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Characterization of disulfide bonds by planned digestion and tandem mass spectrometry.

ABSTRACT: The identification of disulfide bonds provides critical information regarding the structure and function of a protein and is a key aspect in understanding signaling cascades in biological systems. Recent proteomic approaches using digestion enzymes have facilitated the characterization of disulfide-bonds and/or oxidized products from cysteine residues, although these methods have limitations in the application of MS/MS. For example, protein digestion to obtain the native form of disulfide bonds results in short lengths of amino acids, which can cause ambiguous MS/MS analysis due to false positive identifications. In this study we propose a new approach, termed planned digestion, to obtain sufficient amino acid lengths after cleavage for proteomic approaches. Application of the DBond software to planned digestion of specific proteins accurately identified disulfide-linked peptides. RNase A was used as a model protein in this study because the disulfide bonds of this protein have been well characterized. Application of this approach to peptides digested with Asp-N/C (chemical digestion) and trypsin under acid hydrolysis conditions identified the four native disulfide bonds of RNase A. Missed cleavages introduced by trypsin treatment for only 3 hours generated sufficient lengths of amino acids for identification of the disulfide bonds. Analysis using MS/MS successfully showed additional fragmentation patterns that are cleavage products of S-S and C-S bonds of disulfide-linkage peptides. These fragmentation patterns generate thioaldehydes, persulfide, and dehydroalanine. This approach of planned digestion with missed cleavages using the DBond algorithm could be applied to other proteins to determine their disulfide linkage and the oxidation patterns of cysteine residues.

SUBMITTER: Na S

PROVIDER: S-EPMC4410109 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Characterization of disulfide bonds by planned digestion and tandem mass spectrometry.

Na Seungjin S Paek Eunok E Choi Jong-Soon JS Kim Duwoon D Lee Seung Jae SJ Kwon Joseph J

Molecular bioSystems 20150401 4

The identification of disulfide bonds provides critical information regarding the structure and function of a protein and is a key aspect in understanding signaling cascades in biological systems. Recent proteomic approaches using digestion enzymes have facilitated the characterization of disulfide-bonds and/or oxidized products from cysteine residues, although these methods have limitations in the application of MS/MS. For example, protein digestion to obtain the native form of disulfide bonds ...[more]

PMID: 25703060

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Project description:PubChem serves as a comprehensive repository, housing over 100 million unique chemical structures representing the breadth of our chemical knowledge across numerous fields including metabolism, pharmaceuticals, toxicology, cosmetics, agriculture, and many more. Rapid identification of these small molecules increasingly relies on electrospray ionization (ESI) paired with tandem mass spectrometry (MS/MS), particularly by comparison to genuine standard MS/MS data sets. Despite its widespread application, achieving consistency in MS/MS data across various analytical platforms remains an unaddressed concern. This study evaluated MS/MS data derived from one hundred molecular standards utilizing instruments from five manufacturers, inclusive of quadrupole time-of-flight (QTOF) and quadrupole orbitrap "exactive" (QE) mass spectrometers by Agilent (QTOF), Bruker (QTOF), SCIEX (QTOF), Waters (QTOF), and Thermo QE. We assessed fragment ion variations at multiple collisional energies (0, 10, 20, and 40 eV) using the cosine scoring algorithm for comparisons and the number of fragments observed. A parallel visual analysis of the MS/MS spectra across instruments was conducted, consistent with a standard procedure that is used to circumvent the still prevalent issue of mischaracterizations as shown for dimethyl sphingosine and C20 sphingosine. Our analysis revealed a notable consistency in MS/MS data and identifications, with fragment ions' m/z values exhibiting the highest concordance between instrument platforms at 20 eV, the other collisional energies (0, 10, and 40 eV) were significantly lower. While moving toward a standardized ESI MS/MS protocol is required for dependable molecular characterization, our results also underscore the continued importance of corroborating MS/MS data against standards to ensure accurate identifications. Our findings suggest that ESI MS/MS manufacturers, akin to the established norms for gas chromatography mass spectrometry instruments, should standardize the collision energy at 20 eV across different instrument platforms.

Dataset Information

Characterization of disulfide bonds by planned digestion and tandem mass spectrometry.

Publications

Characterization of disulfide bonds by planned digestion and tandem mass spectrometry.

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