Bi-phase extraction method using methanol, methyl-terc butyl ether (MTBE), and water provides two phases: one containing polar metabolites (aka “metabolomics”) and different exposome compounds such as drugs (aka “exposomics”), and the other with complex lipids (aka “lipidomics”). During untargeted analyses, all detectable compounds are acquired using validated LC–MS methods.

Since no single LC–MS method can cover the full metabolome/lipidome/exposome, we use different chromatography modes (reversed-phase liquid chromatography, RPLC and hydrophilic interaction chromatography, HILIC) and electrospray ionization modes to comprehensively characterize these isolated fractions.

A high-resolution mass spectrometer Thermo Q Exactive Plus coupled with a liquid chromatograph (Thermo Vanquish) is routinely used for LIMeX workflow. The mass spectrometer collects full scan MS1 data as well as data-dependent MS/MS spectra for all samples. While MS1 data is used for quantification, MS/MS spectra are used for compound annotation using MS/MS library search or for compound confirmation. The acquired data sets are processed using MS-DIAL 4 software, including annotation of complex lipids, polar metabolites, and other detected compounds using in-house, open-source as well as commercial MS/MS spectral libraries (NIST20, MassBank, MoNA).

LIMeX workflow for untargeted and targeted analysis of complex lipids, polar metabolites, and exposome compounds.

For human cohort studies, we usually report over 500 complex lipids, 100 polar metabolites, and tens of exposome compounds (mainly food components and drugs) using LIMeX−4D (i.e., 4 LC−MS platforms).

All compounds are quantified as peak heights and reported in “arbitrary units”. These values are used as inputs for one-dimensional or multidimensional statistical methods (e.g., t-test, fold-change, heatmap, principal component analysis, partial least squares-discriminant analysis).

The LIMeX workflow uses over 60 different internal standards, which can be used for estimations of absolute concentrations for various metabolites detected in plasma/serum and tissues.

The sample preparation for these low-abundant compounds is more demanding and solid-phase extraction (SPE) is involved in the workflow to remove high-abundant compounds (e.g., phospholipids).

A quadrupole/linear ion trap mass spectrometer (SCIEX QTRAP 5500) operated in multiple reaction monitoring (MRM) coupled with a liquid chromatograph (Dionex/Thermo Ultimate 3000 RSLC) is used for analysis.

Theoretical isotopologues are calculated for annotated metabolites in Python and instrumental files are processed through MRMPROBS software. Peak heights are adjusted for the natural abundance of elements and tracer purity using IsoCor.

The lab is GLP certified for analytical and clinical chemistry testing.

For GLP studies, the following three steps are essential:

A high-resolution mass spectrometer Thermo Q Exactive Plus coupled with a liquid chromatograph (Thermo Vanquish) is routinely used for the analysis of pharmaceutical compounds.

Send this form to Dr. Tomáš Čajka and deliver your samples to:

• Institute of Physiology CAS
• Laboratory of Metabolomics, room E-216
• Videnska 1083, Prague, 14220
• Czech Republic

After receiving your samples, a unique project ID is generated and is used within all platforms, for the final report, and data archiving.

Ing. Jiří Hricko
Research Assistant
LC–MS, GLP

Mgr. Michaela Paučová
Research Assistant
LC–MS, GLP

Mgr. Tatyana Kobets, Ph.D.
Research Assistant
statistics, bioinformatics

Ing. Michaela Nováková
Research Assistant
statistics, bioinformatics

Bc. Aleksandra Shumilova
Research Assistant
statistics, bioinformatics

Mgr. Petra Zedníková (maternity leave)
Research Assistant

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