High Collision-induced Dissociation Research Articles

Sensitive β-substituted aliphatic Ile/Val-Xxx and aromatic Phe/Tyr/His-Xxx residues to form [a]+ ions in high energy CID of peptides

Residue-specific cleavages resulting in the formation of [a]+ ions of peptides have been examined using 20 keV high-energy (high-E) collision-induced dissociation (CID) with a tandem time-of-flight (TOF/TOF) instrument. High-E CID resulted in relatively sensitive cleavage at the Cα-C bond of β-substituted aliphatic Val/Ile-Xxx and aromatic Phe/Tyr/His-Xxx residues when an Arg residue was located on the N-terminal side. The sensitive aromatic Phe/Tyr/His residues and insensitive Gly residue in high-E CID are similar to other methods, such as ultraviolet photodissociation (UVPD) of [M+H]+, low-E CID of [M].+ and MALDI-ISD with a hydrogen-abstracting matrix. The properties of the Val/Ile residues are not necessarily common to the same methods. The high-sensitivity of Val/Ile and Phe/Tyr/His residues to high-E CID may be due to high-energy single collision between target gas and the bulky sidechains of these residues. It is suggested that from high-E CID and MALDI-ISD experiments the formation of [a]+ ions is essentially connected with the loss of atomic hydrogen from protonated peptides [M+H]+, and that high-E CID results in the loss of hydrogen from the backbone amide nitrogen rather than the site of the β-carbon of sidechains.

Characteristics of Ion Activation and Collision Induced Dissociation Using Digital Ion Trap Technology.

Collision induced dissociation (CID) is one of the most established techniques for tandem mass spectrometry analysis. The CID of mass selected ion could be realized by ion resonance excitation with a digital rectangular waveform. The method is simple, and highly efficient CID result could be obtained by optimizing the experimental parameters, such as digital waveform voltage, frequency, and q value. In this work, the relationship between ion trapping waveform voltage and frequency at preselected q value, the relationship between waveform frequency and the q value at certain ion trapping voltage for optimum CID efficiency were investigated. Experiment results showed that the max CID efficiency of precursor reserpine ions can be obtained at different trapping waveform voltage and frequency when q and β are different. Based on systematic experimental analysis, the optimum experimental conditions for high CID efficiency can be calculated at any selected β or q. By using digital ion trap technology, the CID process and efficient fragmentation of parent ions can be realized by simply changing the trapping waveform amplitude, frequency, and the β values in the digital ion trap mass spectrometry. The technology and method are simple. It has potential use in ion trap mass spectrometry. Graphical Abstract ᅟ.

Analysis of Methyl-, Chloro-, Bromo- and Trifluoromethyl-Substituted 1,9-Diphenyl-9H-Fluorene and its Isomers by Gas Chromatography-Ion Trap Multistage Tandem Mass Spectrometry

This work presents a modified method to analyze methyL-, chloro-, bromo- and trifluoromethyl-substituted 1,9-diphenyt-9H-fluorene and their isomers by ion trap mass spectrometry with electron impact ionization (EI). Since MS spectra of 1,9-diphenyl-9H-fluorene with these four groups and their isomers are similar, it is difficult to distinguish them from its isomers. Multistage tandem mass spectrometry analysis involves selecting molecular ions obtained in MS spectra as precursor ions in the MS/MS process, and the fragment [C25H17]+ (m/z 317) obtained in MS/MS spectra as a precursor ion in MS3 processes. Collision-induced dissociation (CID) experiments at different activation energies were done to elucidate possible fragmentation pathways. Proposed fragmentation pathways including m/z 317 and structures of the product ions are acquired simultaneously. At a higher CID voltage, the isomers of C25H17-F3 can be distinguished in MS/MS, while the isomers of C25H17-CH3, C25H17-Cl and C25H17-Br can be distinguished in MS3. This work can provide new and valuable information needed for unambiguous characterization of such substances in complex sample matrices.

A Systematic Investigation into the Nature of Tryptic HCD Spectra

Modern mass spectrometry-based proteomics can produce millions of peptide fragmentation spectra, which are automatically identified in databases using sequence-specific b- or y-ions. Proteomics projects have mainly been performed with low resolution collision-induced dissociation (CID) in ion traps and beam-type fragmentation on triple quadrupole and QTOF instruments. Recently, the latter has also become available with Orbitrap instrumentation as higher energy collisional dissociation (HCD), routinely providing full mass range fragmentation with high mass accuracy. To systematically study the nature of HCD spectra, we made use of a large scale data set of tryptic peptides identified with an FDR of 0.0001, from which we extract a subset of more than 16,000 that have little or no contribution from cofragmented precursors. We employed a newly developed computer-assisted "Expert System", which distills our experience and literature knowledge about fragmentation pathways. It aims to automatically annotate the peaks in high mass accuracy fragment spectra while strictly controlling the false discovery rate. Using this Expert System we determined that sequence specific regular ions covering the entire sequence were present for almost all peptides with up to 10 amino acids (median 100%). Peptides up to 20 amino acid length contained sufficient fragmentation to cover 80% of the sequence. Internal fragments are common in HCD spectra but not in high resolution CID spectra (10% vs 1%). The low mass region contains abundant immonium ions (6% of fragment ion intensity), the characteristic a(2), b(2) ion pair (72% of spectra), side chain fragments and reporter ions for peptide modifications such as tyrosine phosphorylation. B- and y-ions account for only 20% of fragment ions by number but 53% by ion intensity. Overall, 84% of the fragment ion intensity was unambiguously explainable. Thus high mass accuracy HCD and CID data are near comprehensively and automatically interpretable.

Partial De Novo Sequencing and Unusual CID Fragmentation of a 7 kDa, Disulfide-Bridged Toxin

A 7 kDa toxin isolated from the venom of the Texas coral snake (Micrurus tener tener) was subjected to collision-induced dissociation (CID) and electron-transfer dissociation (ETD) analyses both before and after reduction at low pH. Manual and automated approaches to de novo sequencing are compared in detail. Manual de novo sequencing utilizing the combination of high accuracy CID and ETD data and an acid-related cleavage yielded the N-terminal half of the sequence from the reduced species. The intact polypeptide, containing 3 disulfide bridges produced a series of unusual fragments in ion trap CID experiments: abundant internal amino acid losses were detected, and also one of the disulfide-linkage positions could be determined from fragments formed by the cleavage of two bonds. In addition, internal and c-type fragments were also observed.

Thiol metabolomics of endothelial cells using capillary liquid chromatography mass spectrometry with isotope coded affinity tags

Thiol and disulfide levels are critical to maintaining the redox potential of a cell. Perturbations of these levels are important in disease pathogenesis. To improve endogenous mammalian metabolome quantitation, thiol specific tagging, extraction and relative quantitation were undertaken. Reduced and oxidized thiol (disulfide) metabolites from endothelial cells were tagged and extracted using cleavable isotope coded affinity tags (cICAT). Extracted cICAT labeled thiols were analyzed using capillary reverse phase liquid chromatography coupled to mass spectrometry (capLC–MS) with positive mode electrospray ionization. Reactions between thiol metabolite standards and the reactive group of cICAT indicate completion by 8 h at pH 9 with no apparent disulfide formation. cICAT labeled reduced thiols from endothelial cells showed 1–5% RSD using ratiometric quantitation of isotopes and 6–17% RSD based on signal intensity alone. Sample injection was optimized to 16 pmol. Using high mass accuracy MS, 75 putative thiol metabolites were detected in all experimental samples. Treatment of endothelial cells with 2,3-dimethoxy-5-methyl-1,4-benzoquinone (BQ) shows decreased levels in 28 putative reduced thiols and increased levels of 27 putative disulfides. Treatment of endothelial cells with 30 mM glucose resulted in 22 putative reduced thiols with decreased levels and 7 putative disulfides with increased concentration. Thiols were identified based on accurate mass within 3 ppm and analysis of fragmentation patterns. Using higher collision induced dissociation (HCD), shared product ions between different thiols led to the analysis of thiols from the cysteine–glutathione (Cys–GSH) pathway. Specific reduced thiols and disulfides in this pathway revealed changes different from the overall trends of thiols/disulfides. This suggests varying regulation of the Cys–GSH pathway distinct from other thiol-containing pathways and dependence on the type of environmental stimulus. These results indicate the utility of analyzing reduced thiols and disulfides in eukaryotic samples.

Characterization of an ion mobility-multiplexed collision-induced dissociation-tandem time-of-flight mass spectrometry approach

The confidence in peptide (and protein) identifications with ion mobility spectrometry time-of-flight mass spectrometry (IMS–TOFMS) is expected to drastically improve with the addition of information from an efficient ion dissociation step prior to MS detection. High throughput IMS–TOFMS analysis imposes a strong need for multiplexed ion dissociation approaches where multiple precursor ions yield complex sets of fragment ions that are often intermingled with each other in both the drift time and m/ z domains. We have developed and evaluated an approach for collision-induced dissociation (CID) using IMS–TOFMS instrument. It has been shown that precursor ions activated inside an rf-device with an axial dc-electric field produce abundant fragment ions which are radially confined with the rf-field and collisionally cooled at an elevated pressure, resulting in high CID efficiencies comparable or higher than those measured in triple-quadrupole instruments. We have also developed an algorithm for deconvoluting these complex multiplexed tandem MS spectra by clustering both the precursor and fragment ions into matching drift time profiles and by utilizing the high mass measurement accuracy achievable with TOFMS. In a single IMS separation from direct infusion of tryptic digest of bovine serum albumin (BSA), we have reliably identified 20 unique peptides using a multiplexed CID approach downstream of the IMS separation. Peptides were identified based upon the correlation between the precursor and fragment drift time profiles and by matching the profile representative masses to those of in silico BSA tryptic peptides and their fragments. The false discovery rate (FDR) of peptide identifications from multiplexed MS/MS spectra was less than 1%.

Experimental investigations of factors controlling the collision induced dissociation spectra of peptide ions in a tandem hybrid mass spectrometer. I. Leucine enkephalin

A systematic study is reported of the experimental parameters controlling the collision induced dissociation (CID) of protonated Leu-enkephalin ( m/z 556.4) in a tandem hybrid mass spectrometer of BEqQ configuration. For CID reactions occurring in the r.f.-only quadrupole q, with collision energies in the range 5–120 eV (laboratory frame), the fragment ion spectra are functions of not only the collision parameters (collision energy, nature of collision gas, etc.) but also of operating characteristics of the qQ quadrupole assembly. The dependence upon the dynamic ion optical parameters was found to be in accord with established principles, given the significantly higher mass of the precursor ion studied here than in previous evaluations of the performance of XqQ tandem instruments (X = Q or sector analyzer). The intensity ratio for the Y″ 2 and B 3 fragment ions was found to be a sensitive index of the degree of internal excitation of the precursor ions, though no calibration of this intensity ratio vs. internal energy was possible. Comparisons between efficiencies of argon and xenon as target gases, as a function of collision energy, were performed objectively via purity-fit scores obtained by using library comparison algorithms. These data are consistent with a proposal that the momentum transfer mechanism of collisional activation operates at the elastic limit for small organic precursor ions, but that physical size restrictions require an increasingly dominant role for the binary (spectator) limit as the precursor mass is increased. It is proposed that this switch could account for the striking difference between very high CID efficiencies obtained for small organic ions, in XqQ instruments, and the increasingly poor performance as precursor mass is raised to 1000 Da or so. The crucial role of pre-collision internal energy, in the low-energy CID of these larger ions, is emphasized.