Article

Quantitative reactivity profiling predicts functional cysteines in proteomes

The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Nature (Impact Factor: 42.35). 11/2010; 468(7325):790-5. DOI: 10.1038/nature09472
Source: PubMed

ABSTRACT Cysteine is the most intrinsically nucleophilic amino acid in proteins, where its reactivity is tuned to perform diverse biochemical functions. The absence of a consensus sequence that defines functional cysteines in proteins has hindered their discovery and characterization. Here we describe a proteomics method to profile quantitatively the intrinsic reactivity of cysteine residues en masse directly in native biological systems. Hyper-reactivity was a rare feature among cysteines and it was found to specify a wide range of activities, including nucleophilic and reductive catalysis and sites of oxidative modification. Hyper-reactive cysteines were identified in several proteins of uncharacterized function, including a residue conserved across eukaryotic phylogeny that we show is required for yeast viability and is involved in iron-sulphur protein biogenesis. We also demonstrate that quantitative reactivity profiling can form the basis for screening and functional assignment of cysteines in computationally designed proteins, where it discriminated catalytically active from inactive cysteine hydrolase designs.

Download full-text

Full-text

Available from: Sagar D Khare, Jun 30, 2015
0 Followers
 · 
139 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The post-translational modification of proteins by electrophilic oxylipids is emerging as an important mechanism that contributes to the complexity of proteomes. Enzymatic and non-enzymatic oxidation of biological lipids results in the formation of chemically diverse electrophilic carbonyl compounds, such as 2-alkenals and 4-hydroxy alkenals, epoxides, and eicosanoids with reactive cyclopentenone structures. These lipoxidation products are capable of modifying proteins. Originally considered solely as markers of oxidative insult, more recently the modifications of proteins by lipid peroxidation products are being recognized as a new mechanism of cell signaling with relevance to redox homeostasis, adaptive response and inflammatory resolution. The growing interest in protein modifications by reactive oxylipid species necessitates the availability of methods that are capable of detecting, identifying and characterizing these protein adducts in biological samples with high complexity. However, the efficient analysis of these chemically diverse protein adducts presents a considerable analytical challenge. We first provide an introduction into the chemistry and biological relevance of protein adductions by electrophilic lipoxidation products. We then provide an overview of tandem mass spectrometry approaches that have been developed in recent years for the interrogation of protein modifications by electrophilic oxylipid species.
    Mass Spectrometry Reviews 05/2014; 33(3):157-82. DOI:10.1002/mas.21389 · 8.05 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: A novel chemodosimetric fluorescent probe 1 for biothiols was designed and synthesized based on benzothiazolium-quinoline hemicyanine dye with an acrylate group as a functional trigger moiety. Prober 1 exhibited high sensitivity and high selectivity for rapid detection of cysteine (Cys) over homocysteine and glutathione in aqueous environments. The maximal fluorescent response was immediately achieved within 1 min in the presence of only 1 equiv. of Cys, and the color change of the sensing process was discerned visually at the nanomole level. The exceptional high reactivity and specific response of probe 1 for Cys were promoted by the stabilization of the sensing product phenolate anion and the differences in the kinetics of the sensing reaction. Moreover, probe 1 was employed successfully to detect the free Cys residues within protein and monitor Cys in living HeLa cells by fluorescence imaging.
    Sensors and Actuators B Chemical 03/2014; 196:546-554. DOI:10.1016/j.snb.2014.02.052 · 3.84 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: S-nitrosylation, the formation of S-nitrosothiol (SNO), is an important reversible thiol oxidation eventthat that has been increasingly recognized for its role in cell signaling. While many proteins susceptible to S-nitrosylation have been reported, site-specific identification of physiologically relevant SNO modifications remains an analytical challengebecause of the lowabundance and labile nature of thismodification. Herein we presentfurther improvement and optimization of the recentlyreported resin-assisted cysteinyl peptide enrichment protocol for SNO identification and the application to mouse skeletal muscle to identify specific cysteine sites sensitive to S-nitrosylationby a quantitative reactivity profiling strategy.Our results indicate that the protein- and peptide-level enrichment protocols provide comparable specificity and coverage of SNO-peptide identifications.S-nitrosylation reactivity profiling was performed by quantitatively comparing the site-specific SNO modification levels in samples treated with S-nitrosoglutathione (GSNO), an NO donor, at two different concentrations (i.e., 10μM and 100μM).The reactivity profiling experiments led to the identification of 488SNO-modified sites from 197proteins with specificity of ~95% at the uniquepeptidelevel;i.e., ~95% of enriched peptides contain cysteine residuesas the originally SNO-modified sites.Among these sites, 281sites from 145proteins were considered more sensitive to S-nitrosylation based on the ratios of observed SNO levels between the two treatments.These SNO-sensitive sites are more likely to be physiologically relevant.Many of the SNO-sensitiveproteins are localized in mitochondria, contractile fiber, and actin cytoskeleton, suggesting the susceptibility of these subcellular compartments to redox regulation.Moreover, theseobserved SNO-sensitive proteins are primarily involved in metabolic pathways, including TCA cycle, glycolysis/gluconeogenesis, glutathione metabolism, and fatty acid metabolism, suggesting the importance of redox regulation in muscle metabolism and insulin action.
    Free Radical Biology and Medicine 12/2012; 57. DOI:10.1016/j.freeradbiomed.2012.12.010 · 5.71 Impact Factor