Identification of animal Pasteurellaceae by MALDI-TOF mass spectrometry

Species of the family Pasteurellaceae play an important role as primary or opportunistic animal pathogens. In veterinary diagnostic laboratories identification of this group of bacteria is mainly done by phenotypic assays while genetic identification based on housekeeping genes is mostly used for research and particularly important diagnostic samples. MALDI-TOF MS seems to represent a promising alternative to the currently practiced cumbersome, phenotypic diagnostics carried out in many veterinary diagnostic laboratories. We therefore assessed its application for animal associated members of the family Pasteurellaceae. The Bruker Biotyper 3.0 database was complemented with reference spectra of clinically relevant as well as commensal animal Pasteurellaceae species using generally five strains per species or subspecies and tested for its diagnostic potential with additional, well characterized field isolates. About 250 strains comprising 15 genera and more than 40 species and subspecies were included in the study, covering most representatives of the family. A high discrimination at the genus and species level was observed. Problematic discrimination was only observed with some closely related species and subspecies. MALDI-TOF MS was shown to represent a highly potent method for the diagnosis of this group of animal pathogens, combining speed, precision and low running costs.

Testing an alternative search algorithm for compound identification with the 'Wiley Registry of Tandem Mass Spectral Data. MSforID'

A tandem mass spectral database system consists of a library of reference spectra and a search program. State-of-the-art search programs show a high tolerance for variability in compound-specific fragmentation patterns produced by collision-induced decomposition and enable sensitive and specific 'identity search'. In this communication, performance characteristics of two search algorithms combined with the 'Wiley Registry of Tandem Mass Spectral Data, MSforID' (Wiley Registry MSMS, John Wiley and Sons, Hoboken, NJ, USA) were evaluated. The search algorithms tested were the MSMS search algorithm implemented in the NIST MS Search program 2.0g (NIST, Gaithersburg, MD, USA) and the MSforID algorithm (John Wiley and Sons, Hoboken, NJ, USA). Sample spectra were acquired on different instruments and, thus, covered a broad range of possible experimental conditions or were generated in silico. For each algorithm, more than 30,000 matches were performed. Statistical evaluation of the library search results revealed that principally both search algorithms can be combined with the Wiley Registry MSMS to create a reliable identification tool. It appears, however, that a higher degree of spectral similarity is necessary to obtain a correct match with the NIST MS Search program. This characteristic of the NIST MS Search program has a positive effect on specificity as it helps to avoid false positive matches (type I errors), but reduces sensitivity. Thus, particularly with sample spectra acquired on instruments differing in their Setup from tandem-in-space type fragmentation, a comparably higher number of false negative matches (type II errors) were observed by searching the Wiley Registry MSMS.

Whole Proteome Analysis of the Protozoan Parasite Trypanosoma brucei using Stable Isotope Labeling by Amino Acids in Cell Culture and Mass Spectrometry

The single-celled protozoan Trypanosoma brucei spp. is the causative agent of human African trypanosomiasis and nagana in cattle. Quantitative proteomics for the first time allowed for the characterization of the proteome from several different life stages of the parasite (1-3). To achieve this, stable isotope labeling by amino acids in cell culture (SILAC; (4)) was adapted to T. brucei spp. cultures. T. brucei cells grown in standard media with dialyzed fetal calf serum containing heavy isotope-labeled amino acids (arginine and lysine) show efficient incorporation of the labeled amino acids into the whole cell proteome (8-12 divisions) and no detectable amino acid conversions. The method can be applied to both of the major life stages of the parasite and in combination with RNAi or gene knock-out approaches.

Development of micro-electrospray mass spectrometry for ultra high sensitivity and application in the study of drugs of abuse on endogenous \lbrack Met\rbrack $\sp5$-enkephalin release

A micro-electrospray interface was developed specifically for the neurobiological applications described in this dissertation. Incorporation of a unique nano-flow liquid chromatography micro-electrospray "needle" into the micro-electrospray interface (micro-ES/MS) increased the sensitivity of the mass spectrometric assay by $\sim$1000 fold and thus permitted the first analysis of specific neuroactive compounds in brain extracellular fluid collected by in vivo microdialysis (Md).^ Initial in vivo data presented deals with the pharmacodynamics of a novel GABA$\sb{\rm B}$ antagonist and the availability of the compound in its parent (unmetabolized) form to the brain of the anesthetized rat. Next, the first structurally specific endogenous release of (Met) $\sp5$-enkephalin was demonstrated in unanesthetized freely-moving animals (release of $\sim$6.5 fmole of (Met) $\sp5$-enkephalin into the dialysate by direct neuronal depolarization). The Md/micro-ES/MS system was used to test the acute effects of drugs of abuse on the endogenous release of (Met) $\sp5$-enkephalin from the globus pallidus/ventral pallidum brain region in rats. Four drugs known to be abused by man (morphine, cocaine, methamphetamine and diazepam) were tested. Morphine and cocaine both elicited a two-fold or more increase in the release of (Met) $\sp5$-enkephalin over vehicle controls. Diazepam elicited a small decrease in (Met) $\sp5$-enkephalin levels and methamphetamine showed no significant effect on (Met) $\sp5$-enkephalin. These results imply that (Met) $\sp5$-enkephalin may be involved in the reward pathway of certain drugs of abuse. ^

Multiple inert gas elimination technique by micropore membrane inlet mass spectrometry--a comparison with reference gas chromatography.

The mismatching of alveolar ventilation and perfusion (VA/Q) is the major determinant of impaired gas exchange. The gold standard for measuring VA/Q distributions is based on measurements of the elimination and retention of infused inert gases. Conventional multiple inert gas elimination technique (MIGET) uses gas chromatography (GC) to measure the inert gas partial pressures, which requires tonometry of blood samples with a gas that can then be injected into the chromatograph. The method is laborious and requires meticulous care. A new technique based on micropore membrane inlet mass spectrometry (MMIMS) facilitates the handling of blood and gas samples and provides nearly real-time analysis. In this study we compared MIGET by GC and MMIMS in 10 piglets: 1) 3 with healthy lungs; 2) 4 with oleic acid injury; and 3) 3 with isolated left lower lobe ventilation. The different protocols ensured a large range of normal and abnormal VA/Q distributions. Eight inert gases (SF6, krypton, ethane, cyclopropane, desflurane, enflurane, diethyl ether, and acetone) were infused; six of these gases were measured with MMIMS, and six were measured with GC. We found close agreement of retention and excretion of the gases and the constructed VA/Q distributions between GC and MMIMS, and predicted PaO2 from both methods compared well with measured PaO2. VA/Q by GC produced more widely dispersed modes than MMIMS, explained in part by differences in the algorithms used to calculate VA/Q distributions. In conclusion, MMIMS enables faster measurement of VA/Q, is less demanding than GC, and produces comparable results.

On the interference of Kr during carbon isotope analysis of methane using continuous-flow combustion–isotope ratio mass spectrometry

Stable carbon isotope analysis of methane (delta C-13 of CH4) on atmospheric samples is one key method to constrain the current and past atmospheric CH4 budget. A frequently applied measurement technique is gas chromatography (GC) isotope ratio mass spectrometry (IRMS) coupled to a combustion-preconcentration unit. This report shows that the atmospheric trace gas krypton (Kr) can severely interfere during the mass spectrometric measurement, leading to significant biases in delta C-13 of CH4, if krypton is not sufficiently separated during the analysis. According to our experiments, the krypton interference is likely composed of two individual effects, with the lateral tailing of the doubly charged Kr-86 peak affecting the neighbouring m/z 44 and partially the m/z 45 Faraday cups. Additionally, a broad signal affecting m/z 45 and especially m/z 46 is assumed to result from scattered ions of singly charged krypton. The introduced bias in the measured isotope ratios is dependent on the chromatographic separation, the krypton-to-CH4 mixing ratio in the sample, the focusing of the mass spectrometer as well as the detector configuration and can amount to up to several per mil in delta C-13. Apart from technical solutions to avoid this interference, we present correction routines to a posteriori remove the bias.

Highlights in Analytical Chemistry in Switzerland: OMA & OPA - A Software Tool for Mass Spectrometric Sequencing of Nucleic Acids

Oligonucleotides comprising unnatural building blocks, which interfere with the translation machinery, have gained increased attention for the treatment of gene-related diseases (e.g. antisense, RNAi). Due to structural modifications, synthetic oligonucleotides exhibit increased biostability and bioavailability upon administration. Consequently, classical enzyme-based sequencing methods are not applicable to their sequence elucidation and verification. Tandem mass spectrometry is the method of choice for performing such tasks, since gas-phase dissociation is not restricted to natural nucleic acids. However, tandem mass spectrometric analysis can generate product ion spectra of tremendous complexity, as the number of possible fragments grows rapidly with increasing sequence length. The fact that structural modifications affect the dissociation pathways greatly increases the variety of analytically valuable fragment ions. The gas-phase dissociation of oligonucleotides is characterized by the cleavage of one of the four bonds along the phosphodiester chain, by the accompanying loss of nucleases, and by the generation of internal fragments due to secondary backbone cleavage. For example, an 18-mer oligonucleotide yields a total number of 272’920 theoretical fragment ions. In contrast to the processing of peptide product ion spectra, which nowadays is highly automated, there is a lack of tools assisting the interpretation of oligonucleotide data. The existing web-based and stand-alone software applications are primarily designed for the sequence analysis of natural nucleic acids, but do not account for chemical modifications and adducts. Consequently, we developed a software to support the interpretation of mass spectrometric data of natural and modified nucleic acids and their adducts with chemotherapeutic agents.

Combined Secondary Ion Mass Spectrometry Depth Profiling and Focused Ion Beam Analysis of Cu Films Electrodeposited under Oscillatory Conditions

The recrystallization behavior of Cu films electrodeposited under oscillatory conditions in the presence of plating additives was studied by means of secondary ion mass spectrometry (SIMS) and focused ion beam analysis. When combined with bis-(sodium-sulfopropyl)-disulfide (SPS), Imep levelers (polymerizates of imidazole and epichlorohydrin) show characteristic oscillations in the galvanostatic potential/time transient measurements. These are related to the periodic degradation and restoration of the active leveler ensemble at the interface. The leveler action relies on adduct formation between the Imep and MPS (mercaptopropane sulfonic acid)-stabilized CuI complexes that appear as intermediates of the copper deposition when SPS is present in the electrolyte. SIMS depth profiling proves that additives are incorporated into the growing film preferentially under transient conditions during the structural breakdown of the leveler ensemble and its subsequent restoration. In contrast, Cu films electrodeposited in the presence of a structurally intact Imep–CuI–MPS ensemble remain largely contamination free.

Analytic validation of a gas chromatography-mass spectrometry method for quantification of six amino acids in canine serum samples.

OBJECTIVE To analytically validate a gas concentration of chromatography-mass spectrometry (GC-MS) method for measurement of 6 amino acids in canine serum samples and to assess the stability of each amino acid after sample storage. SAMPLES Surplus serum from 80 canine samples submitted to the Gastrointestinal Laboratory at Texas A&M University and serum samples from 12 healthy dogs. PROCEDURES GC-MS was validated to determine precision, reproducibility, limit of detection, and percentage recovery of known added concentrations of 6 amino acids in surplus serum samples. Amino acid concentrations in serum samples from healthy dogs were measured before (baseline) and after storage in various conditions. RESULTS Intra- and interassay coefficients of variation (10 replicates involving 12 pooled serum samples) were 13.4% and 16.6% for glycine, 9.3% and 12.4% for glutamic acid, 5.1% and 6.3% for methionine, 14.0% and 15.1% for tryptophan, 6.2% and 11.0% for tyrosine, and 7.4% and 12.4% for lysine, respectively. Observed-to-expected concentration ratios in dilutional parallelism tests (6 replicates involving 6 pooled serum samples) were 79.5% to 111.5% for glycine, 80.9% to 123.0% for glutamic acid, 77.8% to 111.0% for methionine, 85.2% to 98.0% for tryptophan, 79.4% to 115.0% for tyrosine, and 79.4% to 110.0% for lysine. No amino acid concentration changed significantly from baseline after serum sample storage at -80°C for ≤ 7 days. CONCLUSIONS AND CLINICAL RELEVANCE GC-MS measurement of concentration of 6 amino acids in canine serum samples yielded precise, accurate, and reproducible results. Sample storage at -80°C for 1 week had no effect on GC-MS results.

Wet and dry deposition of 129I in Seville (Spain) measured by accelerator mass spectrometry

Iodine-129 (Full-size image (<1 K)) concentrations have been determined by accelerator mass spectrometry in rainwater samples taken at Seville (southwestern Spain) in 1996 and 1997. This technique allows a reduction in the detection limits for this radionuclide in comparison to radiometric counting and other mass spectrometric methods such as ICP-MS. Typical 129I concentrations range from 4.7×107129I atoms/l (19.2%) to 4.97×109129I atoms/l (5.9%), while 129I depositions are normally in the order of 108–1010 atoms/m2 d. These values agree well with other results obtained for recent rainwater samples collected in Europe. Apart from these, the relationship between 129I deposition and some atmospheric factors has been analyzed, showing the importance of the precipitation rate and the concentration of suspended matter in it.

Development of a rapid column-switching LC-MS/MS method for the quantification of THCCOOH and THCCOOH-glucuronide in whole blood for assessing cannabis consumption frequency

The concentration of 11-nor-9-carboxy-Δ(9)-tetrahydrocannabinol (THCCOOH) in whole blood is used as a parameter for assessing the consumption behavior of cannabis consumers. The blood level of THCCOOH-glucuronide might provide additional information about the frequency of cannabis use. To verify this assumption, a column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the rapid and direct quantification of free and glucuronidated THCCOOH in human whole blood was newly developed. The method comprised protein precipitation, followed by injection of the processed sample onto a trapping column and subsequent gradient elution to an analytical column for separation and detection. The total LC run time was 4.5 min. Detection of the analytes was accomplished by electrospray ionization in positive ion mode and selected reaction monitoring using a triple-stage quadrupole mass spectrometer. The method was fully validated by evaluating the following parameters: linearity, lower limit of quantification, accuracy and imprecision, selectivity, extraction efficiency, matrix effect, carry-over, dilution integrity, analyte stability, and re-injection reproducibility. All acceptance criteria were analyzed and the predefined criteria met. Linearity ranged from 5.0 to 500 μg/L for both analytes. The method was successfully applied to whole blood samples from a large collective of cannabis consumers, demonstrating its applicability in the forensic field.

Characterization and source apportionment of organic aerosol using offline aerosol mass spectrometry

Field deployments of the Aerodyne Aerosol Mass Spectrometer (AMS) have significantly advanced real-time measurements and source apportionment of non-refractory particulate matter. However, the cost and complex maintenance requirements of the AMS make its deployment at sufficient sites to determine regional characteristics impractical. Furthermore, the negligible transmission efficiency of the AMS inlet for supermicron particles significantly limits the characterization of their chemical nature and contributing sources. In this study, we utilize the AMS to characterize the water-soluble organic fingerprint of ambient particles collected onto conventional quartz filters, which are routinely sampled at many air quality sites. The method was applied to 256 particulate matter (PM) filter samples (PM1, PM2:5, and PM10, i.e., PM with aerodynamic diameters smaller than 1, 2.5, and 10 μm, respectively), collected at 16 urban and rural sites during summer and winter. We show that the results obtained by the present technique compare well with those from co-located online measurements, e.g., AMS or Aerosol Chemical Speciation Monitor (ACSM). The bulk recoveries of organic aerosol (60–91 %) achieved using this technique, together with low detection limits (0.8 μg of organic aerosol on the analyzed filter fraction) allow its application to environmental samples. We will discuss the recovery variability of individual hydrocarbon ions, ions containing oxygen, and other ions. The performance of such data in source apportionment is assessed in comparison to ACSM data. Recoveries of organic components related to different sources as traffic, wood burning, and secondary organic aerosol are presented. This technique, while subjected to the limitations inherent to filter-based measurements (e.g., filter artifacts and limited time resolution) may be used to enhance the AMS capabilities in measuring size-fractionated, spatially resolved longterm data sets.