MALDI TOF mass spectrometry

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Characterization of Yersinia using MALDI-TOF mass spectrometry and chemometrics.

Characterization of Yersinia using MALDI-TOF mass spectrometry and chemometrics.

There is an increasing need for new diagnostic technologies which allow rapid, nonsubjective, and accurate identification of microorganisms. These techniques should ideally complement traditional microbiological and PCR-based methods. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) of whole microbial cells, or their extracts, is considered such a method. This technique was known in the past as intact cell mass spectrometry (ICMS) and has evolved in the past 15 years from a niche research procedure into a widely used practical application with the potential to revolutionize the way microorganisms are identified.(12-16) The technique is based on reproducible detection of microbial protein patterns and thus delivers complementary information to classical microbiological or genotyping methods. In a typical approach characteristic mass spectral fingerprints are obtained which subsequently can be compared against validated databases of bacterial reference spectra.(17, 18) Therefore, an extensive knowledge of biomarker identities is not required, which facilitates high-throughput routine analyses in clinical microbiology and the food industry or to assess public health hazards.
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Identification of Adult Fasciola spp. Using Matrix-Assisted Laser/Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

Identification of Adult Fasciola spp. Using Matrix-Assisted Laser/Desorption Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

Abstract: Fascioliasis is a neglected trematode infection caused by Fasciola gigantica and Fasciola hepatica. Routine diagnosis of fascioliasis relies on macroscopic identification of adult worms in liver tissue of slaughtered animals, and microscopic detection of eggs in fecal samples of animals and humans. However, the diagnostic accuracy of morphological techniques and stool microscopy is low. Molecu- lar diagnostics (e.g., polymerase chain reaction (PCR)) are more reliable, but these techniques are not routinely available in clinical microbiology laboratories. Matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a widely-used technique for identification of bacteria and fungi; yet, standardized protocols and databases for parasite detection need to be developed. The purpose of this study was to develop and validate an in-house database for Fasciola species-specific identification. To achieve this goal, the posterior parts of seven adult F. gigantica and one adult F. hepatica were processed and subjected to MALDI-TOF MS to create main spectra profiles (MSPs). Repeatability and reproducibility tests were performed to develop the database. A principal component analysis revealed significant differences between the spectra of F. gigantica and F. hepatica. Subsequently, 78 Fasciola samples were analyzed by MALDI-TOF MS using the previously developed database, out of which 98.7% (n = 74) and 100% (n = 3) were correctly identified as F. gigantica and F. hepatica, respectively. Log score values ranged between 1.73 and 2.23, thus indicating a reliable identification. We conclude that MALDI-TOF MS can provide species-specific identification of medically relevant liver flukes.
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Rapid Discrimination of Salmonella enterica Serovar Typhi from Other Serovars by MALDI-TOF Mass Spectrometry

Rapid Discrimination of Salmonella enterica Serovar Typhi from Other Serovars by MALDI-TOF Mass Spectrometry

Systemic infections caused by Salmonella enterica are an ongoing public health problem especially in Sub-Saharan Africa. Essentially typhoid fever is associated with high mortality particularly because of the increasing prevalence of multidrug- resistant strains. Thus, a rapid blood-culture based bacterial species diagnosis including an immediate sub-differentiation of the various serovars is mandatory. At present, MALDI-TOF based intact cell mass spectrometry (ICMS) advances to a widely used routine identification tool for bacteria and fungi. In this study, we investigated the appropriateness of ICMS to identify pathogenic bacteria derived from Sub-Saharan Africa and tested the potential of this technology to discriminate S. enterica subsp. enterica serovar Typhi (S. Typhi) from other serovars. Among blood culture isolates obtained from a study population suffering from febrile illness in Ghana, no major misidentifications were observed for the species identification process, but serovars of Salmonella enterica could not be distinguished using the commercially available Biotyper database. However, a detailed analysis of the mass spectra revealed several serovar-specific biomarker ions, allowing the discrimination of S. Typhi from others. In conclusion, ICMS is able to identify isolates from a sub-Saharan context and may facilitate the rapid discrimination of the clinically and epidemiologically important serovar S. Typhi and other non-S. Typhi serovars in future implementations.
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Detection of Methicillin Resistance in Staphylococcus aureus From Agar Cultures and Directly From Positive Blood Cultures Using MALDI-TOF Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay

Detection of Methicillin Resistance in Staphylococcus aureus From Agar Cultures and Directly From Positive Blood Cultures Using MALDI-TOF Mass Spectrometry-Based Direct-on-Target Microdroplet Growth Assay

In this study, we focused on the rapid detection of methicillin resistance in S. aureus using MALDI-TOF MS, that is the first study of using MALDI-TOF MS-based DOT-MGA for Gram- positive species, such as S. aureus. Previous studies investigated the use of MALDI-TOF MS for this purpose by comparing acquired spectra of MSSA strains to spectra of MRSA strains to identify unique mass peaks. They found different mass peaks that were applied to differentiate MSSA and MRSA obtaining different sensitivities and specificities of this procedure ( Edwards-Jones et al., 2000 ; Bernardo et al., 2002 ; Du et al., 2002 ; Wang et al., 2013 ). Josten et al. (2014) identified a small peptide (PSM-mec) produced by agr-positive strains that is part of the genomes of health care-associated MRSA. This peptide can be detected in the MALDI-TOF MS spectra (2415 Da) and could be used for rapid identification of this subgroup of MRSA with high specificity and sensitivity. However, a universal approach to determine methicillin susceptibility is still missing. The newly introduced MALDI-TOF MS-based DOT-MGA is a universal phenotypic assay that offers the opportunity to distinguish between susceptible and non-susceptible isolates in the presence of an antibiotic agent. Here, to discriminate between MSSA and MRSA, isolates were incubated in the presence of cefoxitin and growth or no growth was analyzed.
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Quantification of nitroaromatic explosives in contaminated soil using MALDI-TOF mass spectrometry

Quantification of nitroaromatic explosives in contaminated soil using MALDI-TOF mass spectrometry

The LC –MS/MS system (LCMS-8040, Shimadzu, Manchester, UK) consisted of an HPLC system and a triple-quadrupole mass spectrometer. We successively optimized solvents, the gradient, the flow rate, and the column temperature to achieve a good peak separation and fast run time [ 27 ]. HPLC separation was performed with a Nucleodur C 18 HTec column (150 mm × 2 mm, 3 μm; Macherey-Nagel, Düren, Germany) at a flow rate of 0.15 mL/min and a column temperature of 28 °C in gradient mode with water (mobile phase A) and methanol (mobile phase B). The starting methanol concentration was 57% (0–2 min). The methanol concentration was increased to 70% (2–13 min), maintained at 70% (13–14.8 min), reduced to 10% (14.8–15 min), and finally kept at 10% (15–20 min). UV detection and quantification were done at 254 nm. The mass spectrometer was mass-calibrated against a standard sample for LC–MS (Shimadzu). To increase the efficiency of the electrospray ioni- zation (ESI), 5 mM ammonium acetate and 0.001% ammonium hydroxide were added to mobile phase A. For mass detection, negative-ion ESI MS/MS was used to characterize product and specific fragment ions of the explosives (multiple reaction mon- itoring). The technical parameters for the MS measurements were a spray capillary voltage of 3.0 kV, a detector voltage of 2.04 kV, a desolvation line temperature of 250 °C, a heat block temperature of 450 °C, a nebulizing gas flow rate of 3.0 mL/ min, a drying gas flow rate of 15 mL/min, and a collision- induced dissociation gas pressure of 230 kPa. Data analysis was performed with LabSolutions (version 5.65, Shimadzu). Retention times, precursor and product ions, and individual col- lision energies for the five detectable explosives can be found in Table 1 . Calibration curves were prepared by serial dilution of the mixture of explosives and were measured in the range from 0.001 to 10 ng/ μL before sample analysis. The injection volume of the standards and samples was 1 μL.
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Analysis of Lipids in Biological Tissue by LC-MS and MALDI-TOF Mass Spectrometry Imaging

Analysis of Lipids in Biological Tissue by LC-MS and MALDI-TOF Mass Spectrometry Imaging

Lipid lysate samples were delivered in two groups: transgenic and transgenic+treated. LC-MS data of four transgenic+treated and five transgenic samples were acquired. During each of the nine assays, four negative and four positive-ion mode LC-MS runs were conducted. Lysate sample 17348(2743) had to be measured six times in both ion-modes because measurement inaccuracies occurred. For all the ESI-MS analyses of the mouse brain tissue, a preceding methanolic extraction (see section 3.4.1.) was necessary. The obtained lipid mixture was injected into the LC-MS system using a reversed phase column (Eclipse XDB-C18) and a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific Inc.). The solvent composition used for HPLC analysis (see section 3.4.1.) Since LC-MS data using Orbitrap delivered low-ppm mass accurate spectra, lipids could possibly be assigned by comparing the measured mass to the exact calculated one of one species. Utilizing Lipid MAPS Database 5 as an approving settlement, a prior selection of lipid classes, such as PC or PI, was granted. However, a full characterization of single lipid isomers required more detailed knowledge about their structure. Characteristic fragmentations provided informative evidence, comprising the identification of fatty acid chain lengths or functional groups, such as inositol phosphate or phosphocholine. In positive ion-mode fragmentations were manually created using ChemDraw Professional 17.1 (Perkin Elmer) 38 , whereas negative spectra could directly be compared to simulated ones from Lipid MAPS Database 5 . As exemplarily depicted in Figure 5, fragment spectra usually contained the typical functional group of the lipid class, and characteristic peaks signifying the number of carbon and hydrogen atoms in the respective fatty acids.
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Erzeugung von Taq-DNA-Polymerasemutanten und Untersuchung ihrer Substratspezifität mittels MALDI-TOF-Massenspektrometrie

Erzeugung von Taq-DNA-Polymerasemutanten und Untersuchung ihrer Substratspezifität mittels MALDI-TOF-Massenspektrometrie

An efficient high-throughput screening method based on MALDI-TOF mass spectrometry is des- cribed which was used to screen thirteen mutants of Taq DNA polymerase for substrate specificity. The recombinant polymerases were cloned using an established expression system employing E. coli bacteria. Two amino acid positions in the O-helix of the polymerase domain were investigated: phenylalanine at position 667 was exchanged with tyrosine; tyrosine (671) was exchanged with each, phenylalanine, serine, threonine, tryptophane, histidine, and proline. In a second series of ex- periments each variant (Y671) was cloned in combination with a second amino acid exchange F667Y. The Taq DNA polymerase mutants were expressed using standard procedures and isolated by immobilized metal ion affinity chromatography. Preliminary investigations with standard PCR showed that the mutation Y671F was tolerated without loss of activity. None of the other variants was able to produce amounts of PCR product detectable in gel electrophoresis. Therefore the speci- fic activity of selected polymerase mutants was determined by a non-radioactive polymerase assay. The enzyme with the single mutation F667Y displayed 246 % activity compared with the wildtype O-helix enzyme. The single mutation Y671F and the combination Y671F/F667Y retained a specific activity of 14 % and, 18 % respectively.
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Two-dimensional reversed-phase liquid chromatography coupled to MALDI TOF/TOF mass spectrometry : an approach for shotgun proteome analysis

Two-dimensional reversed-phase liquid chromatography coupled to MALDI TOF/TOF mass spectrometry : an approach for shotgun proteome analysis

A total of 1208 proteins were identified by more than one peptide by the application of 2D-RP-HPLC MALDI TOF/TOF to the cytosolic proteome of C. glutamicum but, the analysis time necessary was 375 hours. Although this argument can be considered a drawback of the method, there are a number of improvements which could shorten the analysis time. With the aid of the fast screening method employing MALDI TOF mass spectrometry it is possible to determine the low peptide content fractions and pool them together thus reducing the total number of fractions to analyze. As an example, pooling the first and last five fractions would save up to 30 LC-MALDI runs thus reducing the analysis time by 90 hours. Also the use of an exclusion list across different runs can improve the measurements [97] because peptides showing intensive signals tend to be analyzed repetitively in different runs. In addition the chance of identifying low abundant peptides would be enhanced. The overall orthogonality can be improved by modifying the gradient of the second dimension for the latest fractions which contain the more hydrophobic peptides. The relative tolerance of the MALDI MS to the presence of surfactants and phosphate additives permits the application of alternative chromatography strategies not considered for the on-line LC-ESI-MS/MS. Alternatively, the use of ionic liquid matrices for proteome analysis can be considered [113].
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Mass spectrometry based proteomics in medical diagnostics

Mass spectrometry based proteomics in medical diagnostics

The instability of instrument performance is one of the main reasons why MS based shotgun proteomics has not been as widely adopted as might seem ap- propriate. From Professor Mann’s lab’s experience, the combination of HPLC and Orbitrap mass spectrometers deviate from the desired performance around once per week. A certain expertise and experience is required to resolve these issues [85] and presently most problems occur on the HPLC side [52]. For clinical applications it would be favorable, if all MS-instrumentation operated robustly guaranteeing reliable and reproducible results without the permanent checking of an MS expert. As proteomics becomes more attractive for clinical application, MS manufacturers will probably increase their efforts to develop robust instru- mentation. MALDI-TOF MS serves as a positive example, but it should not be forgotten that it took decades to come up with an instrument that fits clinical needs so well. Therefore, it seems justified to be carefully optimistic that MS instrumentation suitable for non-expert clinical use will be developed in the next decade.
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Anwendbarkeit der Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) für den Nachweis und die Differenzierung von Leptospira spp. im Vergleich zum Multilocus Sequence Typing (MLST)

Anwendbarkeit der Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) für den Nachweis und die Differenzierung von Leptospira spp. im Vergleich zum Multilocus Sequence Typing (MLST)

Lange Zeit konnte die Massenspektrometrie (MS) nur für die Analyse von Atomen oder großen Molekülen verwendet werden, da die Ionisierung der Proben zu unkontrollierbaren Veränderungen in der chemischen Struktur der Analyten führte (SAUER et al., 2010; VESTAL, 2011). Im Jahr 1988 beschrieben Karas und Hillenkamp erstmals eine neue Ionisierungstechnik die keine Strukturänderungen der Moleküle mehr verursachte (KARAS et al., 1988). Mit der Einführung des matrix assisted laser desorption ionization (MALDI)- Prinzips konnten zum ersten Mal große Moleküle wie Proteine und DNA-Oligomere anhand ihrer Größenunterschiede differenziert werden (VESTAL, 2011). Mit der Weiterentwicklung massenspektrometrischer Verfahren in den späten 80er Jahren konnten Peptide und Proteine in biologischen Materialien analysiert werden (SAUER et al., 2010). Lange Zeit wurde MS nur für die Charakterisierung von Proteinen, Lipiden und Zuckern verwendet. Für den Einsatz in der Mikrobiologie war die Technologie lange mit einem zu großen Aufwand verbunden. Erst im Jahr 1996 wurde das Prinzip des MALDI-TOF MS erstmals für die Differenzierung ganzer Zellen von grampositiven und gramnegativen Bakterien angewendet, ohne das die Proteine der Bakterienzelle vorher extrahiert wurden (CLAYDON et al., 1996). MALDI-TOF MS wird seither für die Identifizierung von Bakterien in der Routinediagnostik und der taxonomischen Einteilung von Mikroorganismen verwendet (WELKER (2), 2011). Dabei werden Bakterien- zellen anhand der Unterschiede ihrer Proteinmassen differenziert, die durch ein Massenspektrometer erfasst werden (STEPHAN et al., 2010).
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Editorial: MALDI-TOF MS Application for Susceptibility Testing of Microorganisms

Editorial: MALDI-TOF MS Application for Susceptibility Testing of Microorganisms

Although being a phenotypic approach, matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF MS)-based analysis of the whole-organism protein mass spectra offers the closest approximation to the ribosomal sequence information. This method has been successfully established for the identification and subtyping of bacterial and fungal microorganisms ( Wieser et al., 2012; Clark et al., 2013 ) and, meanwhile, it has become a routine method in many diagnostic laboratories contributing enormously to a reduction of the time- to-result in identification procedures ( Idelevich et al., 2019 ). However, for susceptibility testing, rapid phenotypic approaches feasible for routine applications are still missing ( Schubert and Kostrzewa, 2017 ). As shown in this special issue summarizing the results of the respective Frontiers Research Topic “MALDI-TOF MS Application for Susceptibility Testing of Microorganisms,” this technology is able to provide solutions for rapid determination of antibiotic susceptibilities independently of the underlying resistance phenotype.
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MALDI imaging mass spectrometry in clinical proteomics research of gastric cancer tissues

MALDI imaging mass spectrometry in clinical proteomics research of gastric cancer tissues

tissues might better reflect the underlying pathological state of cancers than gene expression patterns. A few tissue-based reports in gastric cancer have shown that proteomic patterns with surface-enhanced laser desorption/ionization-TOF can distinguish cancer patients from non-cancer patients [109, 110]. A very recent report in gastric cancer demonstrated that protein profiles obtained from endoscopic biopsy samples via MALDI imaging can distinguish pathologic early stage tumors from more advanced tumors [79]. However, none of the mentioned studies performed prognostic evaluations of the protein patterns. This study is the first to show that tissue-based proteomic profiling by MALDI imaging is able to identify protein patterns that predict patient survival in intestinal-type gastric cancer. Previously known and, more importantly, previously unknown protein biomarkers were identified, amongst them HNP-1, CRIP1, and S100-A6. Interestingly, both HNP-1 and CRIP1 have been described in the context of the immune system [111, 112]. It is known from clinical and experimental studies that the immune system is a significant determinant of epithelial tumorigenesis and further development [113].
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Evaluierung des strukturaufklärenden Verfahrens MALDI-TOF-Massenspektrometrie in der Identifizierung von filamentösen Schimmelpilzen / eingereicht von Thuy Huyen Le

Evaluierung des strukturaufklärenden Verfahrens MALDI-TOF-Massenspektrometrie in der Identifizierung von filamentösen Schimmelpilzen / eingereicht von Thuy Huyen Le

In der MALDI Analyse von Schimmelpilzen sind prinzipiell zwei präanalytische Protokolle unterscheidbar, die IC- (Intact Cell MS) Methode und die CL- (Cell Lysis MS) Methode. In der IC-Methode werden zur Analyse Proben von einem Festmedium entnommen und gemeinsam mit einer Matrixlösung direkt auf ein MALDI-Target aufgetragen. Dabei kann die Methode sowohl durch präanalytische Handhabung mit Ameisensäure (Formic Acid, FA), im Sinne einer short on-target Extraktion, präpariert werden oder aber auch direkt ohne Vorbehandlung zur Analyse ausgelesen werden. Vereinzelte IC-Verfahren wurden bereits in der Bestimmung von Schimmelpilzen angewendet, jedoch ist diese Methode überwiegend in der Bakterienidentifikation anzutreffen.
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High-resolution atmospheric-pressure MALDI mass spectrometry imaging workflow for lipidomic analysis of late fetal mouse lungs

High-resolution atmospheric-pressure MALDI mass spectrometry imaging workflow for lipidomic analysis of late fetal mouse lungs

In this study, high mass accuracy (≤2 ppm), high-resolution in mass (140,000 @ m/z 200) and space (10 µm per pixel) atmospheric pressure scanning microprobe MALDI MSI (AP-SMALDI MSI) was used for the first time to characterize the lipid profile of late fetal mouse lungs at day 19 of gestation (E19) in positive- and negative-ion mode. An optimized sample preparation protocol and data analysis workflow were developed for the reliable and reproducible relative quantification of lipids and other cellular metabolites in different tissue sections. To demon- strate the power of our optimized method for relative comparisons of distinct lung sections with alterations in lipid content, knockout (KO) mice with a peroxisomal biogenesis defect (Pex11β−/−) were used in addition to wild type (WT) sections, as an experimental model.
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Systematischer Vergleich von Methoden zur schnellen Identifizierung von Bakterien aus Blutkulturproben mittels MALDI-TOF Massenspektrometrie

Systematischer Vergleich von Methoden zur schnellen Identifizierung von Bakterien aus Blutkulturproben mittels MALDI-TOF Massenspektrometrie

Die Messung erfolgte in einem Bruker Microflex LT Massenspektrometer im linear positiven Modus bei einer Laserimpulsfrequenz von 20 Hz, einer Be- schleunigungsspannung von 20 kV und einer Pulsdauer von jeweils 250 ns. Erfasst wurden Signale mit einem Masse-Ladungsverhältnis im Bereich von 2000 bis 20000 m/z. Für die Kalibrierung des Massenspektrometers wurde ein Proteingemisch im entsprechenden Massebereich verwendet (Bruker Bacterial Test Standard). Von jedem Messfeld wurde ein Summenspektrum aus 280 Einzelspektren akquiriert. Die Weiterverarbeitung der Summen- spektren erfolgte mit der MALDI-Biotyper 2.0 Software. Unter Verwendung der Standardeinstellungen für die Identifizierung von Mikroorganismen wurde für jedes Summenspektrum die Biotyper-ID mit zugehörigem Identi- fikationsscore ermittelt. Die Biotyper-ID entspricht der Speziesbezeichnung Referenzspektrums in der Biotyper-Datenbank mit der größten Ähnlichkeit zum Testspektrum. Der Identifikationsscore ist ein logarithmisch skalierter Ähnlichkeitswert zwischen 0 und 3, der den Grad spektraler Übereinstim- mung mit dem jeweiligen Referenzspektrum bemisst (Sauer et al. 2008). Als Biotyper-ID und Identifikationsscore einer Probe wurden ID und Identi- fikationsscore der Replikatmessung mit dem höchsten Identifikationsscore verwendet.
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Gutenberg Open Science: Untersuchungen zur Position der MALDI-TOF Massenspektrometrie in der Analytik von synthetischen Polymeren

Gutenberg Open Science: Untersuchungen zur Position der MALDI-TOF Massenspektrometrie in der Analytik von synthetischen Polymeren

Ein Vergleich der Verteilungen in Abbildung 62 macht deutlich, dass die beiden Methoden unterschiedliche Verhältnisse der Signalflächen liefern. Im Gegensatz zum HPLC-Chromatogramm nehmen die Signalflächen im MALDI-TOF Spektrum nicht monoton ab; außerdem ergibt sich die höchste Intensität für das Oligomer mit sieben Phenyleneinheiten (siehe Abbildung 63). Für beide Techniken stimmen die Steigungen in dem semilogarithmischen Diagramm ab einer Kettenlänge von circa 11 Phenyleneinheiten interessanterweise überein. Dies ist auch in etwa die Kettenlänge, bei der das Absorptionsspektrum mit dem des Polymers identisch wird (vergleiche Abbildung 58), und die daher auch als effektive Konjugationslänge bezeichnet wurde. Leider können aus dem Massenspektrum nur die Signalflächen der Oligomere bis zu 19 Phenyleneinheiten mit ausreichender Genauigkeit bestimmt werden, da die Signale der höheren Oligomere im Rauschen der Basislinie verschwinden. Aus den vorliegenden Daten ist die Frage, ob die Abhängigkeit der Signalfläche von der Kettenlänge in der HPLC und der MALDI-TOF Massenspektrometrie ab einer Größenordnung von 11 Phenyleneinheiten wirklich identisch wird, oder ob es sich lediglich um einen Zufall in einem beschränkten Massenbereich handelt, nicht zu beantworten.
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Developing mass spectrometry towards applications in clinical proteomics

Developing mass spectrometry towards applications in clinical proteomics

Mass spectrometry is an analytical technology that is used in a variety of fields, including medicine, life sciences, pharmaceutical sciences, organic chemistry and physics 79 . Having such a wide range of applications, the rapidly evolving technique of mass spectrometry has its roots in one of the key experiments of JJ Thomson where he studied the movement of electrons (negatively charged cathode ray particles) and other charged particles in electromagnetic fields 80 . In his experiments, J. J. Thompson demonstrated that the movement of these charged particles in vacuum under the influence of electric and magnetic fields is dependent upon the mass to charge ratio denoted as m/z. Mass spectrometers were commercially available from as early as 1943 and the early reports explaining the principles of time of flight (TOF) and ion cyclotron resonance (ICR) mass spectrometry were published in 1946 and 1948 respectively 81, 82 . The ICR mass spectrometry requires strong magnets and this issue was alleviated when Paul described the use of quadrupole and ion traps as mass analyzers 83, 84 and hence these ion traps are also called Paul traps. Later the idea of tandem mass spectrometry evolved which resulted in the birth of fragmentation mass spectrometry (MS/MS) a hallmark in unambiguous structure determination 85 . This fragmentation or MS/MS turned out to be at the heart of MS-based
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Theoretical Investigations on Cluster Formation in Mass Spectrometry

Theoretical Investigations on Cluster Formation in Mass Spectrometry

QHA results Most promising to tackle the vibrational partition functions of the clusters was the QHA method. Herein, the overall translational and rotational par- tition functions were calculated from standard approximations from quantum me- chanics as found in the GAUSSIAN 16 thermochemistry output [ 96 ]. For the two frag- ments ACN, (ACN)H + as well as for the proton bound cluster (ACN) 2 H + we conducted geometry optimization, NMA and three MD simulations with different starting condi- tions. For the two fragments only the Cartesian coordinates along the MD trajectories were Eckart frame corrected, whereas internal rotation was additionally separated out for the cluster (prior to the Eckart frame correction) according to the numerical approach described in Appendix B . Since both fragments rotate with respect to the fixed coordinate system, the correction was applied for both fragments to prevent an overall rotation of the Eckart frame. Principally the Eckart treatment is designed to manage such situations, however, we noticed severe numerical instabilities for the following reason: When the molecule rotates 60°, the Eckart frame jumps back to its original position due to the C 3 -symmetry of the molecule. At this point small hy- drogen vibrations, independent from the rotation, cause numerous jumps around the 60° mark, which leads to virtually random movement and thus large errors in the transformation to the normal coordinates. Accordingly, it is easier to explicitly avoid the overall rotation by correcting the internal rotation of both fragments. The methyl hydrogen atoms were chosen to determine the center of mass (CoM) of the internal rotor and the axis of rotation was chosen to be the vector drawn from the CoM of the entire molecule to the CoM of the internal rotor R 0 − R. Since in the equilibrium geometry the rotor is balanced, σ 0 was set to zero (see Appendix B ).
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Mass spectrometry imaging in chemical ecology

Mass spectrometry imaging in chemical ecology

Max Planck Institute for Chemical Ecology, Hans-Knöll-Straße 8, 07745 Jena, Germany E-mail: svatos@ice.mpg.de Laterally resolved metabolomics (LRM) is an innovative way studding metabolites in different tissues including plants. A particular area of LRM is mass spectrometry imaging (MSI), where MS data are acquired from predefined spots. Typically an area of interest is scanned as a raster with predefined spot-to-spot steps. Achieved lateral resolution is related to focus of desorption/ionization beam of ions or light. In our labs UV or IR lasers are used for ion desorption/ionization. A commercial SMALDI probe connected to Q-Exactive+ spectrometer combines 2-3 µm lateral resolution with 250,000 mass resolution and attomolar sensitivity. When samples contain substantial amounts of water, mid-IR laser (2940 nm) can be used for ion evaporation from tissue. IR-laser ablated metabolites are further ionized in perpendicular electrospray plume and formed ions are detected in Synapt G1 tandem mass spectrometer. We are operating a prototype of such an LAESI instrument, where profilometry on samples are measured prior MSI and z-coordinates are corrected to achieve constant laser focus on real samples with pronounced topology. Lateral resolution is currently 40 µm sufficient for LRM of individual plant cells. In summary IR-Laser ablation can be guided in the 3 rd dimension to overcome the influence of surface topography on laser focus for consistent laser ablation marks size in mass spectrometry imaging experiments. Diverse chemical can be imaged both in positive/negative ion mode.
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High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora

High-resolution MALDI mass spectrometry imaging of gallotannins and monoterpene glucosides in the root of Paeonia lactiflora

other polyphenolic components since indicators are unspecific (based on all substrates having phenolic groups). In the present work, spatio-chemical information on the distribution of metabolites in the root of P. lactiflora was explored by the combination of a high spatial-resolution of 10 μ m and a high mass resolution of 140,000 at m/z 200. Due to the high quality of the obtained ion images, spatial contexts of individual gallotannins and monoterpenes could be revealed for the first time at the cellular level. High mass accuracy (< 3 ppm root mean square error (RMSE)) and on-tissue MS/MS measurements helped in identifying metabolites. These results are in accordance with earlier histochemical studies conducted on different plant organs 32–34 , but are more detailed.
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