Ion Flight Time Research Articles

MALDI-TOF mass spectrometry from nucleic acid: development and evaluation of a novel platform for identification of mycobacteria and detection of genetic markers of resistance.

Complete identification methods are critical for evaluating nontuberculous mycobacteria (NTM). Here, we describe a novel diagnostic method for identification of eight NTM, Mycobacterium tuberculosis complex, and three drug resistance markers using PCR/matrix-assisted, laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) from cultured organisms. With this technology, a multiplex end-point PCR is performed for targets of interest. Detection probes that are extended in the presence of a target are added. The extended probes have greater molecular weight and can be detected by MALDI-TOF MS. An AFB Primary Panel was designed to differentiate Mycobacterium avium; Mycobacterium intracellulare subsp. chimaera; Mycobacterium avium complex (other); Mycobacterium abscessus subsp. abscessus, bolletii, and massiliense; Mycobacterium kansasii, and M. tuberculosis complex. This design should cover 90% (3,483/3,691) of mycobacteria seen onsite. A development set of unblinded isolates (n = 217) was used to develop PCR primers, detection probes, and probe barcodes. It demonstrated 99.1% (215/217) agreement with reference methods. An evaluation set using blinded isolates (n = 320) showed an overall sensitivity of 94.3% (range by target: 90.0-100%). Overall specificity from negative media, non-target mycobacteria, and bacteria was 99.1% (108/109; range by target: 94.4-100%). Three drug resistance markers erm (41), rrl, and rrs demonstrated 100%, 91%, and 100% sensitivity, respectively, and >99% specificity. Limit of detection per target ranged from 2.2 × 103 to 9.9 × 106 CFU/mL. The AFB Primary Panel allows for mycobacterial speciation, subspeciation, and resistance mutation detection, which is essential for diagnosis, appropriate therapy, identifying outbreaks, and managing treatment-refractory disease. It can perform with high-throughput and high specificity and sensitivity from isolates.IMPORTANCEEven closely related mycobacteria can have unique treatment patterns, but differentiating these organisms is a challenge. Here, we tested an innovative platform that combines two commonly used technologies and creates something new: matrix-assisted, laser-desorption ionization time-of flight mass spectrometry was performed on PCR amplicons instead of on proteins. This created a robust system with the advantages of PCR (high discriminatory power, high throughput, detection of resistance) with the advantages of mass spectrometry (more targets, lower operational cost) in order to identify closely related mycobacterial organisms.

Determination of product ions emerged from dications by varying retarding electric field in reflectron time-of-flight mass spectrometer supported by ion trajectory simulations

Investigation of the metastable dissociations clarifies the characteristics of ionic states. Tandem mass spectrometry using an ion trap mass spectrometer, a time-of-flight mass spectrometer (TOF-MS), and the combination of those spectrometers were used for the analysis of metastable dissociation. However, investigation of the metastable dissociations of multiply charged ions is not straightforward because collision-induced charge transfer reactions to lose charges should be avoided. TOF-MS equipped with a reflectron (refTOF-MS) maintained under high vacuum condition is one of the suitable instruments. However, another difficulty arises due to the working principle of refTOF-MS: The product ions whose m/z is greater than that of multiply charged precursor ions can pass through a reflectron under the experimental conditions for detecting the product ions of a singly charged precursor ion. In this study, we report the ionization of decafluorobiphenyl (DFB) by femtosecond laser pulses and the determination of the product ions emerged from doubly charged precursor ions using a refTOF-MS. Detection of ions behind a reflectron by varying the retarding potential of a reflectron enables us to identify the m/z of product ions in two ways: measurement of the threshold retarding potential reflecting product ion; comparing the relative flight time of the product ions obtained by experiments and ion trajectory simulations. We have reported three and one metastable dissociation channels of DFB+ and DFB2+, respectively, by a conventional product ion analysis, that is the selection of a precursor ion and its product ions using an ion gate followed by the reflection and separation of them by a reflectron. In this study, we further identified the products that passed through a reflectron: charge transfer product of C2+; the product ion of C4F22+ which is a secondary product of DFB ion; the product ion of DFB2+. The detection of the product ions that passed through a reflectron expanded the measurable m/z range of product ions emerged from doubly charged precursor ions.

Ion detector of time-of-flight mass spectrometer with registration of leading and trailing edges.

The accuracy of the ion flight time measurement in the time-of-flight mass spectrometer is critical to achieving high resolution. The pulse amplitude variation of the detector pulses leads to the registration time spread at a given pulse detection threshold. This time spread can be eliminated by determining the position of the pulse apex. To determine the position of the pulse apex, the output of the ion detector is fed simultaneously to the two channels of the time-to-digital converter. In this case, the first channel is set to register the leading edge, and the second channel is set to register the trailing edge of the pulse. Using a simple processing of the received data, the position of the pulse tip is determined. Thus, the dependence of the temporal position of the peak on the pulse amplitude is largely eliminated. Examples are given, and the efficiency of using this algorithm to increase the resolution of time-of-flight mass spectral peak registration is demonstrated.

Isotopic and elemental analysis by electrostatic energy analyzer laser ablated time-of-flight mass spectrometer

In this work, we present a modified setup of the time of flight (TOF) mass spectrometer. It consists of a laser ablation source, and a Q-switched Nd:YAG laser (532 nm, 100 mJ, 7 ns) integrated with a linear TOF mass spectrometer coupled with an electrostatic energy analyzer (EEA). Ions are detected by a channeltron electron multiplier assembled with EEA. The main objective of this work is to resolve the ion’s initial energy problem and improve the reproducibility of the results. Employing this newly developed setup the elemental analysis of a known sample was performed for different laser energies and found no shift in the position of the ions signals on the time scale. In addition to elemental analysis, isotopic compositions of lithium, titanium, copper, and silver samples have been also derived. Our results are in good agreement with their natural abundance. The design parameters of the instrument were optimized. The mass calibration was achieved by measuring the flight time of various ions; Li+, Ti+, Cu+, and Ag+.

Cold-target electron-ion-coincidence momentum-spectroscopy study of electron-impact single and double ionization of N2 and O2 molecules

We report experimental studies of electron-impact ionization of nitrogen (${\mathrm{N}}_{2}$) and oxygen (${\mathrm{O}}_{2}$) molecules using a cold target recoil ion momentum spectrometer (COLTRIMS, reaction microscope). The recoil ion is detected in coincidence with one outgoing electron such that the momentum vectors and consequently the kinetic energies for these final-state particles are determined. The ionization cross sections for producing the doubly and singly charged parent ions were measured as a function of the incident electron energy ranging from 50 to 600 eV. For molecular dications, e.g., ${\mathrm{O}}_{2}^{2+}$, the cross sections are obtained by measuring both the ion time of flight and two-dimensional position spectra, which is demonstrated to be an efficient way to suppress the contribution of the dissociation channels with the ${\mathrm{O}}^{+}$ products as both ions have the identical mass-to-charge ratios. The projectile energy-loss spectra correlated to the final-state ions are obtained, which provide direct evidence of the ionization mechanisms of ${\mathrm{N}}_{2}$ and ${\mathrm{O}}_{2}$ molecules. It is found that the ${\mathrm{O}}_{2}^{2+}$ dication is produced mainly by Auger process after single ionization in $2{\ensuremath{\sigma}}_{g}$ inner-valence shell of ${\mathrm{O}}_{2}$ molecule, while the ${\mathrm{N}}_{2}^{2+}$ dication is generated by the direct removal of two electrons from the outermost $3{\ensuremath{\sigma}}_{g}$ orbital of ${\mathrm{N}}_{2}$ molecule.

A plano-convex thick-lens velocity map imaging apparatus for direct, high resolution 3D momentum measurements of photoelectrons with ion time-of-flight coincidence.

Since their inception, velocity map imaging (VMI) techniques have received continued interest in their expansion from 2D to 3D momentum measurements through either reconstructive or direct methods. Recently, much work has been devoted to the latter of these by relating electron time-of-flight (TOF) to the third momentum component. The challenge is having a timing resolution sufficient to resolve the structure in the narrow (<10ns) electron TOF spread. Here, we build upon the work in VMI lens design and 3D VMI measurement by using a plano-convex thick-lens (PCTL) VMI in conjunction with an event-driven camera (TPX3CAM) providing TOF information for high resolution 3D electron momentum measurements. We perform simulations to show that, with the addition of a mesh electrode to the thick-lens geometry, the resulting plano-convex electrostatic field extends the detectable electron cutoff energy range while retaining the high resolution. This design also extends the electron TOF range, allowing for a better momentum resolution along this axis. We experimentally demonstrate these capabilities by examining above-threshold ionization in xenon, where the apparatus is shown to collect electrons of energy up to ∼7eV with a TOF spread of ∼30ns, both of which are improved compared to a previous work by factors of ∼1.4 and ∼3.75, respectively. Finally, the PCTL-VMI is equipped with a coincident ion TOF spectrometer, which is shown to effectively extract unique 3D momentum distributions for different ionic species in a gas mixture. These techniques have the potential to lend themselves to more advanced measurements involving systems where the electron momentum distributions possess non-trivial symmetries.

꾸지뽕나무 열매 추출물의 Chlorogenic Acid, Rutin 함량 분석 및 생리활성

This study was carried out to very precisely understand the effect of solvent selection on the molecular properties in the investigation of the biological activity according to the molecular size of chitosan. Chitosan hydrolysates derived from the hydrolysis of high molecular weight chitosan (HMWC) were prepared in enzymatic hydrolysis under solvent selective conditions. Chitosan hydrolysates were characterized by HPLC and Matrix-Assisted Laser Desorption/ Ionization-Time of Flight (MALDI-TOF) mass spectrometry analyses, respectively. HPLC analysis did not show a significant difference in the size of hydrolysates according to the type of chitosan. On the other hand, it was confirmed that each molecular size was significantly different from the results presented by HPLC with MALDI-TOF mass spectrometry analysis. In addition, the effect on the antibacterial activity of the hydrolysates of chitosan, designated to “active molecular chitosan (AMC)”, by the solvent selected for the enzymatic reaction also showed a significant difference. Therefore, it is intended to suggest that the selection of solvents for the enzymatic reaction for chitosan hydrolysis and their antibacterial activity is very important.

Inanimate Surfaces and Air Contamination with Multidrug Resistant Species of Staphylococcus in the Neonatal Intensive Care Unit Environment

Background: Contamination of the hospital environment with multi-resistant (MDR) Staphylococcus increases the risk of infection. The aim of this study is to identify the MDR species of Staphylococcus on inanimate surfaces, in air, and in clinical samples, and analyze the risk factors that correlate with the occurrence of infections in a Neonatal Intensive Care Unit. Methods: Samples of inanimate surfaces and air were taken using a premoistened swab (0.9% sodium chloride) and spontaneous air sedimentation, respectively. The clinical isolates were recovered from infected neonates. The isolates (environmental and clinical) were identified by matrix-assisted laser desorption ionization-time of flight and the resistance profile was calculated using the disk diffusion agar technique. Results: In total, 181 isolates were obtained, 93 from (surfaces), 18 from the air, and 70 clinical samples. S. epidermidis was the most frequent species (66.8%), and the failure rate in air cleaning was 100%. More than 60% of the isolates were MDR, and the majority of clinical isolates (60.4%) had a resistance profile identical to that of the environmental isolates. Conclusion: Staphylococcus spp. were found in most of the analyzed samples, with a high frequency of MDR isolates, demonstrating the importance of the hospital environment as a reservoir, and the need for infection control measures, and rational use of antimicrobials.

Bayesian model calibration for vacuum-ultraviolet photoionisation mass spectrometry

We investigate the calibration of an instrument model for a high-pressure chemical reactor and vacuum ultraviolet photoionisation mass spectrometry apparatus, using a collection of ‘static’ mass spectrometry data (i.e. from unreactive samples). A Bayesian calibration method is applied to characterise the uncertainty of the model parameters, the associated model error, and the predicted mass spectrum. A partitioning algorithm is introduced to decompose the mass spectrum into several independent calibration problems, making the analysis of complex mass spectrometry data sets tractable. The a priori mass spectrometer model poorly predicts some portions of the ion time-of-flight spectrum due to both, parametric (i.e. uncertain parameter values) and mechanistic (i.e. missing physics) inadequacies; however, by incorporating an additive model error the prediction model is vastly improved and closely replicates the measured spectrum. We found that the model calibration and additive model error reveals a set of experimental problems, including chemical contamination of the sample, the presence of high harmonics of the ionising radiation, and a systematic fluctuation in ion flight times due to instability of the ion optics power supplies. This approach results in a calibrated instrument model with well-defined parameter uncertainties, which is critical to the analysis of ‘dynamic’ data (i.e. from reacting gas samples).

Ultrafast dissociation of ammonia: Auger Doppler effect and redistribution of the internal energy.

We study vibrationally-resolved resonant Auger (RAS) spectra of ammonia recorded in coincidence with the NH2+ fragment, which is produced in the course of dissociation either in the core-excited 1s-14a11 intermediate state or the first spectator 3a-24a11 final state. Correlation of the NH2+ ion flight times with electron kinetic energies allows directly observing the Auger-Doppler dispersion for each vibrational state of the fragment. The median distribution of the kinetic energy release EKER, derived from the coincidence data, shows three distinct branches as a function of Auger electron kinetic energy Ee: Ee + 1.75EKER = const for the molecular band; EKER = const for the fragment band; and Ee + EKER = const for the region preceding the fragment band. The deviation of the molecular band dispersion from Ee + EKER = const is attributed to the redistribution of the available energy to the dissociation energy and excitation of the internal degrees of freedom in the molecular fragment. We found that for each vibrational line the dispersive behavior of EKERvs. Ee is very sensitive to the instrumental uncertainty in the determination of EKER causing the competition between the Raman (EKER + Ee = const) and Auger (Ee = const) dispersions: increase in the broadening of the finite kinetic energy release resolution leads to a change of the dispersion from the Raman to the Auger one.

Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis.

ABSTRACTStudies involving the pathogenic organism Mycobacterium tuberculosis routinely require advanced biosafety laboratory facilities, which might not be readily available in rural areas where tuberculosis burdens are high. Attempts to adapt heat inactivation techniques have led to inconsistent conclusions, and the risk of protein denaturation due to extensive heating is impractical for subsequent mass spectrometry (MS)-based protein analyses. In this study, 240 specimens with one or two loops of M. tuberculosis strain H37Rv biomass and specific inactivated solutions were proportionally assigned to six heat inactivation methods in a thermal block at 80°C and 95°C for 20, 30, and 90 min. Twenty untreated specimens served as a positive control, and bacterial growth was followed up for 12 weeks. Our results showed that 90 min of heat inactivation was necessary for samples with two loops of biomass. Further protein extraction and a matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) MS assay demonstrated adequate scores for bacterial identification (≥1.7), with the highest score achieved in the 80°C/90 min and 95°C/30 min treatment groups. A proteomics study also confidently identified 648 proteins with ∼93% to 96% consistent protein abundances following heating at 95°C for 20, 30, and 90 min. Heat inactivation at 95°C for 90 min yielded the most quantifiable proteins, and a functional analysis revealed proteins located in the ribosomal subunit. In summary, we proposed a heat inactivation method for the M. tuberculosis strain H37Rv and studied the preservation of protein components for subsequent bacterial identification and protein-related assays.IMPORTANCE Inactivation of Mycobacterium tuberculosis is an important step to guarantee biosafety for subsequent M. tuberculosis identification and related research, notably in areas of endemicity with minimal resources. However, certain biomolecules might be denatured or hydrolyzed because of the harsh inactivation process, and a standardized protocol is yet to be determined. We evaluated distinct heating conditions to report the inactivation efficiency and performed downstream mass spectrometry-based M. tuberculosis identification and proteomics study. The results are important and useful for both basic and clinical M. tuberculosis studies.

Charging and ion ejection dynamics of large helium nanodroplets exposed to intense femtosecond soft X-ray pulses

Ion ejection from charged helium nanodroplets exposed to intense femtosecond soft X-ray pulses is studied by single-pulse ion time-of-flight (TOF) spectroscopy in coincidence with small-angle X-ray scattering. Scattering images encode the droplet size and absolute photon flux incident on each droplet, while ion TOF spectra are used to determine the maximum ion kinetic energy, \(E_{\text {kin}}\), of \(\hbox {He}_{j}^{+}\) fragments (j = 1–4). Measurements span \(\hbox {He}_N\) droplet sizes between \(N\sim 10^{7}\) and \(\sim 10^{10}\) (radii \(R_0\) = 78–578 nm), and droplet charges between \(\sim 9\times 10^{-5}\) and \(\sim 3\times 10^{-3}\) e/atom. Conditions encompass a wide range of ionization and expansion regimes, from departure of all photoelectrons from the droplet, leading to pure Coulomb explosion, to substantial electron trapping by the electrostatic potential of the charged droplet, indicating the onset of hydrodynamic expansion. The unique combination of absolute X-ray intensities, droplet sizes, and ion \(E_{\text {kin}}\) on an event-by-event basis reveals a detailed picture of the correlations between the ionization conditions and the ejection dynamics of the ionic fragments. The maximum \(E_{\text {kin}}\) of He\(^{+}\) is found to be governed by Coulomb repulsion from unscreened cations across all expansion regimes. The impact of ion-atom interactions resulting from the relatively low charge densities is increasingly relevant with less electron trapping. The findings are consistent with the emergence of a charged spherical shell around a quasineutral plasma core as the degree of ionization increases. The results demonstrate a complex relationship between measured ion \(E_{\text {kin}}\) and droplet ionization conditions that can only be disentangled through the use of coincident single-pulse TOF and scattering data.

Determination of Uropathogenic Escherichia coli in Urine by an Immunobiosensor Based Upon Antigen-Antibody Biorecognition with Fluorescence Detection and Bead-Injection Analysis

Uroinfections, which are mainly caused by pathogenic bacteria, especially uro-pathogenic E. coli (UPEC), are among the major health problems in the modern world. The current gold standard for the identification of uroinfection causing pathogens in urine is microbiological cultivation, but uro-pathogens are also determined with quantitative polymerase chain reaction (qPCR) and matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. However, it takes several hours or days to obtain the results. A promising option for rapid identification of pathogens is the application of biosensors. The aim of the current study was to develop a biosensor for the fast detection of UPEC in urine samples allowing achieving a limit of detection below 103 CFU/mL, essential for the diagnostics of recurrent uroinfections and related diseases. First, the pathogens were attached onto a single use column and specifically detected using E. coli antibodies conjugated with a fluorescence marker. The bead-injection analysis platform for fluidics provided E. coli limits of detection and quantification in 150 µL urine samples <3 and <5 cells/mL, respectively. The time of analysis was 17 min. The biosensor results were in good correlation with the results obtained with other methods, indicating that the complex urine matrix of UPEC patients did not affect the biosensor measurements.

Microorganism idenitfication – stability and correctness of mass spectrometry method (MALDI TOF MS)

Introduction: MALDI TOF MS method is increasingly used in routine microbiological diagnostics to identify clinical strains. The result of the identification test is based on the measurement of the mass, charge and flight time of the protein ions. This makes it possible to monitor and supervise the method using a numerical score developed with statistical techniques. Aim: The study aimed to determine the stability and correctness of the mass spectrometry method. Materials and methods: To evaluate the characteristics of the method, microbial identification tests were performed using reference strains. All tests were performed as part of the MALDI TOF MS internal quality control system. Results: All reference strains (100%) were correctly identified to the species level, although the score values were not always within the reliability criteria of the results established by the producer. It was found that the mean values of the score were from 2.000 – 2.299 (49.2%) and 2.300 – 3.000 (50.0%). The coefficient of variation for control tests performed in the consecutive years ranged from 5.18 – 6.56%, which evidence of the high stability of the method. For individual species, reproducibility precision over the 8 years ranged from 2.89% (n = 13) for Enterococcus faecalis ATCC 51299 to 7.02% (n = 28) for Klebsiella pneumoniae ATCC 700603, which proves the high precision of measurements. Conclusions: The mass spectrometry method is characterized by very high stability and correctness. The intra-laboratory quality control system using reference strains is a useful tool for monitoring and supervising the method and laboratory personnel competency performing identification tests during routine microbiological work.

Prevalence of Multidrug-Resistant Tuberculosis Using Phenotypic Drug Susceptibility Testing and GeneXpert MTB/RIF with Characterization of Non-tuberculous Mycobacteria Using MALDI-TOF

Background: Multidrug-resistant tuberculosis (MDR-TB) and infections by nontuberculous mycobacteria (NTM) are diseases of major public health concern. Objective: The aim of the present work is to study the prevalence and patterns of MDRTB as well as the characterization of isolated NTM species. Methodology: All samples (1069) were subjected to smear microscopy, culture on Lowenstein-Jensen (LJ) media, and phenotypic drug susceptibility testing (DST) of MTB to isoniazid (INH), rifampin (RF), streptomycin (S), and ethambutol (E). GeneXpert was used for direct detection of MTB and RF resistance. Matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometry (MS) was utilized for characterizing isolated NTM species. Results: M.tuberculosis (MTB) was isolated at a rate of 95.3% (1019/1069). MDR-TB was detected at rate of 7.16% with significant patterns for INH + RF + S + E (46.5%) and INR + RF (24.6%) (P-value &lt;0.001). RF resistance was detected at a rate of 27.2% by GeneXpert. Seven NTM species (0.6%) were isolated in culture of which M.porcinum and M.fortuitum had confident identification by MALDI-TOF (score ≥1.8). Conclusion: MDR-TB rate was found to be 7.16% with significant dominance for INH + RF + S + E and INR + RF resistance patterns, while NTM rate was 0.6%.

The Identification of Microbial Pathogens Using Matrix Assisted Laser Desorption Ionization Time of Flight in Medical Intensive Care Unit Beni-Suef University Hospital

Background: Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) is a novel technique for identification of microbes. This new method led to a new era in microbial identification because of its rapid, accurate, valid, simple and relatively decreased cost. Objectives: The aim of this study was identification of predominant pathogens by MALDI-TOF technique. Methodology: Pathogens were identified by both conventional methods and MALDI-TOF. Results: From July till December 2018, predominant pathogens were Klebsiella pneumoniae (21%), Pseudomonas aeroguinosa and Candida each constitutes (17%), E-coli (10%), Staph. aureus (9%), Acinetobacter (9%). Identification of isolates (from September to December 2018) by MALDI-TOF revealed a total agreement of (94.1%) with conventional method at genus level, (88.2%) at level of species. Kappa agreement revealed almost perfect correlation between both techniques. Conclusion: The MALDI-TOF results might suggest that its’ usage may be dependable for microbiological identification.

Stimulation of Bacillus sp. by lipopeptide biosurfactant for the degradation of aromatic amine 4-Chloroaniline

This study aims to reveal that the biosurfactant act as a stimulant in aromatic amine 4-Chloroaniline (4-CA) degradation. Isolated degrading strain Bacillus sp. was used for the production of biosurfactant with help of substrate such as engine oil. The surfactant production by the strain was studied by using various screening methods and the results showed best emulsification activity (75%), surface tension reduction activity (28.6 mNm−1) and oil spreading activity (5.9 cm). The obtained surfactant was characterized using Fourier transform infrared spectroscopy (FT-IR), Gas chromatography-Mass Spectrometry (GC-MS), Matrix-Assisted Laser Desorption/ Ionization Time of Flight (MALDI-TOF) which confirmed that the nature of surfactant is lipopeptide. The maximum removal of 4-CA was achieved in different environmental conditions at concentration 100 mg L−1, neutral pH and temperature 30 °C. In the degradation studies, the 4-CA was removed upto 76% by Bacillus sp but in the presence of lipopeptide surfactant, the Bacillus sp removed 4-CA upto 100%. The degraded metabolites were further characterized using High-Pressure Liquid Chromatography (HPLC) and GC-MS. This research indicated that strain Bacillus sp along with the lipopeptide biosurfactant possesses higher potential in the bioremediation of 4-CA compound from the environment.

Concatenated multi-reflection time-of-flight spectra for wide-band mass spectrometry

A series of multi-reflection time-of-flight (MR-ToF) spectra is recorded with increasing ion flight times and added up to a single transient. It shows periodic ToF-signal trains for each ion species revolving between the MR-ToF analyzer’s mirrors. This is used to perform a “comb-sampling” evaluation previously devised for analysis of capacitive-pickup signals (Greenwood et al., Rev. Sci. Instrum. 82 (2011), 043103). The recording and evaluation focuses on simultaneously measuring the full range of trapped ions for wide-band mass spectrometry. The comb-sampling technique is revisited for the present transients, as it is the first time it is used for comparatively background-free single-ion-counting data.

FT-ICR mass spectrometry: Superconducting magnet, external ion source, ion-molecule reactions, and ion-ion traps.

The world of Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry has witnessed, especially in the last 30 years significant advances in many fields of science, such as electronics, magnets, new ICR cell designs, developed ICR event sequences, modern external ionization sources, and linear ion beam guides, as well as modern vacuum technology. In this review, a brief account is given focusing especially on the studies performed in Wanczek's group and ICR research laboratory at the University of Bremen. An FT-ICR mass spectrometer has been developed with a high magnetic field superconducting magnet, operating at 4.7 T. At this magnetic field, a trapping time of 13.5 h was obtained with 30% efficiency. For the tetrachloromethane molecular ion, m/z 166, a mass-resolving power m/Δm = 1.5 × 106 was measured at a pressure of 2 × 10-8 Torr. The transition from magnet sweep to frequency sweep and the application of Fourier-transform has greatly enhanced the ICR technology. External ion sources were invented and differential pumping schemes were developed for enabling ultrahigh vacuum condition for ICR detection, while guiding ions at relatively higher pressures, during their flight to the ICR cell. With the external ion source, a time-of-flight ICR tandem instrument is built. A method to measure the ion flight time and to trap the ions in the ICR cell is described. Many ICR cell characteristics such as z-axis ion ejection and coupling of radial and axial ion motions in a superposed homogeneous magnetic and inhomogeneous trapping electric field were extensively studied. Gas-phase ion-molecule reactions of several reactive inorganic compounds with a focus on phosphorous and sulfur as well as silicon chemistry were also studied in great detail. The gas-phase ion chemistry of several trifluoromethyl-reagents such as trifluoromethyltrimethylsilane and tris(trifluoromethyl)phosphine were also investigated in ICR. Dual polarities multisegmented ICR cells were invented and deeply characterized. Sophisticated ICR pulse event programs were developed to enable long-range ion-ion interactions between simultaneously trapped positive and negative ions.

Identification of Cytoplasmic and Conserved Hypothetical Mycobacterium tuberculosis proteins based on mass spectrometry and iedb analysis resource as potent biomarkers for therapeutic strategies or valuable new TB vaccine

Aims and objectives: Tuberculosis (TB) remains a major challenge to public health. The disease burden caused by TB is falling globally, in all WHO regions, and in most countries, but not fast enough to reach the first, 2020, milestones of the End TB Strategy. By 2020, the TB incidence rate needs to be falling at 4–5% per year, and the proportion of people with Tuberculosis who die from the disease needs to fall to 10%. These deaths are in part due to late or missed diagnosis and could be prevented with appropriate treatment. The aim of this study was to identify new relevant cytoplasmic and conserved hypothetical protein biomarkers as TB diagnostic or therapeutic targets. Methods: TB Protein contents were prepared by detergent phase separation method. Two dimensional gel electrophoresis (2DE) was performed using the Ettan IPGphor 3 isoelecteric focusing (GE Healthcare). Comprehensive genomic and proteomic data were retrieved from MycoBrowser portal (http://www.mycobrowser.epfl.ch) followed by matrix assisted laser desorption ionization time-of flight Mass Spectrometry. The predictive of the MHC I peptide binding is carried out by the IEDB analysis resource (http://www.iedb.org). Results: At least six purified proteins with assigned putative functions were determined that corresponded to aldehyde dehydrogenase (Rv0147), iron regulated H-NS (Rv3597c), translocase SecE2 (Rv0379), methyltransferase (Rv3699), adenosylmethionin (Rv1392) and conserved hypothetical protein of Rv0443. Within the category of cytoplasm, membrane and conserved hypothetical patterns, six identified proteins were shown high expression during exponential growth in middlebrook 7H9 broth media. The MHC I peptide binding predictions of these protein spots on the SDS page gels were valued by the Immune Epitope Database tools. The IEDB-AR prediction result is given based on IC50nM value and percentile grad. Principally, a lower score, specified higher affinity. Peptides with IC50 estimation &lt; 50 nM are regarded as high affinity. Conclusions: The output scores of the predicting peptides have been shown strong binding for each of identified peptides. Rank of the predicted output affinity of Rv0443 compared to the two others, Rv0147 and Rv0379, presumably leads to stronger binding affinity. The conserved hypothetical proteins, Rv3699 and Rv0443 displayed significant homology with several other TB proteins. These proteins can also be included in TB vaccine constructs or subunit vaccine mixtures combined with a potent adjuvant. We considered these protein profiles to represent reputed biomarker as a new TB vaccine or therapeutic strategies against TB.