Reiko Kiyonami (Presenter)
Thermo Fisher Scientific
Bio: Senior strategic marketing specialist in Thermo Fisher Scientific. The main research areas are focused on developing new Omics workflows (untargeted omics profiling approaches and targeted protein/peptide and metabolite quantitation techniques) on state of art HRAM Orbitrap-based mass spectrometers for supporting biomarker discovery, validation and system biology studies.
Authorship: Reiko Kiyonami and David Peak
Thermo Fisher Scientific, San Jose, CA 95134
| Short Abstract Lipids play a key role in cell, tissue and organ physiology with diseases such as diabetes which involve disruption of their metabolic enzymes and pathways. Identification of unique lipid biomarkers to distinguish healthy humans compared to those with a disease can have an impact on the early detection of diseases and personalized medicine. Here we demonstrate that HRAM LC MSn approach on a high filed hybrid quadrupole Orbitrap mass spectrometer enables rapid putative biomarker discovery through lipidomics profiling experiments. Two phenotypes of the rat (ZDF vs. lean wild type) plasma samples were used as a model case. |
Long Abstract
Lipids are known to play a key role in cell, tissue and organ physiology and have been implicated in many diseases such as cancer and diabetes. Lipids may also play a role as potential markers for the presence or absence of certain disease states. The identification of such unique biomarkers may aid in detecting potential risk of disease in certain individuals. Although recent advances in HPLC-MS platforms have allowed the rapid and sensitive detection of a variety of lipid species with minimal sample preparation, many challenges remain. The diversity in the structural and physical / chemical properties of the lipidome requires the combination of HPLC separation technique with a high resolution, accurate mass spectrometer for accurate lipid molecular ion determination. Moreover, the molecular weight information only may not be sufficient for identifying each isomer of individual lipid species. MS/MS information is further required for unambiguous identification of each individual lipid species in biological samples.
The new released Thermo Scientific™ Q Exactive™ HF hybrid quadrupole-Orbitrap mass spectrometer features an ultra-high-field Orbitrap analyzer which doubles its speed and resolution compared to first generation of Orbitrap analyzer (Figure 1). The ultra-high resolution (up to 240,000) of the Q Exactive HF MS allows accurate mass measurement with less than 3 ppm accuracy and the faster scan speed (up to 18 Hz) of the Q Exactive HF MS results in higher number of precursor ions triggered for MS/MS, allowing more lipid identification in a single HPLC/MS/MS run with improved productivity. The Q Exactive HF MS also offers several high resolution accurate mass approaches (Full MS; SIM: selected ion monitoring; PRM: parallel reaction monitoring) for highly sensitive and highly selective quantification of individual lipid species of interest.
In this study, a Q Exactive HF MS was used to carry out a large scale lipid profile analysis of two phenotypes of the rat (ZDF vs. lean wild type) . Per each type of the rat model, three different lots of rat plasmas were used. The lipids were extracted from the plasmas using solvents of Chloroform, Methanol and water. A Thermo Scientific™ Dionex™ UltiMate™ 3000 Rapid Separation LC (RSLC) system was used for lipid separation. Mobile phase A was 60:40 Acetonitrile / Water and mobile phase B was 90:10 IPA / Acetonitrile; both A and B contained 10mM ammonium formate and 0.1% formic acid. The column was an Accucore C18 (Thermo Scientific, 2.1 x 150mm, 2.6µm) column operated at 55°C with flow rate of 260 µL/min.
The Q Exactive HF mass spectrometer was operated using data dependent LC MS/MS method in both positive mode and negative mode, respectively. Each full MS scan (120,000 R at 200m/z) was followed by 15 MS/MS (30,000 R at 200 m/z) scans. The cycle time was 2.4 seconds providing sufficient scans across the chromatographic peak profile for accurate relative quantification using the HR/AM precursor ion while simultaneously acquiring dd-MS/MS spectra for lipid identification. Lipid Search 4.1 software was used for lipid identification and quantification.
The Q Exactive HF MS system delivers high mass accuracy and resolution combined with faster scan speed, enabling faster and deeper lipidome coverage. Approximately more than 1000 lipid species were simultaneously identified and quantified with high confidence and great analytical precision from the two types of rat plasma. Moreover, significant concentration increases of triglycerides and phospholipids were observed from the ZDF rat plasmas.
References & Acknowledgements:
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