combined multiple strategies among items i-vii to maximize the coverage of endogenous tyrosine nitration sites in a proteome.
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