In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography

Ghesquière, Bart; Colaert, Niklaas; Helsens, Kenny; Dejager, Lien; Vanhaute, Caroline; Verleysen, Katleen; Kas, Koen; Timmerman, Evy; Goethals, Marc; Libert, Claude; Vandekerckhove, Joël; Gevaert, Kris

doi:10.1074/mcp.m900259-mcp200

Cited by 85 publications

(108 citation statements)

References 48 publications

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“…In this study, the nitropeptides were reduced to aminopeptides followed by acetylation in order to improve fragmentation and identification. Using the COFRADIC approach, Ghesquiére et al [34] observed only one out of four peptides coinciding with our results. This most likely reflects irreproducibility of the in vitro nitration procedure rather than of the methods for isolation and identification of the nitrated peptides.…”

Section: Resultssupporting

confidence: 80%

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Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Larsen

Bache

Gramsbergen

et al. 2011

J. Am. Soc. Mass Spectrom.

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Protein nitration take place on tyrosine residues under oxidative stress conditions and may influence a number of processes including enzyme activity, protein-protein interactions and phospho-tyrosine signalling pathways. Nitrated proteins have been identified in a number of diseases, however, the study of these proteins has been compromised by the lack of good methods for identifying nitrated proteins, their nitration sites and the level of nitration. Here, we present a method for identification of nitrated peptides that allows the site specific assignment of nitration, is easy to use and reproducible, and opens up for the possibility to quantify the level of nitration of specific peptides as function of different oxidative conditions, namely combined fractional diagonal chromatography (COFRADIC) in combination with off-line nano-LC-MALDI. We identify six nitrated peptides from in vitro nitrated bovine serum albumin and propose that automated COFRADIC using nano-LC and off-line MALDI-MS might be a possibility for identification of tyrosine nitrated proteins and the nitration sites in complex samples.

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Section: Resultssupporting

confidence: 80%

“…showed that the COFRADIC approach could also be used to identify nitrotyrosine containing peptides in both simple and complex samples [34].…”

Section: Introductionmentioning

confidence: 99%

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Larsen

Bache

Gramsbergen

et al. 2011

J. Am. Soc. Mass Spectrom.

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“…Excess radicals are scavenged by serum proteins, leading to protein modifications such as tyrosine nitration (28). Using our COFRADIC approach we next investigated whether this oxidative burst led to oxidation of methionine in serum proteins of mice infected with Salmonella.…”

Section: A Cofradic Methods For Isolating Peptides Containingmentioning

confidence: 99%

“…Following the COFRADIC sorting procedure we analyzed the obtained fractions onto an Ultimate 3000 nano-HPLC system in-line connected to a LTQ Orbitrap XL mass. Mass spectrometric analysis was performed as described (28).…”

Section: Reduction Of a Methionine Sulfoxide Peptide Using Msra Andmentioning

confidence: 99%

Redox Proteomics of Protein-bound Methionine Oxidation

Ghesquière

Jonckheere

Colaert

et al. 2011

Molecular & Cellular Proteomics

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127

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We here present a new method to measure the degree of protein-bound methionine sulfoxide formation at a proteome-wide scale. In human Jurkat cells that were stressed with hydrogen peroxide, over 2000 oxidationsensitive methionines in more than 1600 different proteins were mapped and their extent of oxidation was quantified. Meta-analysis of the sequences surrounding the oxidized methionine residues revealed a high preference for neighboring polar residues. Using synthetic methionine sulfoxide containing peptides designed according to the observed sequence preferences in the oxidized Jurkat proteome, we discovered that the substrate specificity of the cellular methionine sulfoxide reductases is a major determinant for the steady-state of methionine oxidation. This was supported by a structural modeling of the MsrA catalytic center. Finally, we applied our method onto a serum proteome from a mouse sepsis model and identi- Reactive oxygen species (ROS)1 are involved in a broad range of processes including signal transduction and gene expression (1), receptor activation (2), antimicrobial and cytotoxic actions of immune cells (3), and aging and age-related degenerative diseases (4). Cellular oxidative stress is associated with increased levels of reactive oxygen species and the molecular damages they cause (5). Of interest here is that some reactive oxygen species specifically modify targeted biomolecules, whereas others cause nonspecific damage. Peroxides for instance are generally more selective compared with hydroxyl radicals (6). Major ROS targets are proteins, with oxidation occurring both at the peptide backbone and at amino acid side-chains (6). The major oxidation product of protein-bound methionine is methionine sulfoxide, further oxidation of which can lead to methionine sulfone, albeit to a much lesser extent (7). The (patho)physiological importance of this modification is reflected by the methionine sulfoxide reductases (Msr) that are present in nearly all organisms (8, 9): decreased activity of these enzymes was associated with aging and Alzheimer disease (10), and abnormal dopamine signaling was recently found in the methionine sulfoxide reductase A knockout mouse (11). Oxidation of methionine can lead to loss of enzyme activity as shown for a brain voltagedependent potassium channel (12). Other studies suggest that methionine oxidation prevents methylation (13) or has an effect on phosphorylation on serines and threonines proximate to the oxidized site (14). In this respect, protein kinases are also targeted by methionine oxidation affecting their activity (e.g. (15)). Further, oxidation of surface methionines increases the protein surface hydrophobicity (16) and may perturb native protein folding, and such oxidized proteins further often become targets for degradation by the proteasome (17).Although methionines are utmost susceptible to oxidation by several types of ROS (18), no adequate proteomic methodologies exist to characterize the exact sites of oxidation and quantify the degree of oxidation. ...

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“…In a typical experiment, a sample is fractionated by HPLC, fractions are collected, dried, treated to modify a residue or PTM and again subjected to HPLC analysis. This technique has been used to study modifications including phosphorylation, nitration and N-glycosylation [27][28][29][30]. The latest development in this field is an automated enzyme-based approach involving capillary electrophoresis, in which the distal end of the first capillary incorporates an enzyme-based microreactor [31].…”

Section: Diagonal Separationmentioning

confidence: 99%

Advances in purification and separation of posttranslationally modified proteins

Černý

Skalák

Cerná

et al. 2013

Journal of Proteomics

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Posttranslational modifications (PTMs) of proteins represent fascinating extensions of the dynamic complexity of living cells' proteomes. The results of enzymatically catalyzed or spontaneous chemical reactions, PTMs form a fourth tier in the gene -transcript -protein cascade, and contribute not only to proteins' biological functions, but also to challenges in their analysis. There have been tremendous advances in proteomics during the last decade. Identification and mapping of PTMs in proteins have improved dramatically, mainly due to constant increases in the sensitivity, speed, accuracy and resolution of mass spectrometry (MS). However, it is also becoming increasingly evident that simple gel-free shotgun MS profiling is unlikely to suffice for comprehensive detection and characterization of proteins and/or protein modifications present in low amounts. Here, we review current approaches for enriching and separating posttranslationally modified proteins, and their MS-independent detection. First, we discuss general approaches for proteome separation, fractionation and enrichment. We then consider the commonest forms of PTMs (phosphorylation, glycosylation and glycation, lipidation, methylation, acetylation, deamidation, ubiquitination and various redox modifications), and the best available methods for detecting and purifying proteins carrying these PTMs.

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In Vitro and in Vivo Protein-bound Tyrosine Nitration Characterized by Diagonal Chromatography

Cited by 85 publications

References 48 publications

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Identification of Nitrotyrosine Containing Peptides using Combined Fractional Diagonal Chromatography (COFRADIC) and Off-Line Nano-LC-MALDI

Redox Proteomics of Protein-bound Methionine Oxidation

Advances in purification and separation of posttranslationally modified proteins

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