Characterization of Isomeric VX Nerve Agent Adducts on Albumin in Human Plasma Using Liquid Chromatography–Tandem Mass Spectrometry
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This study includes the characterization of isomeric VX organophosphorus adducts on albumin in human plasma using liquid chromatography-tandem mass spectrometry (LC-MS/MS). VX or its structural isomers were spiked into a vial containing plasma in order to obtain phosphorylated albumin. After pronase and trypsin digestion, the resulting solutions were analyzed to confirm adduct formation with the amino acid tyrosine on the albumin in human plasma. The LC-MS/MS experiments show that VX and its isomers can be attached to tyrosine on the albumin in human blood. Mass spectrometric studies revealed some interesting fragmentation pathways during the ionization process, such as ethylene, formic acid and ammonia elimination and an intermolecular electrophilic aromatic substitution reaction. The proposed mechanisms for the formation of the fragments were confirmed through the analysis of fragments of deuterated adducts.Keywords:
Human serum albumin
Fragmentation
Isobaric labeling
Tandem mass tag
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Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.
Bottom-up proteomics
Top-down proteomics
Tandem mass tag
Isobaric labeling
Peptide mass fingerprinting
Capillary electrophoresis–mass spectrometry
Fragmentation
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A recently developed methodology for the characterization of complex proteomes, top-down Fourier transform mass spectrometry (FTMS), is applied for the first time to a plant proteome, that of the model plant Arabidopsis thaliana. Of the 3000 proteins predicted by the genome sequence, 97 were recently identified in two separate "bottom-up" mass spectrometry studies in which the proteins were purified and digested and in which the mass spectrometry-measured mass values of the resulting peptides matched against those expected from the DNA-predicted proteins. In the top-down approach applied here, molecular ions from a protein mixture are purified, weighed exactly (+/-1 Da), and fragmented in the FTMS. Of the 22 molecular weight values found in three isolated mixtures, 7 were chosen, and their primary structures were fully characterized; in only one case was the bottom-up structure in full agreement. The top-down technique is not only efficient for identification of the DNA-predicted precursors, such as that of a protein present as a 5% mixture component, but also for characterization of the primary structure of the final protein. For two proteins the previously predicted cleavage site for loss of the signal peptide was found to be incorrect. Two 27-kDa proteins are fully characterized, although they are found to differ by only 12 residues and 6 Da in mass in a 3:1 ratio; the bottom-up studies did not distinguish these proteins. Direct tandem mass spectrometry dissociation of two 15-kDa molecular ions showed >90% sequence similarity, whereas three-stage mass spectrometry traced their +14-Da molecular mass discrepancies to an unusual N-methylation on the N-terminal amino group; the bottom-up approach identified only one precursor protein. The high potential of the top-down FTMS approach for characterization as well as identification of complex plant proteomes should provide a real incentive for its further automation.
Top-down proteomics
Proteome
Bottom-up proteomics
Tandem mass tag
Isobaric labeling
Molecular mass
Peptide mass fingerprinting
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This chapter contains sections titled: Introduction Electrospray Ionization Tandem Mass Spectrometry Q–TOF and Q–FT–ICR Systems Resolution Mass Accuracy Peptide Sequencing by Electrospray Tandem Mass Spectrometry Protein Modifications and their MS/MS Reactions Detection of Protein Modifications by MS and MS/MS Phosphorylation Tyrosine Sulfation Redox-related Modifications Myristoylation Acetylation Methylation Glycosylation Ubiquitination Isoaspartate Formation Summary and Outlook References
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Abstract In those cases where the information obtained by peptide mass fingerprinting or matrix‐assisted laser desorption/ionization tandem mass spectrometry (MALDI‐MS/MS) is not sufficient for unambiguous protein identification, nano‐electrospray ionization (nano‐ESI) and/or electrospray ionization tandem mass spectrometry (ESI‐MS/MS) analysis must be performed. The sensitivity of nano‐ESI/MS, however, is lower than that of MALDI‐MS, especially at very low analyte concentrations and/or in the presence of contaminants, such as salt and detergents. Moreover, to perform ESI‐MS/MS, the peptide masses of the precursor ions must be known. The approach described in this paper, MALDI‐directed nano‐ESI‐MS/MS, makes use of information obtained from the more sensitive MALDI‐MS experiments in order to direct subsequent nano‐ESI‐MS/MS experiments. Peptide molecular ions found in the MALDI‐MS analysis are then selected, as their (+2) precursor ions, for nano‐ESI‐MS/MS sequencing, even though these ions cannot be detected in the ESI‐MS spectra. This method, originally proposed by Tempst et al . ( Anal. Chem. 2000, 72: 777–790), has been extended to provide better sensitivity and shorter analysis times; also, a comparison with liquid chromatography/tandem mass spectrometry (LC/MS/MS) has been performed. These experiments, performed using quadrupole time‐of‐flight instruments equipped with commercially available nano‐ESI sources, have allowed the unambiguous identification of in‐gel digested proteins at levels below their ESI‐MS detection limits, even in the presence of salts and detergents. Copyright © 2003 John Wiley & Sons, Ltd.
Desorption electrospray ionization
Top-down proteomics
Capillary electrophoresis–mass spectrometry
Extractive electrospray ionization
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Phenolphthalein
Sibutramine
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Abstract The molecular ion of intact β‐endorphin bovine, 1–31 (BE), which was extracted from bovine pituitary, was determined by electrospray ionization mass spectrometry. Liquid secondary ion mass spectrometry determined the molecular masses of three peptides produced by trypsin digestion of BE, and tandem mass spectrometry was used to determine the amino acid sequence of the tryptic peptide BE 20–24 . These data, in combination, were used to characterize BE in bovine pituitary.
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Top-down proteomics
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Tandem mass spectrometry has been used to obtain information related to portions of the primary sequence for an intact protein, bovine ribonuclease A. Multiply charged molecular ions, generated by electrospray ionization, were collisionally dissociated at low energies in a triple quadrupole mass spectrometer to yield singly and multiply charged fragment ions that can be assigned to the known sequence of the protein. Dissociation of the highly charged molecular ions resulted in pairs of complementary product ions. The higher order (gas-phase) protein structure affects the dissociation processes, as observed in comparisons of tandem mass spectra of the native and disulfide-reduced forms of ribonuclease A.
Top-down proteomics
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We investigated the effect of N-terminal amino group and carboxyl group methylation on peptide analysis by electrospray mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Permethylation of the N-terminal amino group and the carboxyl groups can reduce metal ion adducts but does not enhance sensitivity in electrospray as previously observed for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. N-terminal trimethylated peptides exhibit collision-induced dissociation (CID) tandem mass spectra that differ from their unmodified analogs; the results support the mobile proton hypothesis of peptide fragmentation. A permanent positive charge at the N-terminus leads to competition between permanent-charge directed processes and loss of the N-terminal trimethyl amino group. Carboxyl methylation has no effect on fragmentation behavior other than to shift the mass of fragments containing methylated carboxyl groups. Comparison of regular and tandem mass spectra of different methylated peptides allowed probing the location of incomplete methylation, the proton displaced by alkali metal ions and the purity of a mass-selected methylated peptide ion.
Collision-induced dissociation
Fragmentation
Fast atom bombardment
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An Introduction to Protein Sequencing Using Tandem Mass Spectrometry The Primary Structure of Proteins and a Historical Overview of Protein Sequencing Fundamental Mass Spectrometry Collisionally Induced Dissociation of Protonated Peptide Ions and the Interpretation of Product Ion Spectra Basic Polyacrylamide Gel Electrophoresis The Preparation of Protein Digests for Mass Spectrometric Sequencing Experiments Mass Spectrometric Analysis of Tryptic Digests Protein Identification by Database Searching Sequence Analysis of Novel Proteins The Characterization of Post-Translationally Modified Proteins Using Tandem Mass Spectrometry Index.
Top-down proteomics
Tandem mass tag
Peptide mass fingerprinting
Isobaric labeling
Bottom-up proteomics
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