Mapping native disulfide bonds at a proteome scale

Lu, Shan; Fan, Shidong; Yang, Bing; Li, Yuxin; Meng, Jia-Ming; Wu, Long; Пин, Ли; Zhang, Kun; Zhang, Meijun; Fu, Yan; Luo, Jincai; Sun, Rui-Xiang; He, Shan; Dong, Meng‐Qiu

doi:10.1038/nmeth.3283

Cited by 134 publications

(136 citation statements)

References 34 publications

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“…The software was also adapted to enable mapping of disulfide bonds within numerous proteins, termed 'pLink-SS' [120]. Using pLink-SS along with MS analysis, 199 disulfide bonds in 150 proteins of E. coli have been identified by comparing disulfide proteomes in dsbA À , dsbC À (DsbA and DsbC are thiol-disulfide oxidoreductases) and wild-type BW25113 strains [121].…”

Section: Oxmrmmentioning

confidence: 99%

“…In addition, 309 disulfide bonds and 1738 glutathionylated sites were identified in A549 cells under diamide treatment. Moreover, when monitoring the distance of two cysteine residues forming disulfide bonds, they found that CXXC is the most prominent mode in disulfide bonds-formation in E. coli, and the formation of disulfide bonds could occasionally alter protein conformations by expelling metal ions [120]. Disulfide proteome analysis provides new concepts for exploring redox-sensitive proteins and mapping disulfide bonds critical for protein function.…”

Section: Oxmrmmentioning

confidence: 99%

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High-throughput screening of cellular redox sensors using modern redox proteomics approaches

Jiang

Wang

Nice

et al. 2015

Expert Review of Proteomics

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Cancer cells are characterized by higher levels of intracellular reactive oxygen species (ROS) due to metabolic aberrations. ROS are widely accepted as second messengers triggering pivotal signaling pathways involved in the process of cell metabolism, cell cycle, apoptosis, and autophagy. However, the underlying cellular mechanisms remain largely unknown. Recently, accumulating evidence has demonstrated that ROS initiate redox signaling through direct oxidative modification of the cysteines of key redox-sensitive proteins (termed redox sensors). Uncovering the functional changes underlying redox regulation of redox sensors is urgently required, and the role of different redox sensors in distinct disease states still remains to be identified. To assist this, redox proteomics has been developed for the high-throughput screening of redox sensors, which will benefit the development of novel therapeutic strategies for cancer treatment. Highlighted here are recent advances in redox proteomics approaches and their applications in identifying redox sensors involved in tumor development.

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Section: Oxmrmmentioning

confidence: 99%

Section: Oxmrmmentioning

confidence: 99%

High-throughput screening of cellular redox sensors using modern redox proteomics approaches

Jiang

Wang

Nice

et al. 2015

Expert Review of Proteomics

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“…(2014)pLink-SSHCD or high-resolution ETDHCD MS2A: Automatic workflow with FDR estimation, disulfide-specific ions and internal ions are considered, works for complex samples;L: highly complex forms such as three peptides inter-linked through two disulfide bonds cannot be identifiedLu et al . (2015)…”

Section: Introductionmentioning

confidence: 99%

“…This method was published in 2015 (Lu et al . 2015). Since then, to our knowledge, it has been used successfully in dozens of studies and seven of them have been published (Hartman et al .…”

Section: Introductionmentioning

confidence: 99%

Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures

Cao

Fan

et al. 2018

Biophys Rep

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Disulfide bonds are vital for protein functions, but locating the linkage sites has been a challenge in protein chemistry, especially when the quantity of a sample is small or the complexity is high. In 2015, our laboratory developed a sensitive and efficient method for mapping protein disulfide bonds from simple or complex samples (Lu et al. in Nat Methods 12:329, 2015). This method is based on liquid chromatography–mass spectrometry (LC–MS) and a powerful data analysis software tool named pLink. To facilitate application of this method, we present step-by-step disulfide mapping protocols for three types of samples—purified proteins in solution, proteins in SDS-PAGE gels, and complex protein mixtures in solution. The minimum amount of protein required for this method can be as low as several hundred nanograms for purified proteins, or tens of micrograms for a mixture of hundreds of proteins. The entire workflow—from sample preparation to LC–MS and data analysis—is described in great detail. We believe that this protocol can be easily implemented in any laboratory with access to a fast-scanning, high-resolution, and accurate-mass LC–MS system.

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“…It follows that modification or mutation of these key cysteines must be avoided. Finally, an increasing number of proteins are now recognized as possessing conserved disulfide bonds (Lu et al, 2015). Although thiol-specific probes will generally not react with cysteines that are involved in a disulfide linkage, the addition of extra cysteines into recombinant proteins can potentially lead to aberrant, nonphysiological disulfide formation.…”

Section: Introductionmentioning

confidence: 99%

Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

Evans

Millhauser

2015

Methods in Enzymology

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Site-directed spin labeling (SDSL) is a powerful tool for the characterization of protein structure and dynamics; however, its application in many systems is hampered by the reliance on unique and benign cysteine substitutions for the site-specific attachment of the spin label. An elegant solution to this problem involves the use of genetically encoded unnatural amino acids (UAAs) containing reactive functional groups that are chemically orthogonal to those of the 20 amino acids found naturally in proteins. These unique functional groups can then be selectively reacted with an appropriately functionalized spin probe. In this chapter, we detail the genetic incorporation of the ketone-bearing amino acid p-acetyl phenylalanine (pAcPhe) into recombinant proteins expressed in E. coli. Incorporation of pAcPhe is followed by chemoselective reaction of the ketone side chain with a hydroxylamine-functionalized nitroxide to afford the spin-labeled side chain “K1,” and we present two protocols for successful K1 labeling of proteins bearing site-specific pAcPhe. We outline the basic requirements for pAcPhe incorporation and labeling, with an emphasis on practical aspects that must be considered by the researcher if high yields of UAA incorporation and efficient labeling reactions are to be achieved. To this end, we highlight recent advances that have led to increased yields of pAcPhe incorporation, and discuss the use of aniline-based catalysts allowing for facile conjugation of the hydroxylamine spin label under mild reaction conditions. To illustrate the utility of K1 labeling in proteins where traditional cysteine-based SDSL methods are problematic, we site-specifically K1 label the cellular prion protein at two positions in the C-terminal domain and determine the interspin distance using double electron–electron resonance EPR. Recent advances in UAA incorporation and ketone-based bioconjugation, in combination with the commercial availability of all requisite reagents, should make K1 labeling an increasingly viable alternative to cysteine-based methods for SDSL in proteins.

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Mapping native disulfide bonds at a proteome scale

Cited by 134 publications

References 34 publications

High-throughput screening of cellular redox sensors using modern redox proteomics approaches

High-throughput screening of cellular redox sensors using modern redox proteomics approaches

Mapping disulfide bonds from sub-micrograms of purified proteins or micrograms of complex protein mixtures

Genetic Incorporation of the Unnatural Amino Acid p-Acetyl Phenylalanine into Proteins for Site-Directed Spin Labeling

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