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Dive into the research topics where Martin F. Almstetter is active.

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Featured researches published by Martin F. Almstetter.


Analytical and Bioanalytical Chemistry | 2011

Metabolite extraction from adherently growing mammalian cells for metabolomics studies: optimization of harvesting and extraction protocols.

Katja Dettmer; Nadine Nürnberger; Hannelore Kaspar; Michael Gruber; Martin F. Almstetter; Peter J. Oefner

Trypsin/ethylenediaminetetraacetic acid (EDTA) treatment and cell scraping in a buffer solution were compared for harvesting adherently growing mammalian SW480 cells for metabolomics studies. In addition, direct scraping with a solvent was tested. Trypsinated and scraped cell pellets were extracted using seven different extraction protocols including pure methanol, methanol/water, pure acetone, acetone/water, methanol/chloroform/water, methanol/isopropanol/water, and acid–base methanol. The extracts were analyzed by GC-MS after methoximation/silylation and derivatization with propyl chloroformate, respectively. The metabolic fingerprints were compared and 25 selected metabolites including amino acids and intermediates of energy metabolism were quantitatively determined. Moreover, the influence of freeze/thaw cycles, ultrasonication and homogenization using ceramic beads on extraction yield was tested. Pure acetone yielded the lowest extraction efficiency while methanol, methanol/water, methanol/isopropanol/water, and acid–base methanol recovered similar metabolite amounts with good reproducibility. Based on overall performance, methanol/water was chosen as a suitable extraction solvent. Repeated freeze/thaw cycles, ultrasonication and homogenization did not improve overall metabolite yield of the methanol/water extraction. Trypsin/EDTA treatment caused substantial metabolite leakage proving it inadequate for metabolomics studies. Gentle scraping of the cells in a buffer solution and subsequent extraction with methanol/water resulted on average in a sevenfold lower recovery of quantified metabolites compared with direct scraping using methanol/water, making the latter one the method of choice to harvest and extract metabolites from adherently growing mammalian SW480 cells.


Journal of Dairy Science | 2010

Nuclear magnetic resonance and mass spectrometry-based milk metabolomics in dairy cows during early and late lactation

Matthias S. Klein; Martin F. Almstetter; Gregor Schlamberger; Nadine Nürnberger; Katja Dettmer; Peter J. Oefner; Heinrich H. D. Meyer; Steffi Wiedemann; Wolfram Gronwald

Milk production in dairy cows has dramatically increased over the past few decades. The selection for higher milk yield affects the partitioning of available nutrients, with more energy being allocated to milk synthesis and less to physiological processes essential to fertility and fitness. In this study, the abundance of numerous milk metabolites in early and late lactation was systematically investigated, with an emphasis on metabolites related to energy metabolism. The aim of the study was the identification and correlation of milk constituents to the metabolic status of the cows. To investigate the influence of lactation stage on physiological and metabolic variables, 2 breeds of different productivity were selected for investigation by high-resolution nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. We could reliably quantify 44 different milk metabolites. The results show that biomarkers such as acetone and beta-hydroxybutyrate are clearly correlated to the metabolic status of the individual cows during early lactation. Based on these data, the selection of cows that cope well with the metabolic stress of early lactation should become an option.


Analytical and Bioanalytical Chemistry | 2012

Comprehensive two-dimensional gas chromatography in metabolomics.

Martin F. Almstetter; Peter J. Oefner; Katja Dettmer

One of the major objectives in metabolomics is the identification of subtle changes in metabolite profiles as affected by genetic or environmental factors. Comprehensive two-dimensional gas chromatography (GC × GC) hyphenated to a fast-acquisition mass spectrometer is a well-established analytical technique to study the composition of complex samples due to its enhanced separation capacity, sensitivity, peak resolution, and reproducibility. This review reports applications of GC × GC to metabolomics studies of sample of different types (biofluid, cells, tissue, bacteria, yeast, plants), and discusses its advantages and limitations.


Analytical Chemistry | 2009

Integrative normalization and comparative analysis for metabolic fingerprinting by comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry.

Martin F. Almstetter; Inka J. Appel; Michael Gruber; Claudio Lottaz; Birgit Timischl; Rainer Spang; Katja Dettmer; Peter J. Oefner

Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC x GC-TOF-MS) was applied to the comparative metabolic fingerprinting of a wild-type versus a double mutant strain of Escherichia coli lacking the transhydrogenases UdhA and PntAB. Using peak lists generated with the Leco ChromaTOF software as input, we developed retention time correction and data alignment tools (INCA). The accuracy of peak alignment and detection of 1.1- to 4-fold changes in metabolite concentration was validated by a spike-in experiment with 20 standard compounds. A list of 48 significant features that differentiated the two E. coli strains was obtained with an estimated false discovery rate (FDR) of <0.05. A total of 27 metabolites, mainly from the citrate cycle, were identified. That the signal intensity of the m/z 73 trace of the trimethylsilyl (TMS) group reflected true differences in metabolite abundance was confirmed by quantification of pyruvate, fumarate, malate, succinate, alpha-ketoglutarate, citrate, cis-aconitate, myo-inositol, and glucose-6-phosphate using compound specific fragment ions and stable isotope labeled standards. Relative standard deviations for metabolite extraction and GC x GC-TOF-MS analysis of those analytes ranged from 13.2 to 26.3% for the universal m/z 73 trace and 7.4 to 24.5% for the analyte specific fragment ion trace.


Journal of Chromatography A | 2011

Improved enantiomer resolution and quantification of free d-amino acids in serum and urine by comprehensive two-dimensional gas chromatography–time-of-flight mass spectrometry

Magdalena C. Waldhier; Martin F. Almstetter; Nadine Nürnberger; Michael Gruber; Katja Dettmer; Peter J. Oefner

The potential of comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOFMS) in the quantitative analysis of amino acid enantiomers (AAEs) as their methyl chloroformate (MCF) derivatives in physiological fluids was investigated. Of the two column sets tested, the combination of an Rt-γDEXsa chiral column with a polar ZB-AAA column provided superior selectivity. Twenty AAEs were baseline resolved including L-Leu and D-Ile, which had failed separation by one-dimensional chiral GC-quadrupole-MS (GC-qMS). Lower limits of quantification (LLOQ) were in the range of 0.03-2 μM. Reproducibility of the analysis of a serum specimen in octaplicate ranged from 1.3 to 16.6%. The GC×GC-TOFMS method was validated by analyzing AAEs in 48 urine and 43 serum specimens, respectively, and by comparing the results with data obtained by a previously validated GC-qMS method. Mean recoveries ranged from 78.4% for D-Leu to 116.4% for D-Pro in urine and 72.2% for L-Thr to 129.4% for L-Ile in serum. The method was applied to the comparison of AAE serum levels in patients suffering from liver cirrhosis to a control group. Significantly increased D-AA concentrations were found for the patient group, whereas L-AA levels were slightly decreased.


Electrophoresis | 2010

Comparison of serum versus plasma collection in gas chromatography – Mass spectrometry‐based metabolomics

Katja Dettmer; Martin F. Almstetter; Inka J. Appel; Nadine Nürnberger; Gregor Schlamberger; Wolfram Gronwald; Heinrich H. D. Meyer; Peter J. Oefner

Bovine serum, EDTA‐plasma and EDTA‐plasma fortified with acetylsalicylic acid (ASA) as antioxidant were compared with regard to their suitability for metabolomic studies. Metabolic fingerprints were generated from GC‐TOF‐MS data using the Leco ChromaTOF® software in combination with the in‐house retention time correction and data alignment tool INCA. A total of 6, 9 and 21 significant features with a false discovery rate of <0.05 were identified by INCA upon comparing EDTA‐ versus EDTA‐ASA‐plasma, EDTA‐plasma versus serum and EDTA‐ASA‐plasma versus serum, respectively. To confirm that the observed signal intensities in the GC‐TOF‐MS fingerprints reflected true metabolite abundances, 19 amino acids, glucose and 6 organic acids were quantified by means of GC‐MS using stable‐isotope‐labeled internal standards. As observed with the fingerprints, only the concentrations of lactate and citrate were found to be significantly lower in EDTA‐plasma and serum, respectively, whereas the concentrations of the other metabolites were similar among the three sample types investigated.


Analytical Chemistry | 2011

Performance Evaluation of Gas Chromatography–Atmospheric Pressure Chemical Ionization–Time-of-Flight Mass Spectrometry for Metabolic Fingerprinting and Profiling

Christian J. Wachsmuth; Martin F. Almstetter; Magdalena C. Waldhier; Michael Gruber; Nadine Nürnberger; Peter J. Oefner; Katja Dettmer

Gas chromatography-atmospheric-pressure chemical ionization-time-of-flight mass spectrometry (GC-APCI-TOFMS) was compared to GC × GC-electron ionization (EI)-TOFMS, GC-EI-TOFMS, GC-chemical ionization (CI)-quadrupole mass spectrometry (qMS), and GC-EI-qMS in terms of reproducibility, dynamic range, limit of detection, and quantification using a mix of 43 metabolites and 12 stable isotope-labeled standards. Lower limits of quantification for GC-APCI-TOFMS ranged between 0.06 and 7.81 μM, and relative standard deviations for calibration replicates were between 0.4% and 8.7%. For all compounds and techniques, except in four cases, R(2) values were above 0.99. Regarding limits of quantification, GC-APCI-TOFMS was inferior to only GC × GC-EI-TOFMS, but outperformed all other techniques tested. GC-APCI-TOFMS was further applied to the metabolic fingerprinting of two Escherichia coli strains. Of 45 features that differed significantly (false discovery rate < 0.05) between the strains, 25 metabolites were identified through highly accurate and reproducible (Δm ± SD below 5 mDa over m/z 190-722) mass measurements. Starting from the quasimolecular ion, six additional metabolites were identified that had not been found in a previous study using GC × GC-EI-TOFMS and an EI mass spectral library for identification purposes. Silylation adducts formed in the APCI source assisted the identification of unknown compounds, as their formation is structure-dependent and is not observed for compounds lacking a carboxylic group.


Journal of Proteome Research | 2013

Correlations between milk and plasma levels of amino and carboxylic acids in dairy cows.

Matthias S. Klein; Martin F. Almstetter; Nadine Nürnberger; Gregor Sigl; Wolfram Gronwald; Steffi Wiedemann; Katja Dettmer; Peter J. Oefner

The objective of this study was to investigate the relationship between the concentrations of 19 amino acids, glucose, and seven carboxylic acids in the blood and milk of dairy cows and their correlations with established markers of ketosis. To that end, blood plasma and milk specimens were collected throughout lactation in two breeds of dairy cows of different milk yield. Plasma concentrations of glucose, pyruvate, lactate, α-aminobutyrate, β-hydroxybutyrate (BHBA), and most amino acids, except for glutamate and aspartate, were on average 9.9-fold higher than their respective milk levels. In contrast, glutamate, aspartate, and the Krebs cycle intermediates succinate, fumarate, malate, and citrate were on average 9.1-fold higher in milk than in plasma. For most metabolites, with the exception of BHBA and threonine, no significant correlations were observed between their levels in plasma and milk. Additionally, milk levels of acetone showed significant direct relationships with the glycine-to-alanine ratio and the BHBA concentration in plasma. The marked decline in plasma concentrations of glucose, pyruvate, lactate, and alanine in cows with plasma BHBA levels above the diagnostic cutoff point for subclinical ketosis suggests that these animals fail to meet their glucose demand and, as a consequence, rely increasingly on ketone bodies as a source of energy. The concomitant increase in plasma glycine may reflect not only the excessive depletion of protein reserves but also a potential deficiency of vitamin B6.


Methods of Molecular Biology | 2012

Metabolic Fingerprinting Using Comprehensive Two-Dimensional Gas Chromatography – Time-of-Flight Mass Spectrometry

Martin F. Almstetter; Peter J. Oefner; Katja Dettmer

Comprehensive two-dimensional gas chromatography-time-of-flight mass spectrometry (GC × GC-TOF-MS) is applied to the comparative metabolic fingerprinting of physiological fluids. Stable isotope-labeled internal standards plus norvaline serve as extraction standards and are added to the blanks, controls and patient samples prior to protein precipitation with methanol. The extracts are evaporated to complete dryness and derivatized in two steps using methoximation with methoxylamine hydrochloride (MeOx) and silylation with N-methyl-N-trimethylsily-trifluoroacetamide (MSTFA). Between derivatization steps a second internal standard containing odd-numbered, saturated straight chain fatty acids is added for quality control and to normalize retention time shifts. After GC × GC-TOF-MS analysis raw data are processed, aligned, and combined in one data matrix for subsequent statistical evaluation. Both a custom-made and the NIST 05 library are used to preliminarily identify significant metabolites. For verification purposes, commercial standards are run individually. Absolute quantification of selected metabolites is achieved by using a multi-point calibration curve and isotope-labeled internal standards.


The Handbook of Plant Metabolomics | 2013

Comprehensive Two‐Dimensional Gas Chromatography for Metabolomics

Katja Dettmer; Martin F. Almstetter; Christian J. Wachsmuth; Peter J. Oefner

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Katja Dettmer

University of Regensburg

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Michael Gruber

University of Regensburg

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Inka J. Appel

University of Regensburg

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