Selective Bridging of Bis-Cysteinyl Residues by Arsonous Acid Derivatives as an Approach to the Characterization of Protein Tertiary Structures and Folding Pathways by Mass Spectrometry

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240

In the post-genomic era, a wealth of structural information has been amassed for proteins from NMR and crystallography. However, static protein structures alone are not a sufficient description: knowledge of the dynamic nature of proteins is essential to understand their wide range of functions and behavior during the life cycle from synthesis to degradation. Furthermore, few proteins have the ability to act alone in the crowded cellular environment. Assemblies of multiple proteins governed by complex signaling pathways are often required for the tasks of target recognition, binding, transport, and function. Mass spectrometry has emerged over the past several years as a powerful tool to address many of these questions. Recent improvements in "soft" ionization techniques have enabled researchers to study proteins and biomolecular complexes, both directly and indirectly. Likewise, continuous improvements in instrumental design in recent years have resulted in a dramatic expansion of the m/z range and resolution, enabling observation of large multi-protein assemblies whose structures are retained in the gas phase. In this article, we discuss some of the mass spectrometric techniques applied to investigate the nature of the conformations and dynamical properties that govern protein function.

Section: Biomolecular Conformationsmentioning

confidence: 99%

Studies of biomolecular conformations and conformational dynamics by mass spectrometry

Kaltashov

Eyles

2002

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240

“…The implicit assumption in these experiments is that Cys residues are unreactive when in a disulfide form; however, special control experiments are necessary to distinguish between Cys residues that form disulfides and those that are simply buried in the protein’s interior. Glocker and co-workers used melarsen oxide, an arsonous acid derivative, to selectively bridge bis-cysteinyl residues to characterize the tertiary structure of partially reduced bovine pancreatic trypsin inhibitor (Happersberger, Przybylski, & Glocker, 1998). MALDI-TOF MS peptide mapping revealed that only one of the three disulfide bonds, Cys14-Cys38, was predominantly reduced.…”

Section: Amino Acid-specific Labels and Examples Of Their Applicmentioning

confidence: 99%

Probing protein structure by amino acid‐specific covalent labeling and mass spectrometry

Mendoza

Vachet

2008

325

445

For many years, amino acid-specific covalent labeling has been a valuable tool to study protein structure and protein interactions, especially for systems that are difficult to study by other means. These covalent labeling methods typically map protein structure and interactions by measuring the differential reactivity of amino acid side chains. The reactivity of amino acids in proteins generally depends on the accessibility of the side chain to the reagent, the inherent reactivity of the label and the reactivity of the amino acid side chain. Peptide mass mapping with ESI- or MALDI-MS and peptide sequencing with tandem MS are typically employed to identify modification sites to provide site-specific structural information. In this review, we describe the reagents that are most commonly used in these residue-specific modification reactions, details about the proper use of these covalent labeling reagents, and information about the specific biochemical problems that have been addressed with covalent labeling strategies.

“…MS analysis of the fluorescent cross-linked peptides was performed to identify and determinate the interaction sites. Arsonous acid compounds were used to derivatize dithiol groups to characterize protein tertiary structures with MS (Kussmann & Przybylski, 1995;Happersberger, Przybylski, & Glocker, 1998).…”

Section: Protein Cross-linking and Probing Of Cysteine Accessibilitymentioning

confidence: 99%

Cysteine tagging for MS‐based proteomics

Giron

Dayon

Sanchez

2010

Amino acid-tagging strategies are widespread in proteomics. Because of the central role of mass spectrometry (MS) as a detection technique in protein sciences, the term "mass tagging" was coined to describe the attachment of a label, which serves MS analysis and/or adds analytical value to the measurements. These so-called mass tags can be used for separation, enrichment, detection, and quantitation of peptides and proteins. In this context, cysteine is a frequent target for modifications because the thiol function can react specifically by nucleophilic substitution or addition. Furthermore, cysteines present natural modifications of biological importance and a low occurrence in the proteome that justify the development of strategies to specifically target them in peptides or proteins. In the present review, the mass-tagging methods directed to cysteine residues are comprehensively discussed, and the advantages and drawbacks of these strategies are addressed. Some concrete applications are given to underline the relevance of cysteine-tagging techniques for MS-based proteomics.