Sample Preparation for Peptide Mapping— A Pharmaceutical Quality-Control Perspective

Lundell, Niklas; Schreitmüller, Thomas

doi:10.1006/abio.1998.2919

Cited by 134 publications

(96 citation statements)

References 101 publications

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“…3C, Table 1). Although no free Cys is available on the SrxS-SSrx dimer, it was observed with two and four carbamidomethyl adducts, which can probably be attributed to increased His and Met reactivity toward IAM for this mutant Srx (26,27).…”

Section: S-sprx) Disulfide Bondsmentioning

confidence: 91%

Evidence for the Formation of a Covalent Thiosulfinate Intermediate with Peroxiredoxin in the Catalytic Mechanism of Sulfiredoxin

Roussel

Béchade

Kriznik

et al. 2008

Journal of Biological Chemistry

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The typical 2-Cys peroxiredoxins are thiol-peroxidases involved in the physiology of hydrogen peroxide not only as a toxic but also as a signaling molecule. Coordination of these functions depends on the sulfinylation of the catalytic Cys, a modification reversed by ATP-dependent sulfiredoxin, which specifically reduces the sulfinic acid group of overoxidized 2-Cys peroxiredoxins into a sulfenic acid. Sulfiredoxin was originally proposed to operate by covalent catalysis, with formation of a peroxiredoxin-sulfiredoxin intermediate linked by a thiosulfinate bond between the catalytic Cys of both partners, a hypothesis rejected by a study of the human enzyme. To settle the argument, we investigated the catalytic mechanism of Saccharomyces cerevisiae sulfiredoxin, by the characterization of the nature and kinetics of formation of the protein species formed between sulfiredoxin and its substrate in the presence of ATP, using mutants of the non-essential Cys residues of both proteins. We observed the formation of a dithiothreitol-reducible peroxiredoxin-sulfiredoxin species using SDS-PAGE and Western blot analysis, and its mass was shown to correspond to a thiosulfinate complex by high resolution mass spectrometry coupled to liquid chromatography. We next measured indirectly and directly a rate constant of formation of the thiosulfinate species of ϳ2 min ؊1 , for both wild-type and mutant sulfiredoxins, at least equal to the steady-state rate constant of the reaction, with a stoichiometry of 1:1 relative to peroxiredoxin. Taken altogether, our results strongly argue in favor of the formation of a covalent thiosulfinate peroxiredoxin-sulfiredoxin species as an intermediate on the catalytic pathway.

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Section: S-sprx) Disulfide Bondsmentioning

confidence: 91%

Evidence for the Formation of a Covalent Thiosulfinate Intermediate with Peroxiredoxin in the Catalytic Mechanism of Sulfiredoxin

Roussel

Béchade

Kriznik

et al. 2008

Journal of Biological Chemistry

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“…2b,c) 16 . Indeed, iodoacetamide is used routinely for capping before digestion for protein sequencing 17 . Notably, some derivatives react with N-nucleophiles 18 .…”

Section: Modifications Of Natural Amino Acidsmentioning

confidence: 99%

Selective chemical protein modification

2014

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“…Furthermore, these conditions lead to an increase in digestion artifacts such as transpeptidation and non-specific cleavage 148 and deamidation/oxidation, 136,149 which complicate analysis and reduce confidence in the identification of proteomic analytes.…”

Section: Introductionmentioning

confidence: 99%

A Digital Microfluidic Approach to Proteomic Sample Processing

2009

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Proteome profiling is the identification and quantitation of all proteins in biological samples. An important application of proteome profiling that has received much attention is clinical proteomics, a field that promises the discovery of biomarkers that will be useful for early diagnosis and prognosis of diseases. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from complex biological fluids and (ii) extensive biochemical processing (reduction, alkylation and enzymatic digestion) prior to analysis. However, the lack of standardized sample handling and processing in proteomics is a major limitation for the field. The conventional macroscale manual sample handling requires multiple containers and transfers, which often leads to sample loss and contamination. For clinical proteomics to be adopted as a gold standard for clinical measures, the issue of irreproducibility needs to be addressed. A potential solution to this problem is to form integrated systems for sample handling and processing, and in this dissertation, I describe my work towards realizing this goal using digital microfluidics (DMF). DMF is a technique characterized by the manipulation of discrete droplets (100 nL -10 L) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. This thesis demonstrates how DMF can be a powerful tool capable of automating several protein handling and processing steps used in proteomics.iii Methods for implementing proteomic multi-step solution-phase processes (reduction, alkylation, and digestion) on DMF were developed. This was accompanied by the development of heterogenous enzymatic microreactors for efficient protein digestion. Strategies for reducing non-specific adsorption were developed. Finally, in proof-of-concept work, a combination of protein extraction and protein processing was demonstrated on DMF devices.iv ACKNOWLEDGEMENTS

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Sample Preparation for Peptide Mapping— A Pharmaceutical Quality-Control Perspective

Cited by 134 publications

References 101 publications

Evidence for the Formation of a Covalent Thiosulfinate Intermediate with Peroxiredoxin in the Catalytic Mechanism of Sulfiredoxin

Evidence for the Formation of a Covalent Thiosulfinate Intermediate with Peroxiredoxin in the Catalytic Mechanism of Sulfiredoxin

Selective chemical protein modification

A Digital Microfluidic Approach to Proteomic Sample Processing

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