Stefan Neubauer

U13C cell extract of Pichia pastoris – a powerful tool for evaluation of sample preparation in metabolomics

Quantitative metabolic profiling is preceded by dedicated sample preparation protocols. These multistep procedures require detailed optimization and thorough validation. In this work, a uniformly (13)C-labeled (U(13)C) cell extract was used as a tool to evaluate the recoveries and repeatability precisions of the cell extraction and the extract treatment. A homogenous set of biological replicates (n = 15 samples of Pichia pastoris) was prepared for these fundamental experiments. A range of less than 30 intracellular metabolites, comprising amino acids, nucleotides, and organic acids were measured both in monoisotopic (12)C and U(13)C form by LC-MS/MS employing triple quadrupole MS, reversed phase chromatography, and HILIC. Recoveries of the sample preparation procedure ranging from 60 to 100% and repeatability precisions below 10% were obtained for most of the investigated metabolites using internal standardization approaches. Uncertainty budget calculations revealed that for this complex quantification task, in the optimum case, total combined uncertainty of 12% could be achieved. The optimum case would be represented by metabolites, easy to extract from yeast with high and precise recovery. In other cases the total combined uncertainty was significantly higher.

Speciation analysis of orthophosphate and myo-inositol hexakisphosphate in soil- and plant-related samples by high-performance ion chromatography combined with inductively coupled plasma mass spectrometry

A novel method based on high-performance ion chromatography inductively coupled plasma mass spectrometry employing strong anion exchange chromatography with HNO3 gradient elution for simultaneous analysis of orthophosphate and myo-inositol hexakisphosphate (IP6 ) in soil solution and plant extracts is presented. As inductively coupled plasma mass spectrometry analysis of phosphorus at m/z 31 is hampered by N-based interferences, (31)P was measured as (31)P(16)O(+) at m/z 47 employing dynamic reaction cell technique with O2 as reaction gas. Orthophosphate and IP6 were separated within a total chromatographic run-time of 12 min revealing a limit of detection of 0.3 μmol/L. The coefficients of determination obtained in a working range of 1-100 and 1-30 μmol/L were 0.9991 for orthophosphate and 0.9968 for IP6, respectively. The method was successfully applied to extracts from three different soils as well as root and shoot extracts of Brassica napus L. The precision of three independently prepared soil extracts was in the range of 4-10% relative standard deviation for PO4 (3-) and 3-8% relative standard deviation for IP6. Soil adsorption/desorption kinetics for IP6/orthophosphate were performed for investigating the sorption behavior of the two P species in the experimental soils.

ICT: isotope correction toolbox

SUMMARY Isotope tracer experiments are an invaluable technique to analyze and study the metabolism of biological systems. However, isotope labeling experiments are often affected by naturally abundant isotopes especially in cases where mass spectrometric methods make use of derivatization. The correction of these additive interferences--in particular for complex isotopic systems--is numerically challenging and still an emerging field of research. When positional information is generated via collision-induced dissociation, even more complex calculations for isotopic interference correction are necessary. So far, no freely available tools can handle tandem mass spectrometry data. We present isotope correction toolbox, a program that corrects tandem mass isotopomer data from tandem mass spectrometry experiments. Isotope correction toolbox is written in the multi-platform programming language Perl and, therefore, can be used on all commonly available computer platforms. AVAILABILITY AND IMPLEMENTATION Source code and documentation can be freely obtained under the Artistic License or the GNU General Public License from: https://github.com/jungreuc/isotope_correction_toolbox/ CONTACT {christian.jungreuthmayer@boku.ac.at,juergen.zanghellini@boku.ac.at} SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Metabolomics sampling of Pichia pastoris revisited: rapid filtration prevents metabolite loss during quenching.

Metabolomics can be defined as the quantitative assessment of a large number of metabolites of a biological system. A prerequisite for the accurate determination of intracellular metabolite concentrations is a reliable and reproducible sample preparation method, which needs to be optimized for each organism individually. Here, we compare the performance of rapid filtration and centrifugation after quenching of Pichia pastoris cells in cold methanol. During incubation in the quenching solution, metabolites are lost from the cells with a half-life of 70-180 min. Metabolites with lower molecular weights showed lower half-lifes compared to metabolites with higher molecular weight. Rapid filtration within 2 min after quenching leads to only minor losses below 2%, and is thus the preferred method for cell separation.

Enantioselective multiple heartcut two-dimensional ultra-high-performance liquid chromatography method with a Coreshell chiral stationary phase in the second dimension for analysis of all proteinogenic amino acids in a single run

A multiple heartcut (MHC) 2D-UHPLC method with UV detection has been developed for the enantioselective analysis of complex amino acid mixtures in a single run. The MHC method is based on an achiral gradient RPLC separation with 1.8 μm C18 phase (100 × 2.1 mm ID column) in the first dimension (1D) and enantioselective isocratic separation on a tert-butylcarbamoylquinine-based 2.7 μm Coreshell particle column (50 × 3 mm ID) in the second dimension (2D). Pre-column derivatization has been performed with Sangers reagent (2,4-dinitrofluorobenzene) yielding chromogenic 2,4-dinitrophenylated amino acids (DNP-AAs). Heartcuts of 40 μL fractions of the 1D peaks were sampled into the 2D system via a two-position four-port dual valve connected to two loop decks each equipped with six 40 μL parking loops. Using this setup, 25 amino acids (20 proteinogenic plus allo-Thr, allo-Ile, homoserine (Hse), Orn, β-Ala) have been analyzed enantioselectively in a fully automated manner with a single chiral column within 130 min total run time (1D and 2D). All 2D separations together took 101.5 min (29 cuts with 3.5 min run time each) and thus the total analysis time was quite efficiently utilized. Faster separations were restricted by some software constraints which did not allow to adjust run times in 2D individually. The practical utility of this enantioselective MHC method is documented by application for the absolute configuration determination of the amino acids in gramicidin and bacitracin. Further optimizations should lead to a generic enantioselective amino acid analyzer for the quality control of synthetic peptides and the structural characterization of non-ribosomal peptides.