Selective detection and identification of phosphopeptides by dansyl MS/MS/MS fragmentation

Amoresano, Angela; Monti, Gianluca; Cirulli, Claudia; Marino, Gennaro

doi:10.1002/rcm.2461

Cited by 25 publications

(18 citation statements)

References 11 publications

(15 reference statements)

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“…A proposed method for detection of carbonyl groups uses biotinhydrazide for carbonyl labeling; labeled proteins can be analyzed exploiting the specific interaction biotin‐avidin; this strategy has been used in combination with an affinity chromatography step and mass spectrometric analysis to identify specific sites of carbonylation in proteins 25–31…”

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Dansyl labeling and bidimensional mass spectrometry to investigate protein carbonylation

Palmese

Rosa

Marino

et al. 2010

Rapid Comm Mass Spectrometry

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Carbonylation is a non-enzymatic irreversible post-translational modification. The adduction of carbonyl groups to proteins is due to the presence of excess of ROS in cells. Carbonylation of specific amino acid side chains is one of the most abundant consequences of oxidative stress; therefore, the determination of carbonyl groups content in proteins is regarded as a reliable way to estimate the cellular damage caused by oxidative stress. This paper reports a novel RIGhT (Reporter Ion Generating Tag) (A. Amoresano, G. Monti, C. Cirulli, G. Marino. Rapid Commun. Mass Spectrom. 2006, 20, 1400) approach for selective labeling of carbonyl groups in proteins using dansylhydrazide, coupled with selective analysis by bidimensional mass spectrometry. We first applied this approach to ribonuclease A and lysozyme as model proteins. According to the so-called 'gel-free procedures', the analysis is carried out at the level of peptides following tryptic digest of the whole protein mixture. Modified RNaseA was analyzed in combined MS(2) and MS(3) scan mode, to specifically select the dansylated species taking advantage of the dansyl-specific fragmentation pathways. This combination allowed us to obtain a significant increase in signal/noise ratio and a significant increase in sensitivity of analysis, due to the reduction of duty cycle of the mass spectrometer. The unique signal obtained was correlated to peptide 1-10 of RNaseA carbonylated and labeled by dansylhydrazide. This strategy represents the first method leading to the direct identification of the carbonylation sites in proteins, thus indicating the feasibility of this strategy to investigate protein carbonylation in a proteomic approach.

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Dansyl labeling and bidimensional mass spectrometry to investigate protein carbonylation

Palmese

Rosa

Marino

et al. 2010

Rapid Comm Mass Spectrometry

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“…Using this method, Kanski et al have shown that nitration appears to increase in metabolic proteins during cardiac aging. Amoresano et al (11) have developed an assay called reporter ion generation tag (RIGhT) for the detection of nitrotyrosine-containing proteins (10). This method involves the selective modification of nitrotyrosine to dansylaminotyrosine using dansyl chloride and then monitoring proteins by generating specific ions that are unique to dansyl derivatives by MS. Another quantitative proteomics approach to detect 3-nitrotyrosine developed by Sharov et al (255) is based on the selective reduction of 3-nitrotyrosine to 3-aminotyrosine.…”

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Cardiovascular Redox and Ox Stress Proteomics

Kumar

Calamaras

Haeussler

et al. 2012

Antioxidants & Redox Signaling

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Significance: Oxidative post-translational modifications (OPTMs) have been demonstrated as contributing to cardiovascular physiology and pathophysiology. These modifications have been identified using antibodies as well as advanced proteomic methods, and the functional importance of each is beginning to be understood using transgenic and gene deletion animal models. Given that OPTMs are involved in cardiovascular pathology, the use of these modifications as biomarkers and predictors of disease has significant therapeutic potential. Adequate understanding of the chemistry of the OPTMs is necessary to determine what may occur in vivo and which modifications would best serve as biomarkers. Recent Advances: By using mass spectrometry, advanced labeling techniques, and antibody identification, OPTMs have become accessible to a larger proportion of the scientific community. Advancements in instrumentation, database search algorithms, and processing speed have allowed MS to fully expand on the proteome of OPTMs. In addition, the role of enzymatically reversible OPTMs has been further clarified in preclinical models. Critical Issues: The identification of OPTMs suffers from limitations in analytic detection based on the methodology, instrumentation, sample complexity, and bioinformatics. Currently, each type of OPTM requires a specific strategy for identification, and generalized approaches result in an incomplete assessment. Future Directions: Novel types of highly sensitive MS instrumentation that allow for improved separation and detection of modified proteins and peptides have been crucial in the discovery of OPTMs and biomarkers. To further advance the identification of relevant OPTMs in advanced search algorithms, standardized methods for sample processing and depository of MS data will be required. Antioxid. Redox Signal. 17, 1528-1559.

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“…Dansyl chloride (DNS‐Cl) is a fluorescent reagent which is widely used in biochemistry to modify the primary amine group of proteins and peptides for N‐terminal sequencing14 and in selective proteomics to perform advanced MS analyses 15–17. The synthesis of the 13 C heavy labeled form of DNS‐Cl allowed also relative quantitation in metabolomics to be performed 18…”

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Dansyl‐peptides matrix‐assisted laser desorption/ionization mass spectrometric (MALDI‐MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI‐MS/MS analysis of the proteome

Chiappetta

Ndiaye

Demey

et al. 2010

Rapid Comm Mass Spectrometry

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Peptide tagging is a useful tool to improve matrix-assisted laser desorption/ionization tandem mass spectrometric (MALDI-MS/MS) analysis. We present a new application of the use of the dansyl chloride (DNS-Cl). DNS-Cl is a specific primary amine reagent widely used in protein biochemistry. It adds a fluorescent dimethylaminonaphthalene moiety to the molecule. The evaluation of MALDI-MS and MS/MS analyses of dansylated peptides shows that dansylation raises the ionization efficiency of the most hydrophilic species compared with the most hydrophobic ones. Consequently, higher Mascot scores and protein sequence coverage are obtained by combining MS and MS/MS data of native and tagged samples. The N-terminal DNS-Cl sulfonation improves the peptide fragmentation and promotes the generation of b-fragments allowing better peptide sequencing. In addition, we set up a labeling protocol based on the microwave chemistry. Peptide dansylation proved to be a rapid and cheap method to improve the performance of liquid chromatography (LC)/MALDI-MS/MS analysis at the proteomic scale in terms of peptide detection and sequence coverage.

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Selective detection and identification of phosphopeptides by dansyl MS/MS/MS fragmentation

Cited by 25 publications

References 11 publications

Dansyl labeling and bidimensional mass spectrometry to investigate protein carbonylation

Dansyl labeling and bidimensional mass spectrometry to investigate protein carbonylation

Cardiovascular Redox and Ox Stress Proteomics

Dansyl‐peptides matrix‐assisted laser desorption/ionization mass spectrometric (MALDI‐MS) and tandem mass spectrometric (MS/MS) features improve the liquid chromatography/MALDI‐MS/MS analysis of the proteome

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