12 results on '"Wang, Dongxia"'
Search Results
2. Identification of protein ubiquitylation by electrospray ionization tandem mass spectrometric analysis of sulfonated tryptic peptides
- Author
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Wang, Dongxia, Kalume, Dario, Pickart, Cecile, Pandey, Akhilesh, and Cotter, Robert J.
- Subjects
Mass spectrometry -- Usage ,Amino acid sequence -- Analysis ,Cellular proteins -- Structure ,Cellular proteins -- Chemical properties ,Ionization -- Analysis ,Chemistry - Abstract
We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carded out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitinconjugated proteins using IC/MS/MS.
- Published
- 2006
3. Approach for determining protein ubiquitination sites by MALDI-TOF mass spectrometry
- Author
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Wang, Dongxia and Cotter, Robert J.
- Subjects
Ubiquitin -- Research ,Chemistry, Analytic -- Research ,Proteins -- Research ,Chemistry - Abstract
Protein ubiquitination plays an important role in the degradation and other functional regulation of cellular proteins in organisms ranging from yeasts to mammals. Trypsin digestion of ubiquitin conjugated proteins produces diglycine branched peptides in which the C-terminal Gly-Gly fragment of ubiquitin is attached to the [epsilon]-amino group of a modified lysine residue within the peptide. This provides a platform for mapping ubiquitination sites using mass spectrometry. Here we report the development of a novel strategy for determining post-traslational protein ubiquitination based on the N-terminal sulfonation of diglycine branched peptides. In contrast to conventional tandem MS spectra of native tryptic peptides, MALDI MS/MS analysis of a sulfonated tryptic peptide containing a diglycine branch generates a unique spectrum composed of a signature portion and a sequence portion. The signature portion of the spectrum consists of several intense ions resulting from the elimination of the tags, the N-terminal residues at the peptide and the branch, and their combination. This unique ion distribution pattern can distinguish ubiquitination modificatons from others and can identify the first N-terminal residues of the peptides as well. The sequence portion consists of an exclusive series of y-type ions and y' ions (differing by the loss of one glycine residue from the sulfonated diglycine branch) that can directly reveal the amino acid sequence of the peptide and the precise location of the ubiquitination site. The technique is demonstrated for a series of synthetic peptides and is validated by a model protein, tetraubiquitin. Our results show that the MALDI MS/MS analysis of sulfonated tryptic pepfides can provide a highly effective method for the determination of ubiquitination substrates, ubiquitination sites on protein targets, and modification sites on ubiquitins themselves.
- Published
- 2005
4. Protein tyrosine kinase Csk-catalyzed phosphorylation of Src containing unnatural tyrosine analogues
- Author
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Wang, Dongxia and Cole, Philip A.
- Subjects
Phosphorylation -- Observations ,Tyrosine -- Research ,Chemistry - Abstract
The incorporation of five unnatural tyrosine analogues into Src instead of natural tail tyrosine residue was undertaken using expressed protein ligation, and the substrates were determine as Csk substrates.
- Published
- 2001
5. Molecular determinants for Csk-catalyzed tyrosine phosphorylation of the Src tail
- Author
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Wang, Dongxia, Huang, Xin-Yun, and Cole, Philip A.
- Subjects
Biochemistry -- Research ,Molecules -- Research ,Tyrosine -- Physiological aspects ,Phosphorylation -- Physiological aspects ,Proteins -- Research ,Gene mutations -- Physiological aspects ,Biological sciences ,Chemistry - Abstract
Research has been conducted on the critical tail tyrosine residue phosphorylation in Src. The use of the purified recombinant Csk and Src proteins and mutants in investigating this phosphorylation event is discussed.
- Published
- 2001
6. Comprehensive Analysis of the Glycan Complement of SARS-CoV‑2 Spike Proteins Using Signature Ions-Triggered Electron-Transfer/Higher-Energy Collisional Dissociation (EThcD) Mass Spectrometry.
- Author
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Wang, Dongxia, Baudys, Jakub, Bundy, Jonathan L., Solano, Maria, Keppel, Theodore, and Barr, John R.
- Published
- 2020
- Full Text
- View/download PDF
7. SnO 2 @C@VO 2 Composite Hollow Nanospheres as an Anode Material for Lithium-Ion Batteries.
- Author
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Guo W, Wang Y, Li Q, Wang D, Zhang F, Yang Y, and Yu Y
- Abstract
Porous SnO
2 @C@VO2 composite hollow nanospheres were ingeniously constructed through the combination of layer-by-layer deposition and redox reaction. Moreover, to optimize the electrochemical properties, SnO2 @C@VO2 composite hollow nanospheres with different contents of the external VO2 were also studied. On the one hand, the elastic and conductive carbon as interlayer in the SnO2 @C@VO2 composite can not only buffer the huge volume variation during repetitive cycling but also effectively improve electronic conductivity and enhance the utilizing rate of SnO2 and VO2 with high theoretical capacity. On the other hand, hollow nanostructures of the composite can be consolidated by the multilayered nanocomponents, resulting in outstanding cyclic stability. In virtue of the above synergetic contribution from individual components, SnO2 @C@VO2 composite hollow nanospheres exhibit a large initial discharge capacity (1305.6 mAhg-1 ) and outstanding cyclic stability (765.1 mAhg-1 after 100 cycles). This design of composite hollow nanospheres may be extended to the synthesis of other nanomaterials for electrochemical energy storage.- Published
- 2018
- Full Text
- View/download PDF
8. Improved Sensitivity for the Qualitative and Quantitative Analysis of Active Ricin by MALDI-TOF Mass Spectrometry.
- Author
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Wang D, Baudys J, Barr JR, and Kalb SR
- Abstract
Ricin is a highly toxic protein which causes cell death by blocking protein synthesis and is considered a potential bioterrorism agent. Rapid and sensitive detection of ricin toxin in various types of sample matrices is needed as an emergency requirement for public health and antibioterrorism response. An in vitro MALDI TOF MS-based activity assay that detects ricin mediated depurination of synthetic substrates was improved through optimization of the substrate, reaction conditions, and sample preparation. In this method, the ricin is captured by a specific polycolonal antibody followed by hydrolysis reaction. The ricin activity is determined by detecting the unique cleavage product of synthetic oligomer substrates. The detection of a depurinated substrate was enhanced by using a more efficient RNA substrate and optimizing buffer components, pH, and reaction temperature. In addition, the factors involved in mass spectrometry analysis, such as MALDI matrix, plate, and sample preparation, were also investigated to improve the ionization of the depurinated product and assay reproducibility. With optimized parameters, the limit of detection of 0.2 ng/mL of ricin spiked in buffer and milk was accomplished, representing more than 2 orders of magnitude enhancement in assay sensitivity. Improving assay's ruggeddness or reproducibility also made it possible to quantitatively detect active ricin with 3 orders of magnitude dynamic range.
- Published
- 2016
- Full Text
- View/download PDF
9. A two-stage multiplex method for quantitative analysis of botulinum neurotoxins type A, B, E, and F by MALDI-TOF mass spectrometry.
- Author
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Wang D, Baudys J, Krilich J, Smith TJ, Barr JR, and Kalb SR
- Subjects
- Botulinum Toxins classification, Botulinum Toxins analysis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods
- Abstract
In this publication, we report on the development of a quantitative enzymatic method for the detection of four botulinum neurotoxin (BoNT) serotypes responsible for human botulism by MALDI-TOF mass spectrometry. Factors that might affect the linearity and dynamic range for detection of BoNT cleavage products were initially examined, including the amount of peptide substrate and internal standard, the timing of cleavage reaction, and the components in the reaction solution. It was found that a long incubation time produced sensitive results, but was not capable of determining higher toxin concentrations, whereas a short incubation time was less sensitive so that lower toxin concentrations were not detected. In order to overcome these limitations, a two-stage analysis strategy was applied. The first stage analysis involved a short incubation period (e.g., 30 min). If no toxin was detected at this stage, the cleavage reaction was allowed to continue and the samples were analyzed at a second time point (4 h), so that toxin levels lower than 1 mouse LD50 or 55 attomoles per milliliter (55 amol/mL) could be quantified. By combining the results from two-stage quantification, 4 or 5 orders of magnitude in dynamic range were achieved for the detection of the serotypes of BoNT/A, BoNT/B, BoNT/E, or BoNT/F. The effect of multiplexing the assay by mixing substrates for different BoNT serotypes into a single reaction was also investigated in order to reduce the numbers of the cleavage reactions and save valuable clinical samples.
- Published
- 2014
- Full Text
- View/download PDF
10. Subtyping botulinum neurotoxins by sequential multiple endoproteases in-gel digestion coupled with mass spectrometry.
- Author
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Wang D, Baudys J, Rees J, Marshall KM, Kalb SR, Parks BA, Nowaczyk L 2nd, Pirkle JL, and Barr JR
- Subjects
- Amino Acid Sequence, Botulinum Toxins chemistry, Botulinum Toxins classification, Chromatography, Liquid, Endopeptidases metabolism, Gels, Guanidine chemistry, Molecular Sequence Data, Neurotoxins chemistry, Neurotoxins classification, Proteomics, Sequence Alignment, Tandem Mass Spectrometry, Botulinum Toxins isolation & purification, Clostridium botulinum chemistry, Endopeptidases chemistry, Molecular Typing methods, Neurotoxins isolation & purification
- Abstract
Botulinum neurotoxin (BoNT) is one of the most toxic substances known. BoNT is classified into seven distinct serotypes labeled A-G. Among individual serotypes, researchers have identified subtypes based on amino acid variability within a serotype and toxin variants with minor amino acid sequence differences within a subtype. BoNT subtype identification is valuable for tracing and tracking bacterial pathogens. A proteomics approach is useful for BoNT subtyping since botulism is caused by botulinum neurotoxin and does not require the presence of the bacteria or its DNA. Enzymatic digestion and peptide identification using tandem mass spectrometry determines toxin protein sequences. However, with the conventional one-step digestion method, producing sufficient numbers of detectable peptides to cover the entire protein sequence is difficult, and incomplete sequence coverage results in uncertainty in distinguishing BoNT subtypes and toxin variants because of high sequence similarity. We report here a method of multiple enzymes and sequential in-gel digestion (MESID) to characterize the BoNT protein sequence. Complementary peptide detection from toxin digestions has yielded near-complete sequence coverage for all seven BoNT serotypes. Application of the method to a BoNT-contaminated carrot juice sample resulted in the identification of 98.4% protein sequence which led to a confident determination of the toxin subtype.
- Published
- 2012
- Full Text
- View/download PDF
11. Multiple roles for acetylation in the interaction of p300 HAT with ATF-2.
- Author
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Karanam B, Wang L, Wang D, Liu X, Marmorstein R, Cotter R, and Cole PA
- Subjects
- Acetylation, Activating Transcription Factor 2 chemistry, Amino Acid Sequence, Animals, COS Cells, Cell Cycle Proteins chemistry, Chlorocebus aethiops, Histone Acetyltransferases chemistry, Humans, Leucine Zippers, Molecular Sequence Data, Protein Structure, Tertiary, Transcription Factors chemistry, Transcription, Genetic, p300-CBP Transcription Factors, Activating Transcription Factor 2 metabolism, Cell Cycle Proteins metabolism, Histone Acetyltransferases metabolism, Transcription Factors metabolism
- Abstract
The transcriptional coactivator paralogues p300 and CBP contain acetyltransferase domains (HAT) and catalyze the lysine acetylation of histones and other proteins as an important aspect of their functions. Prior studies revealed that the basic leucine zipper domain (b-ZIP) of transcription factor ATF-2 (also called CRE-BP1) can interact with the CBP HAT domain. In this study, we have examined the ATF-2 b-ZIP interaction with the p300 HAT domain and shown that p300 HAT autoacetylation can enhance the binding affinity. Pull-down assays revealed that hyperacetylated p300 HAT is more efficiently retained by immobilized ATF-2 b-ZIP than hypoacetylated p300 HAT. Loop deleted p300 HAT lacking autoacetylation was retained about as well as hyperacetylated p300 HAT, suggesting that the loop and ATF-2 compete for p300 HAT binding. While ATF-2 b-ZIP is a weak inhibitor of hypoacetylated p300 HAT acetylation of a histone H4 peptide, hyperacetylated p300 HAT is much more potently inhibited by ATF-2 b-ZIP. Moreover, we showed that ATF-2 b-ZIP could serve as an acetyltransferase substrate for p300 HAT. Using mass spectrometry, two p300 HAT lysine acetylation sites were mapped in ATF-2 b-ZIP. Immunoprecipitation-Western blot analysis with anti-acetyl-lysine antibody revealed that ATF-2 can undergo reversible acetylation in vivo. Mutational analysis of the two ATF-2 b-ZIP acetylation sites revealed their potential contributions to ATF-2-mediated transcriptional activation. Taken together, these studies suggest multiple roles for protein acetylation in the regulation of transcription by p300/CBP and ATF-2.
- Published
- 2007
- Full Text
- View/download PDF
12. Direct identification of ubiquitination sites on ubiquitin-conjugated CHIP using MALDI mass spectrometry.
- Author
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Wang D, Xu W, McGrath SC, Patterson C, Neckers L, and Cotter RJ
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Chromatography, High Pressure Liquid, Glycine chemistry, Guanidine chemistry, Ions, Mass Spectrometry, Molecular Sequence Data, Peptides chemistry, Protein Binding, Protein Structure, Tertiary, Rabbits, Trypsin chemistry, Trypsin pharmacology, Drosophila Proteins chemistry, Nuclear Proteins chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Ubiquitin chemistry
- Abstract
The study of protein ubiquitination, a post-translational modification by ubiquitin, has emerged as one of the most active areas in biology because of the important role of this type of modification on the regulation of various cellular proteins. Advances in techniques for the determination and site mapping of protein ubiquitination can facilitate the elucidation of molecular mechanisms of this modification. We have recently described a novel method for identifying peptides containing ubiquitinated amino acid residues, based on the MALDI-MS/MS analysis of tryptic peptide derivatives. In particular, we have utilized N-terminal sulfonation of these peptides to provide a unique fragmentation pattern that leads to the direct identification and sequencing of ubiquitin modified peptides. Here we present an application of this new method on the characterization of ubiquitin conjugated C-terminal Hsc70-interacting protein (CHIP), a recently identified U-box containing E3 enzyme. Three peptides bearing ubiquitination sites have been identified from the digest of ubiquitinated CHIP; one of these was a site on CHIP, while the other two were found on the ubiquitin molecules, demonstrating that sulfonation of tryptic peptides is a general and efficient method for characterizing protein ubiquitination.
- Published
- 2005
- Full Text
- View/download PDF
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