FIMM - Institute for Molecular Medicine FinlandP.O. Box 20, FI-00014 University of Helsinki, FinlandBiomedicum Helsinki 2U, Tukholmankatu 8, 00290 Helsinki, Finland

www.fimm.fi

A High-throughput Mass Spectrometry Based Method for the Quantitative Targeted Metabolomics

IntroductionMetabolites are the small chemical entities involved in the cellular metabolism. Metabolomics is a discovery-driven field of science, which aims at the comprehensive quantitative analysis of all metabolites in any biological system or a specific physiological state.

Unlike other “omics” studies metabolomics provide the functional readout of a cellular phenotype.

Fig1: Schematic of the “omic” hierarchy. The flow of information from genes to metabolites

ObjectiveTo develop a high throughput quantitative method in plasma and serum sample for targeted metabolite profiling across different classes of metabolites in a single run.

Experimental methodsStandard Solution and Quality Control SampleCalibration standard solution (multi-analytes and multi-concentrations) containing 94 polar metabolites at their individual concentration ranges were prepared at 80/20 Acetonitrile/Water+1% formic acid.Pooled blood plasma were used as quality controls during the analysis.

Automated Sample PreparationPolar metabolites were extracted from the human plasma by protein precipitation method in 96 well plate. Extraction method was automated using Hamilton Star liquid handling system to increase the throughput and decrease the inter-assay variations .

ResultsWe have developed and optimized a method for quantitative analysis of targeted metabolites in a high throughput manner. This targeted method allow;

1. Analysis of 96 samples per 24 hours2. Small sample portion size 100 ul3. High selectivity provided by MRM 4. High sensitivity for trace level

applications (up to pmol)5. Quantification of 94 metabolites

covering 15 classes) by using labeled internal standards or through external standards

Fig4:Pie chart representing the number of metabolite classes

Method validationLinearityLinearity was determined with 11 different concentration levels of the diluted standards covering 3 orders of magnitude.

Precision (repeatability) and Process Efficiency (PE):Precision was carried out using five replicates (QC’s). All within-day RSD were below 10%. PE was calculated by adding known concentrations of spike to the sample before and after extraction. Figure 5 represents PE ranging from 53-122 % for metabolites for which labeled internal standards were used.

Fig5: Precision and process efficiency of the method

ConclusionOur developed method has an ability to detect large number of polar metabolites in wide range of concentrations in a single analysis. This targeted metabolite profiling method has high potential for the discovery of the biomarkers for the disease risk, to meet the clinical demands.

UPLC MethodLiquid chromatography was performed on an Acquity UPLC system (Waters corporation, Milford) coupled with a Hydrophilic Interaction Chromatography (HILIC) column. HILIC in general is commonly used due to the high compatibility with mass spectrometry and is powerful technique for the retention of polar analytes that offers higher selectivity, higher sensitivity and improved efficiency for quantification of metabolites in biological matrices.

Mass Spectrometry Method:LC/MS/MS analysis was performed on an Waters XEVO-TQ S triple quadrupole mass spectrometry by polarity switching electro spray ionization (ESI) using multiple reaction monitoring (MRM) method.

Fig2: : Experimental protocol for metabolite extraction using robot

Fig3: Waters XEVO-TQS triple quadrupole mass spectrometer and Hamilton star liquid handling system

Vasudev Kantae 1, Jean-Christophe Yorke 1,Vidya R. Velagapudi* 1, Matej Orešič* 2

1 Metabolomics Unit, Technology Centre, Institute for Molecular Medicine Finland FIMM, Helsinki, Finland2 VTT Technical Research Centre of Finland, Helsinki, Finland * Equal contribution