Mass spectrometry最新文献

Isotope labeling measurements using mass spectrometry can provide informative insights on the metabolic systems of various organisms. The detailed identification of carbon positions included in the fragment ions of dicarboxylic and tricarboxylic acids in central carbon metabolism is needed for precise interpretation of the metabolic states. In this study, fragment ions containing the carbon backbone cleavage of dicarboxylic and tricarboxylic in the Krebs cycle were investigated by using gas chromatography (GC)-electron ionization (EI)-MS and GC-EI-MS/MS. The positions of decarboxylation in the dicarboxylic and tricarboxylic acids were successfully identified by analyses using position-specific ¹³C-labeled standards prepared by in vitro enzymatic reactions. For example, carboxyl groups of C1 and C6 of trimethylsilyl (TMS)- and tert-butyldimethylsilyl (TBDMS)-derivatized malic and citric acids were primarily cleaved by EI. MS/MS analyses were also performed, and fragment ions of TBDMS-citric and α-ketoglutaric acids (αKG) with the loss of two carboxyl groups in collision-induced dissociation (CID) were observed.

Typical modes of bond cleavages of organic compounds in mass spectrometry are briefly summarized. Although these fragmentation rules can be quite useful for interpreting mass spectra of simple compounds, application to structurally complex molecules that contain multiple hetero atoms such as nitrogen or oxygen becomes increasingly difficult, because the exact location of an unpaired electron or positive or negative charges becomes obscure in precursor ions. About a decade ago, we proposed "a rule of mass shift," which correctly predicts the m/z for observed peaks corresponding to singly charged even-electron fragment ions. The basis of the rule postulates that ions observed as peaks in an ordinary mass spectrum should be sufficiently stable to survive during the flight path in a mass spectrometer. The important recognition is that each atom in a stable ion should be in an ordinary valence state, and no free valence should be allowed. Therefore, if the cleavage of a bond leads to an ion with an unstable structure, some structural changes must take place in order for the ion to be observed in the mass spectrum. Such structural changes can be the addition of hydrogen atom(s) and/or a proton for positive ions, and the addition of a hydrogen atom and/or the elimination of two hydrogen atoms in the case of negative ions. These required structural changes in each case are schematically depicted and discussed in detail. Two typical examples are shown, in which m/z's of the observed peaks are correctly predicted. The scope and limitations, as well as the significance of the rule for analyzing fragmentations in organic mass spectrometry are also discussed.

Hydrogen/deuterium exchange (HDX) coupled with pepsin digestion is useful for rapidly analyzing the kinetic properties of small amounts of protein. However, the analysis of HDX by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is time-consuming due to a lack of dedicated software. Currently available software programs mainly calculate average mass shifts, even though the isotopic distribution width contains information regarding multiple protein conformations. Moreover, HDX reaction samples are typically composed of peptides that contain various numbers of deuterium atoms, which also hinders the rapid and comprehensive analysis of protein dynamics. We report here on the development of a software program "Scipas DX" that can be used to automatically analyze the hydrogen-deuterium isotopic distribution in peaks in HDX spectra and calculate the average number of atoms exchanged, the average deuteration ratio, the abundance ratio for exchanged atoms, and their fitted spectra with a high degree of accuracy within a few minutes. Analysis of the abundance ratio for exchanged atoms of a model protein, adenylate kinase 1, using Scipas DX indicate that the local structure at residues 83-106 and 107-117 are in a slow equilibrium, suggesting that these regions adopt multiple conformations that are involved in the stability and in switching between the active and inactive forms. Furthermore, precise HDX kinetics of the average deuteration ratio both confirmed the known induced conformations of two regions (residues 46-75 and 131-165) that are responsible for ligand binding and verified the novel structural dynamics of residues 107-117 and 166-196 following ligand binding to ligand-binding pockets 1 and 2, respectively. Collectively, these results highlight the usefulness and versatility of Scipas DX in MALDI-MS HDX-based analyses of protein dynamics.

Lipids, a class of biomolecules, play a significant role in the physiological system. In this study, gas-phase hydroxyl radicals (OH·) and atomic oxygens (O) were introduced into the collision cell of a triple quadruple mass spectrometer (TQ-MS) to determine the positions of the double bond in unsaturated phospholipids. A microwave-driven compact plasma generator was used as the OH·/O source. The reaction between OH·/O and the precursor ions passing through the collision cell generates product ions that correspond to the double bond positions in the fatty acyl chain. This double bond position specific fragmentation process initiated by the attachment of OH·/O to the double bond of a fatty acyl chain is a characteristic of oxygen attachment dissociation (OAD). A TQ-MS incorporating OAD, in combination with liquid chromatography, permitted a high throughput analysis of the double bond positions in complex biomolecules. It is important to know the precise position of double bonds in lipids, since these molecules can have widely different functionalities based on the position of the double bonds. The assignment of double bond positions in a mixture of eight standard samples of phosphatidylcholines (phospholipids with choline head groups) with multiple saturated fatty acyl chains attached was successfully demonstrated.

Herein, a dark-current discharge state created by combining argon flow with a needle electrode in ambient air is described that has an ionization efficiency and mechanism comparable to those of conventional helium direct analysis in real time (DART), without requiring dopants and DART glow discharge. Using this method, polar compounds such as α-amino acids were ionized in the dark-current argon discharge via (de)protonation, molecular anion formation, fragmentation, (de)protonation with the attachment of oxygen, deprotonation with hydrogen loss and negative ion attachment. In contrast, nonpolar compounds (e.g., n-alkanes) were detected as positive ions via hydride abstraction and oxidation. Major background ions observed were H₃O⁺(H₂O) _n , O₂ ^·+, O₂ ^·-(H₂O) _n and CO₃ ^·-. These results indicate that the present dark-current discharge efficiently generates resonance-state argon with an internal energy of ∼14.2 eV, higher than that of the well-known metastable state (∼11.6 eV). It is therefore suggested that ionization reactions occurring there can be attributed to the Penning ionization of atmospheric H₂O and O₂ by resonance-state argon, analogous to helium DART.

The influence of solvent composition and surface tension on the signal intensity of deprotonated molecules [M-H]^- in electrospray ionization mass spectrometry (ESI MS) was evaluated using alanine (Ala), threonine (Thr) and phenylalanine (Phe), which have differing levels of hydrophobicity. The surface tension of the ESI solution was varied by changing the ratio of the organic solvents methanol (MeOH) and acetonitrile (MeCN) in water (H₂O). In ESI MS, the signal intensity of all the amino acids was increased with decreasing surface tension for the two solutions, H₂O/MeOH and H₂O/MeCN. The use of H₂O/MeCN was more favorable for achieving a strong signal for the analytes compared to H₂O/MeOH. The smaller vaporization enthalpy of MeCN compared to MeOH was proposed as one of the most plausible explanation for this. The order of the signal intensity of amino acids was Phe>Thr>Ala, the same order as their hydrophobicity. It can be practically concluded that the use of solutions with lower surface tensions and lower vaporization enthalpies would result in higher signal intensities in ESI MS.