Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:4.1.1.6 (CAD)
 4,420 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

For proteins of < 20 kDa, this new radical site dissociation method cleaves different and many more backbone bonds than the conventional MS/MS methods (e.g., collisionally activated dissociation, CAD) that add energy directly to the even-electron ions. A minimum kinetic energy difference between the electron and ion maximizes capture; a 1 eV difference reduces capture by 10(3). Thus, in an FTMS ion cell with added electron trapping electrodes, capture appears to be achieved best at the boundary between the potential wells that trap the electrons and ions, now providing 80 +/- 15% precursor ion conversion efficiency. Capture cross section is dependent on the ionic charge squared (z2), minimizing the secondary dissociation of lower charge fragment ions. Electron capture is postulated to occur initially at a protonated site to release an energetic (approximately 6 eV) H. atom that is captured at a high-affinity site such as -S-S- or backbone amide to cause nonergodic (before energy randomization) dissociation. Cleavages between every pair of amino acids in mellitin (2.8 kDa) and ubiquitin (8.6 kDa) are represented in their ECD and CAD spectra, providing complete data for their de novo sequencing. Because posttranslational modifications such as carboxylation, glycosylation, and sulfation are less easily lost in ECD than in CAD, ECD assignments of their sequence positions are far more specific.
 ...
PMID:Electron capture dissociation for structural characterization of multiply charged protein cations. 1069 43

The free radical initiator Vazo 68 is coupled to a peptide and electrosprayed into an ion trap mass spectrometer. On collisional activation, the Vazo 68-peptide conjugate generates a free radical, which can be collisionally activated to cleave the peptide backbone. Mostly z-type fragments are formed, as in CAD of other radical peptides and ECD fragmentation. We present data for the Angiotensin II-Vazo 68 conjugate and discuss possible sites of H atom abstraction from the peptide. This experimental methodology for generating peptide fragments is a useful step toward the development of a completely gas-phase approach to protein sequencing.
 ...
PMID:Bioconjugates for tunable peptide fragmentation: free radical initiated peptide sequencing (FRIPS). 1614 60

The conventional approach in modern proteomics to identify proteins from limited information provided by molecular and fragment masses of their enzymatic degradation products carries an inherent risk of both false positive and false negative identifications. For reliable identification of even known proteins, complete de novo sequencing of their peptides is desired. The main problems of conventional sequencing based on tandem mass spectrometry are incomplete backbone fragmentation and the frequent overlap of fragment masses. In this work, the first proteomics-grade de novo approach is presented, where the above problems are alleviated by the use of complementary fragmentation techniques CAD and ECD. Implementation of a high-current, large-area dispenser cathode as a source of low-energy electrons provided efficient ECD of doubly charged peptides, the most abundant species (65-80%), in a typical trypsin-based proteomics experiment. A new linear de novo algorithm is developed combining efficiency and speed, processing on a conventional 3 GHz PC, 1000 MS/MS data sets in 60 s. More than 6% of all MS/MS data for doubly charged peptides yielded complete sequences, and another 13% gave nearly complete sequences with a maximum gap of two amino acid residues. These figures are comparable with the typical success rates (5-15%) of database identification. For peptides reliably found in the database (Mowse score > or = 34), the agreement with de novo-derived full sequences was >95%. Full sequences were derived in 67% of the cases when full sequence information was present in MS/MS spectra. Thus the new de novo sequencing approach reached the same level of efficiency and reliability as conventional database-identification strategies.
 ...
PMID:Proteomics-grade de novo sequencing approach. 1633 84

A new hybrid electrospray quadrupole Fourier transform mass spectrometry (FTMS) instrument design is shown and characterized. This instrument involves coupling an electrospray source and mass-resolving quadrupole, ion accumulation, and collision cell linear ion trap system developed by MDS Sciex with a home-built ion guide and ion cyclotron resonance (ICR) cell. The iterative progression of this design is shown. The final design involves a set of hexapole ion guides to transfer the ions from the accumulation/collision trap through the magnetic field gradient and into the cell. These hexapole ion guides are separated by a thin gate valve and two conduction limits to maintain the required <10(-9) mbar vacuum for FTICR. Low-attomole detection limits for a pure peptide are shown, 220 000 resolving power in broadband mode and 820 000 resolving power in narrow-band mode are demonstrated, and mass accuracy in the <2 ppm range is routinely available provided the signal is abundant, cleanly resolved, and internally calibrated. This instrument design provides high experimental flexibility, allowing Q2 CAD, SORI-CAD, IRMPD, and ECD experiments with selected ion accumulation as well as experiments such as nozzle skimmer dissociation. Initial top-down mass spectrometry experiments on a protein is shown using ECD.
 ...
PMID:A new hybrid electrospray Fourier transform mass spectrometer: design and performance characteristics. 1635 30

The c-subunit of ATP synthase (AtpH) is an 8 kD integral membrane protein with two transmembrane domains; we set out to demonstrate it amenable to top-down electrospray-ionization Fourier-transform mass spectrometry (FT-MS) using both collision activated and electron capture dissociation (CAD/ECD). Thermal activation concomitant with electron delivery was necessary for efficient ECD (activated-ion ECD; aiECD), yielding complementary information and greater sequence coverage in the transmembrane domains in comparison with CAD.
 ...
PMID:Increased coverage in the transmembrane domain with activated-ion electron capture dissociation for top-down Fourier-transform mass spectrometry of integral membrane proteins. 1744 48

Small neutral losses from charge-reduced species [M + nH] (( n-1)+* ) is one of the most abundant fragmentation channels in both electron capture dissociation, ECD, and electron transfer dissociation, ETD. Several groups have previously studied these losses on particular examples. Now, the availability of a large (11 491 entries) SwedECD database ( http://www.bmms.uu.se/CAD/indexECD.html) of high-resolution ECD data sets on doubly charged tryptic peptides has made possible a systematic study involving statistical evaluation of neutral losses from [M + 2H] (+ * ) ions. Several new types of losses are discovered, and 16 specific (>94%) losses are characterized according to their specificity and sensitivity, as well as occurrence for peptides of different lengths. On average, there is more than one specific loss per ECD mass spectrum, and two-thirds of all MS/MS data sets in SwedECD contain at least one specific loss. Therefore, specific neutral losses are analytically useful for improved database searching and de novo sequencing. In particular, N and GG isomeric sequences can be distinguished. The pattern of neutral losses was found to be remarkably dissimilar with the losses from radical z* fragment ions: e.g., there is no direct formation of w ions from the reduced species. This finding emphasizes the difference in fragmentation behaviors of hydrogen-abundant and hydrogen-deficient species.
 ...
PMID:Analytical utility of small neutral losses from reduced species in electron capture dissociation studied using SwedECD database. 1883 16

High-performance liquid chromatography (HPLC) is a very powerful and versatile chromatographic technique for the separation of natural products (NPs) in complex matrices, such as crude extracts for selective detection and quantification or general profiling. The method is widespread and has been adapted to the analysis of a broad range of NPs generally without the need for complex sample preparation. The choice of the appropriate detection method in HPLC is crucial because of the diversity of NPs and the fact that there is no single technique for their efficient detection. In this review both qualitative and quantitative applications of HPLC with UV, DAD, FD, ECD, RID, FID, CL, ESLD, CAD, MS, MS-MS, and NMR are covered to provide a general, rather than an exhaustive, overview. The potential and limitations as well as some new trends in HPLC hyphenation are discussed.
 ...
PMID:HPLC in natural product analysis: the detection issue. 1914 52

Mass spectrometry faces considerable difficulties in de novo sequencing of long non-tryptic peptides with S-S bonds. Long disulfide-containing peptides brevinins 1E and 2Ec from frog Rana ridibunda were reduced and alkylated with nine novel and three known derivatizing agents. Eight of the novel reagents are maleimide derivatives. Modified samples were subjected to MS/MS studies on FT-ICR and Orbitrap mass spectrometers using CAD/HCD or ECD/ETD techniques. Procedures, fragmentation patterns, and sequence coverage for two peptides modified with 12 tags are described. ECD/ETD and CAD fragmentation revealed complementary sequence information. Higher-energy collisionally activated dissociation (HCD) sufficiently enhanced y-ions formation for brevinin 1E, but not for brevinin 2Ec. Some novel tags [N-benzylmaleimide, N-(2,6-dimethylphenyl)maleimide] along with known N-phenylmaleimide and iodoacetic acid showed high total sequence coverage taking into account combined ETD and HCD fragmentation. Moreover, modification of long (34 residues) brevinin 2Ec with N-benzylmaleimide or N-(2,6-dimethylphenyl)maleimide yielded high sequence coverage and full C-terminal sequence determination with ECD alone.
 ...
PMID:Novel cysteine tags for the sequencing of non-tryptic disulfide peptides of anurans: ESI-MS study of fragmentation efficiency. 2197 73

Integral membrane proteins pose challenges to traditional proteomics approaches due to unique physicochemical properties including hydrophobic transmembrane domains that limit solubility in aqueous solvents. A well resolved intact protein molecular mass profile defines a protein's native covalent state including post-translational modifications, and is thus a vital measurement toward full structure determination. Both soluble loop regions and transmembrane regions potentially contain post-translational modifications that must be characterized if the covalent primary structure of a membrane protein is to be defined. This goal has been achieved using electrospray-ionization mass spectrometry (ESI-MS) with low-resolution mass analyzers for intact protein profiling, and high-resolution instruments for top-down experiments, toward complete covalent primary structure information. In top-down, the intact protein profile is supplemented by gas-phase fragmentation of the intact protein, including its transmembrane regions, using collisionally activated and/or electron-capture dissociation (CAD/ECD) to yield sequence-dependent high-resolution MS information. Dedicated liquid chromatography systems with aqueous/organic solvent mixtures were developed allowing us to demonstrate that polytopic integral membrane proteins are amenable to ESI-MS analysis, including top-down measurements. Covalent post-translational modifications are localized regardless of their position in transmembrane domains. Top-down measurements provide a more detail oriented high-resolution description of post-transcriptional and post-translational diversity for enhanced understanding beyond genomic translation.
 ...
PMID:Profiling of integral membrane proteins and their post translational modifications using high-resolution mass spectrometry. 2198 82