Technologien & Ressourcen

Gas chromatography - mass spectrometry (GC-MS)

Using a combination of a LECO HT time-of-flight mass spectrometer, an AGILENT 7890A gas chromatograph equipped with a GERSTEL MPS2XL autosampler, we are able to quantify a large array of polar plant metabolites. Typically, these comprise a number of 60-100 known metabolites like amino acids, organic acids, polyamins, sugars, sugar phosphates etc. and twice the number of metabolites with unknown chemical structure. Routinely we apply in-line derivatization (derivatization of each individual sample prior injection) to improve the quality of our measurements. This enables us to quantify metabolites from large sample numbers in a highly reproducible manner.

Publications containing GC-MS data:

 

Casartelli, A., D. Riewe, H.M. Hubberten, T. Altmann, R. Hoefgen, S. Heuer: Exploring traditional aus-type rice for metabolites conferring drought tolerance. Rice (2017), accepted/in press.

 

Knoch, D., Riewe, D., Meyer, R.C., Boudichevskaia, A., Schmidt, R., Altmann, T. (2017) Genetic dissection of metabolite variation in Arabidopsis seeds: evidence for mQTL hotspots and a master regulatory locus of seed metabolism. J. Exp. Bot. 68 1655-1667. [link]10.1093/jxb/erx049 (open access).

 

Radchuk V, Tran V, Radchuk R, Diaz-Mendoza M, Weier D, Riewe D, Fuchs J, Hensel G, Kumlehn J, Munz E, Heinzel N, Rolletschek, Martinez M, Borisjuk, L. (2017) Vacuolar processing enzyme 4 contributes to maternal control of grain size in barley by executing programmed cell death in pericarp. New Phytologist (2017) Epub ahead of print. DOI: [link]10.1111/nph.14729.

 

Radchuk V, Riewe D, Peukert M, Matros A, Strickert M, Radchuk R, Weier D, Steinbiß H, Sreenivasulu N, Weschke W, Weber H. Down-regulated sucrose transporters HvSUT1, HvSUT2 affects sucrose homeostasis along its delivery path in barley grains. Journal of Experimental Botany (2017) Epub ahead of print. DOI: [link]10.1093/jxb/erx266.

 

Riewe, D., Jeon, H.J., Lisec, J., Heuermann, M.C., Schmeichel, J., Seyfarth, M., Meyer, R.C., Willmitzer, L. and Altmann, T. (2016) A naturally occurring promoter polymorphism of the Arabidopsis FUM2 gene causes expression variation, and is associated with metabolic and growth traits. Plant J, 88(5), 826-838. [link]10.1111/tpj.13303.

 

Muscolo, A., Junker, A., Klukas, C., Weigelt-Fischer, K., Riewe, D., Altmann, T. (2015) Phenotypic and metabolic responses to drought and salinity of four contrasting lentil accessions. Journal of Experimental  Botany. 66(18):5467-80. [link]10.1093/jxb/erv208 (open access).

 

Junker, A., Muraya, M.M., Weigelt-Fischer, K., Arana-Ceballos, F., Klukas, C., Melchinger, A.E., Meyer, R.C., Riewe, D. and Altmann, T. (2014) Optimizing experimental procedures for quantitative evaluation of crop plant performance in high throughput phenotyping systems. Front Plant Sci, 5, 770. [link]10.3389/fpls.2014.00770 (open acces).

 

Kohl, S., Hollmann, J., Erban, A., Kopka, J., Riewe, D., Weschke, W. and Weber, H. (2015) Metabolic and transcriptional transitions in barley glumes reveal a role as transitory resource buffers during endosperm filling. J Exp Bot, 66, 1397-1411. [link]10.1093/jxb/eru492 (open access).

 

Kempe, K., Rubtsova, M., Riewe, D. and Gils, M. (2013) The production of male-sterile wheat plants through split barnase expression is promoted by the insertion of  introns and flexible peptide linkers. Transgenic Res, 22, 1089-1105. [link]10.1007/s11248-013-9714-7.

 

Riewe, D., Koohi, M., Lisec, J., Pfeiffer, M., Lippmann, R., Schmeichel, J., Willmitzer, L. and Altmann, T. (2012) A tyrosine aminotransferase involved in tocopherol synthesis in Arabidopsis. Plant J, 71, 850-859. [link]10.1111/j.1365-313X.2012.05035.x (open access).

 

Thiel, J., Riewe, D., Rutten, T., Melzer, M., Friedel, S., Bollenbeck, F., Weschke, W. and Weber, H. (2012) Differentiation of endosperm transfer cells of barley: a comprehensive analysis at the micro-scale. Plant J, 71, 639-655. [link]10.1111/j.1365-313X.2012.05018.x (open access).

 

Brotman, Y., Riewe, D., Lisec, J., Meyer, R.C., Willmitzer, L. and Altmann, T. (2011) Identification of enzymatic and regulatory genes of plant metabolism through QTL analysis in Arabidopsis. J Plant Physiol, 168, 1387-1394. [link]10.1016/j.jplph.2011.03.008.

High resolution liquid chromatography - mass spectrometry (LC-MS, BRUKER Maxis 2 QTOF-MS)

A high resolution Bruker Maxis 2 quadrupole time-of-flight mass spectrometer (R = 85000, accuracy = 0.6 ppm) hyphenated with an AGILENT 1290 liquid chromatograph (max 1200 bar) and equipped with a GERSTEL autosampler is used for high throughput metabolite profiling of semi-polar compounds like secondary metabolites, phytosterols, vitamins, hormones... and hydrophobic analytes like lipids, tocoperols, pigments... Typically, several Hundreds to Thousands non-redundant chromatographic features (mz) are quantitatively recorded which can be partially annotated by database queries (KNApSAcK, KEGG) using accurate mass, isotopic pattern and MSMS fragment spectra of the analytes. The liquid chromatograph can be coupled to a mikroplate fraction collector (ADVION Nanomate) to comprehensively isolate separated metabolites for in-depth characterization. The analysis time ranges from 5 min (secondary metabolite profiling, lipid profiles of oil storing organs) to 30 min (comprehensive profiling of semi-polar metabolites, lipid profiling of samples with complex lipidomic composition) and allows reproducible measurements of a large number of samples.

Publications containing LC-MS data:

Riewe, D., J. Wiebach, T. Altmann (2017) Structure annotation and quantification of wheat seed oxidized lipids by high resolution LC-MS/MS. Plant Physiology, epub ahead of print (open access) [link]10.1104/pp.17.00470.

 

 

Metabolomics Server

A Linux server running RStudio ([link]https://www.rstudio.com/) is used for the efficient processing and analysis of large metabolomics datasets. 1TB RAM and 144 cpus allow for fast and parallelized computation of tasks in R from mass spectrometry raw file to result. Practically, we produce peaktables from raw data of our mass spectrometer (GC-MS and high resolution LC-MS) using the R packages TargetSearch (Cuadros-Inostroza et al., 2009), xcms (Smith et al., 2006) and CAMERA (Kuhl et al., 2012). To interpret mass spectral data beyond the possibilities provided by commercial software, tasks like database queries, de-novo sum formula annotation, structure determination by processing and automatized interpretation of MS/MS spectra or statistical cross-omics analyses combining metabolomic with genomic, transcriptional and phenomic data are executed using innovative self-developed R-scripts.

Example publications containing advanced analysis of mass spectrometry data:

Riewe, D., J. Wiebach, T. Altmann (2017) Structure annotation and quantification of wheat seed oxidized lipids by high resolution LC-MS/MS. Plant Physiology, epub ahead of print. [link]10.1104/pp.17.00470 (open access).

 

Knoch, D., Riewe, D., Meyer, R.C., Boudichevskaia, A., Schmidt, R., Altmann, T. (2017) Genetic dissection of metabolite variation in Arabidopsis seeds: evidence for mQTL hotspots and a master regulatory locus of seed metabolism. J. Exp. Bot. 68 1655-1667. [link]10.1093/jxb/erx049 (open access).

 

 

Biomek® FXP workstation

Currently a new Biomek FXP pipetting workstation from Beckman is installed at the IPK in the Department of Molecular Genetics (MOG) of Prof. Dr. Altmann. 
With its large temperature-controlled deck capacity it expands the future needs for flexible laboratory solutions in all aspects of high throughput liquid handling – including pipetting, dilution, dispensing and integration – into a single, automated system. 
The workstation contains a dual-pod pipetting system with a fixed 96-well multichannel pod and an independent span-8 flexible pod that can be used in parallel.
As one of the future central component in the MOG department the workstation is designed as a complete automated system, including extended labware storage, a BRT robotic labware transport system, plate incubation (0°C-70°C), plate washing and Bar-code reading for plates.
Additional components, like a fast capper-decapper-System (CapitAll), a WASP plate heat sealer, a magnetic beads station, a coolable Sigma zentrifuge and a separate and independend accessible Tecan Infinite M200 photometer are integrated and controlled by SAMI® Workstation EX software to maximize capacity and independend through-put of future assay handling.