1. FIMM - Institute for Molecular Medicine Finland
P.O. Box 20, FI-00014 University of Helsinki, Finland
Biomedicum Helsinki 2U, Tukholmankatu 8, 00290 Helsinki, Finland
www.fimm.fi
A Hi gh- t hr oughput Mas s
Spect r omet r y Bas ed Met hod
f or t he Quant i t at i ve
Tar get ed Met abol omi cs
Introduction
Metabolites 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
Objective
To 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 methods
Standard Solution and Quality Control Sample
Calibration 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 Preparation
Polar 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 .
Results
We 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 hours
2. Small sample portion size 100 ul
3. 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 validation
Linearity
Linearity 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
Conclusion
Our 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 Method
Liquid 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, Finland
2 VTT Technical Research Centre of Finland, Helsinki, Finland * Equal contribution