Laser-induced electron impact ionization in a reflectron time-of-flight mass spectrometer

Some details of the generation of electrons by impinging a laser beam on a metal surface are described. It is shown that highly efficient electron generation is observed only during the laser pulse. Therefore, this technique delivers intense pulses of electrons. The process is investigated and different ion source set-ups are discussed. In conjunction with a time-of-flight mass spectrometer this technique can be used to produce mass spectra of different samples ranging from simple organic molecules to peptides.     

Rapid analysis of polychlorinated biphenyls in the gas phase with resonance-enhanced two-photon ionization: optimal injection of ions into the ion-trap storage/time-of-flight mass spectrometer.

LI-IT-TOFMS (laser ionization/ion-trap storage/time-of-flight mass spectrometry) is expected to be a powerful tool for environmental monitoring. In the research reported here, real-time LI-IT-TOFMS measurements were carried out on gaseous 2-4 chlorinated PCBs in order to evaluate the applicability of an environmental monitoring method. With respect to ion-trap storage for PCBs, we found that the effect was due to the driving RF voltage on the ring electrode in the ion trap. For PCBs ions produced by laser irradiation, we observed that it was more efficient to reach the center of the ion trap by using a gated RF voltage rather than by using a continuous RF voltage. The ion trajectories in the ion trap were simulated by SIMION 7.0. We found that the voltage of the exit end cap electrode affected both the number of ions trapped and the orbit of ions inside the trap cell. Optimization of this parameter was performed using both simulated and experimental results. The achievable PCBs sensitivity for real-time (1 min) measurement using the LI-IT-TOFMS method was found to be in the pptV range (<0.01 mg/m3N) by means of a comparison with the conventional gas sampling/GS-MS method. A satisfactory proportional relationship was confirmed between the laser-based and conventional results.     

Selective determination of 2,4-xylenol by gas chromatography/supersonic jet/resonance-enhanced multiphoton ionization/time-of-flight mass spectrometry

Gas chromatography/supersonic jet/resonance-enhanced multiphoton ionization/time-of-flight mass spectrometry (GC/SSJ/REMPI/TOF-MS) was employed for isomer-selective determination of 2,4-xylenol in river and seawater samples. The sample containing 2,4-xylenol was measured using argon, rather than helium, as the GC carrier gas to cool the analyte molecule sufficiently. The instrumental detection limit (IDL) achieved at a flow rate of 1 mLmin(-1) was 14 pg. Although this value was comparable to the value (ca. 10 pg) obtained by gas chromatography/electron impact/quadrupole mass spectrometry (GC/EI/QMS). When the flow rate was increased to 8 mLmin(-1), interference from the 2,5-xylenol isomer was completely suppressed. The IDL was degraded to 83 or 160 pg at a flow rate of 5 or 8 mLmin(-1), respectively. The recovery of 2,4-xylenol from the river and the seawater samples was 85 and 93%, respectively. The time for analysis was only 10 min per one sample in GC/SSJ/REMPI/TOF-MS. These results suggest that GC/SSJ/REMPI/TOF-MS is useful for the selective measurement of 2,4-xylenol, which has been designated a Class I chemical substance in the Pollutant Release and Transfer Register (PRTR).     

Tandem time-of-flight mass spectrometer for photodissociation of biopolymer ions generated by matrix-assisted laser desorption ionization (MALDI-TOF-PD-TOF) using a linear-plus-quadratic potential reflectron

A tandem time-of-flight mass spectrometer for the study of photodissociation of biopolymer ions generated by matrix-assisted laser desorption ionization was designed and constructed. A reflectron with linear and quadratic (LPQ) potential components was used. Characteristics of the LPQ reflectron and its utility as the second stage analyzer of the tandem mass spectrometer were investigated. Performance of the instrument was tested by observing photodissociation of [M + H](+) from angiotensin II, a prototype polypeptide. Quality of the photodissociation tandem mass spectrum was almost comparable to that of the post-source decay spectrum. Monoisotopic selection of the parent ion was possible, which was achieved through the ion beam-laser beam synchronization. General theoretical considerations needed for a successful photodissociation of large biopolymer ions are also presented.     

Detection of femtomole and sub-femtomole levels of peptides by tandem magnetic sector/reflectron time-of-flight mass spectrometry and matrix-assisted laser desorption ionization

This article describes results of low-level (sub-femtomole) detection of peptides by matrix-assisted laser desorption ionization. The matrix-assisted laser desorption ionization method can be used for low-level detection of the parent ion, either [M + H]⁺ or [M + Na]⁺, and collision-induced dissociation of the parent ion can be performed at the picomole level. The instrument used for these studies is a novel high-performance magnetic sector (electric(E)/magnetic(B) sector)/reflectron time-of-flight (TOP) tandem mass spectrometer (EB/TOF).     

Sensitive and selective analysis of polychlorinated dibenzo-p-dioxins/dibenzofurans and their precursors by supersonic jet/resonance-enhanced multiphoton ionization/time-of-flight mass spectrometry.

Polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs), some of which are extremely toxic, are present at low concentrations in incinerator flue gases. The on-line real-time measurement of these compounds is necessary, since these concentrations fluctuate drastically with the process conditions used. Supersonic jet/resonance-enhanced multiphoton ionization/time-of-flight mass spectrometry (SSJ/REMPI/TOF-MS), which provides both high selectivity and sensitivity, appears suitable for application to the on-line real-time monitoring of PCDD/Fs. In this review, the resonant ionization wavelengths of PCDD/Fs and their precursors are given. Moreover, improvements in SSJ/REMPI/TOF-MS that lead to enhanced sensitivity and selectivity are presented. That is, novel ionization sources and advanced ionization schemes are reported for the sensitive measurement of PCDD/Fs and their precursors. Those techniques are advantageous for the trace analysis of these compounds.     

Detection of specific Bacillus anthracis spore biomarkers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was applied for the characterization of Bacillus anthracis spore biomarkers. B. anthracis spores were extracted under a simple procedure, followed by linear mode analysis, using sinapinic acid as the matrix. Several markers with a mass range of 4-7 kDa were detected in three B. anthracis strains: Vollum, Sterne and V770-NP1-R. Similar spectra were also obtained for spore extracts of two members of the B. cereus group: B. thuringiensis and B. cereus, but not for B. mycoides, B. subtilis or B. licheniformis, suggesting that these markers are specific to closely related members of the B. cereus group. When alpha-cyano-4-hydroxycinnamic acid was used as the matrix, at least four additional new markers within a mass range of 2-4 kDa could be detected in all B. anthracis spore extracts. These markers, corresponding to a molecular weight of 2528.3, 2792.4, 3077.4, and 3590.7 Da, have not been observed in extracts of the three closely related Bacillus species - B. cereus, B. thuringiensis and B. mycoides. These unique B. anthracis biomarkers, which were isotopically resolved and reproducibly detected in the highly accurate MALDI-TOFMS reflectron mode, may be useful as a basis for rapid and specific identification of B. anthracis strains.     

Applications of laser ionization/fourier-transform mass spectrometry to the study of metal ions and their clusters in the gas phase

Recent software and hardware modifications of the Nicolet FTMS-1000 Fourier-transform mass spectrometer have made it possible to conduct research in what can be termed a “complete gas-phase chemical laboratory”. Selected ions of interest can be mixed with various reagents and their detailed chemistries monitored through a series of as many as eight reaction sequences. At any point in these sequences, ion structures can be elucidated and fundamental kinetic and thermodynamic parameters of the reactions can be determined. These powerful new techniques have been applied to examine the gas-phase chemistry and photochemistry of metal ions, metal ion clusters, and metal ion complexes, all of which have a bearing on the fundamentals of organometallic chemistry and catalysis.     

Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine

A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H](2+) ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme.     

Multiphoton ionization and photodissociation at the second-order space focus in a time-of-flight mass spectrometer: The amino acid tryptophan

Neutral IR-desorbed molecules of the amino acid tryptophan entrained in a supersonic beam were post-ionized by multiphoton ionization (MUPI) in the gas phase. Variations in the intensity of the ionization laser produced different degrees of fragmentation observed in the time-of-flight mass spectra of tryptophan according to the ladder-switching model for MUPI fragmentation. From the number of absorbed photons the upper values for the appearance energies of specific fragment ions were obtained. A complete fragmentation pathway of tryptophan was established by using photodissociation of molecular ions and fragment ions formed by MUPI. Photodissociation was successfully performed by secondary excitation with a UV laser at the second-order space focus of a reflection time-of-flight instrument. A fragmentation tree for a laser-desorbed molecule obtained in a laser tandem time-of-flight instrument is shown for the first time.     

Comparison of mass spectra of peptides in different matrices using matrix-assisted laser desorption/ionization and a multi-turn time-of-flight mass spectrometer, MULTUM-IMG.

The mass spectra of peptides obtained with different matrices were compared using a matrix-assisted laser desorption/ionization (MALDI) ion source and a multi-turn time-of-flight (TOF) mass spectrometer, MULTUM-IMG, which has been developed at Osaka University. Two types of solid matrices, alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), and a liquid matrix made from a mixture of 3-aminoquinoline and CHCA were used. When measuring the peak signal intensity of human angiotensin II [M+H]+ from a fixed sample position, the liquid matrix produced a stable signal over 1000 laser shots, while the signal obtained with CHCA and DHB decayed after about 300 and 100 shots, respectively. Significant differences in the mass resolving power were not observed between the spectra obtained with the three matrices. Signal peak areas were measured as a function of the cycle number in a multi-turn ion trajectory, i.e., the total flight time over a millisecond time scale. For both [M+H]+ of human angiotensin II and bovine insulin, the decay of the signal peak area was the most significant with CHCA, while that measured with DHB was the smallest. The results of the mean initial ion velocity measurements suggested that the extent of metastable decomposition of the analyte ions increased in order of DHB, the liquid matrix, and CHCA, which is consistent with the difference in the decay of the signal peak area as the total flight time increased.     

Use of matrix clusters and trypsin autolysis fragments as mass calibrants in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Trypsin autolysis fragments and matrix clusters are often observed as intense peaks in mass spectra of protein digests. It is demonstrated that these can be exploited to improve the mass calibration of a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectrometer. Interpretation of some of the autolysis masses is complicated by the existence of disulfide bonds. Surprisingly large matrix clusters are often visible for alpha-cyano-4-hydroxy-cinnamic acid. The fractional part of their masses differentiates them from protein digestion fragments.     

Early stages of styrene-isoprene copolymerization in gas phase clusters probed by resonance enhanced multiphoton ionization

We present direct evidence for the formation of the covalent bonded styrene (isoprene)(2) oligomer and the isoprene dimer ions following resonance ionization of the gas phase styrene-isoprene binary clusters. The application of resonance ionization to study polymerization reactions in clusters provides new information on the structure and mechanism of formation of the early stages of polymerization and holds considerable promise for the discovery of new initiation mechanisms and for the development of novel materials with unique properties.     

Stability of disubstituted copper complexes in the gas phase analyzed by electrospray ionization mass spectrometry

A series of nitrogen ligand (L)/copper complexes of the type [Cu^IL]⁺, [Cu^IIL(X)]⁺ and [Cu^IL₂]⁺ (X = Cl^–, BF, acac^–, CH₃COO^– and SO₃CF) was studied in the gas phase by electrospray ionization mass spectrometry. The following ligands (L) were employed: 1,12‐diazaperylene (dap), 1,1′‐bisisoquinoline (bis), 2,2′‐bipyridine (bpy), 1,10‐phenanthroline (phen), 2,11‐disubstituted 1,12‐diazaperylenes (dap), 3,3′‐disubstituted 1,1′‐ bisisoquinoline (bis), 5,8‐dimethoxy‐substituted diazaperylene (meodap), 6,6′‐ dimethoxy‐substituted bisisoquinoline (meobis) and 2,9‐dimethyl‐1,10‐phenanthroline (dmphen). Collision‐induced decomposition measurements were applied to evaluate the relative stabilities of the different copper complexes. The influence of the spatial arrangement of the ligands, of the type of substituents and of the counter ion of the copper salts employed for the complexation was examined. Correlations were found between the binding constants of the [ML₂]⁺ complexes in solution and the relative stabilities of the analogous complexes in the gas phase. Furthermore, complexation with the ligands 2,11‐dialkylated 1,12‐diazaperylenes [alkyl = ethyl (dedap) and isopropyl (dipdap)] was studied in the solvents CH₃OH and CH₃CN. Copyright © 2010 John Wiley & Sons, Ltd.     

Electrospray ionization with ambient pressure ion mobility separation and mass analysis by orthogonal time-of-flight mass spectrometry.

Rapid screening and identification of drug and other mixtures are possible using a novel ambient pressure high-resolution ion mobility (APIMS) orthogonal reflector time-of-flight mass spectrometer (TOFMS). Departing ions from the APIMS drift tube traversed a pressure interface between the APIMS and TOFMS where they were subjected to numerous gas collisions that could produce selective fragmentation. By increasing the accelerating field in the pressure interface region, the ions generated using water-cooled electrospray ionization (ESI) underwent collision-induced dissociation (CID). Mixtures of ESI ions were separated by APIMS based on their respective size-to-charge (s/z) ratios while CID and analysis of mass-to-charge (m/z) ratios occurred in the pressure interface and TOFMS. Product ions that were formed in this pressure interface region could be readily assigned to precursor ions by matching the mobility drift times. This process was demonstrated by the examination of a mixture of amphetamines and the resulting fragmentation patterns of the mobility-separated precursor ion species [M + H](+).     

Real-time monitoring of monochlorinated benzene and polychlorinated biphenyls in the gas phase with on-line detection: High chemical selectivity of resonance-enhanced two-photon ionization.

The development of molecular spectroscopy has enabled us to select chlorinated aromatic hydrocarbons very rapidly. In particular, the laser ionization TOFMS (time-of-flight mass spectrometry) method is expected to be useful as an on-line, selective, and sensitive method. In the present work, real-time laser ionization TOFMS measurements were carried out on gaseous chlorinated aromatic hydrocarbons. The laser ionization method used resonance-enhanced two-photon ionization with the direct introduction of gas into the vacuum chamber. This method for analyzing aromatic hydrocarbons was developed using a pulsed supersonic molecular beam method. In the context of developing a highly selective and sensitive method, excitation of monochlorinated benzene at lambda = 263.07 nm was found to be effective in the wavelength region from 263 nm to 265 nm. Also the excitation of polychlorinated biphenyls at lambda = 266 nm was found to be substantially more effective than at lambda = 280, 300 or 320 nm. The achievable sensitivity for real-time (1 min) measurements using the laser ionization TOFMS technique was found to be in the ppbV range.     

On-line capillary liquid chromatography tandem mass spectrometry on an ion trap/ reflectron time-of-flight mass spectrometer using the sequence tag database search approach for peptide sequencing and protein identification

Capillary high-performance liquid chromatography has been coupled on-line with an ion trap storage/reflectron time-of-flight mass spectrometer to perform tandem mass spectrometry for tryptic peptides. Selection and fragmentation of the precursor ions were performed in a three-dimensional ion trap, and the resulting fragment ions were pulsed out of the trap into a reflectron time-of-flight mass spectrometer for mass analysis. The stored waveform inverse Fourier transform waveform was applied to perform ion selection and an improved tickle voltage optimization scheme was used to generate collision-induced dissociation. Tandem mass spectra of various doubly charged tryptic peptides were investigated where a conspicuous y ion series over a certain mass range defined a partial amino acid sequence. The partial sequence was used to determine the identity of the peptide or even the protein by database search using the sequence tag approach. Several peptides from tryptic digests of horse heart myoglobin and bovine cytochrome c were selected for tandem mass spectrometry (MS/MS) where it was demonstrated that the proteins could be identified based on sequence tags derived from MS/MS spectra. This approach was also utilized to identify protein spots from a two-dimensional gel separation of a human esophageal adenocarcinoma cell line.     

Distinguishing of linkage isomers of lactotetra oligosaccharides by using the relative ion intensity analysis of post-source decay fragment ions in curved-field reflectron matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

The native oligosaccharides of lacto-N-neotetraose (Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc; LNnT) and lacto-N-tetraose (Gal beta1-3GlcNAc beta1-3Gal beta1-4Glc; LNT) were analyzed by using curved-field reflectron matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). Since a curved-field reflectron TOFMS enables a simultaneous focusing of a wide mass range of metastable fragment ions, the relative ion intensities in the post-source decay (PSD) mass spectra can be discussed. The PSD mass spectra of LNnT and LNT were distinguishable in their relative ion intensities. In the case of LNT, beta-elimination could occur in the N-acetyl glucosamine (GlcNAc) at the C-3 position, which was bonded by galactose (Gal); however, it did not occur in LNnT. The 3-O elimination caused a difference in the relative ion intensities in the PSD mass spectra of LNnT and LNT. The beta1-3 glycosyl linkage cleaved more easily than the beta1-4 glycosyl linkage in the MALDI-PSD fragmentation. An analysis of the relative ion intensities in the MALDI-PSD mass spectra of oligosaccharides was very useful for distinguishing the linkage isomers and for characterizing the types of glycosyl linkages.