Generation of a Universal Human Complement Source by Large-Scale Depletion of IgG and IgM from Pooled Human Plasma

Constant improvements to the Orbitrap mass analyzer, such as acquisition speed, resolution, dynamic range and sensitivity have strengthened its value for the large-scale identification and quantification of metabolites in complex biological matrices. Here, we report the development and optimization of Data Dependent Acquisition (DDA) and Sequential Window Acquisition of all THeoretical fragment ions (SWATH-type) Data Independent Acquisition (DIA) workflows on a high-field Orbitrap FusionTM TribridTM instrument for the robust identification and quantification of metabolites in human plasma. By using a set of 47 exogenous and 72 endogenous molecules, we compared the efficiency and complementarity of both approaches. We exploited the versatility of this mass spectrometer to collect meaningful MS/MS spectra at both high- and low-mass resolution and various low-energy collision-induced dissociation conditions under optimized DDA conditions. We also observed that complex and composite DIA-MS/MS spectra can be efficiently exploited to identify metabolites in plasma thanks to a reference tandem spectral library made from authentic standards while also providing a valuable data resource for further identification of unknown metabolites. Finally, we found that adding multi-event MS/MS acquisition did not degrade the ability to use survey MS scans from DDA and DIA workflows for the reliable absolute quantification of metabolites down to 0.05 ng/mL in human plasma.

Download Full-text

Influence of ω-Aminoacids on the Activation of the First Component of Complement at Low Ionic Strength

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650135 ◽

1981 ◽

Vol 45 (01) ◽

pp. 086-089 ◽

Cited By ~ 5

Author(s):

Yumiko Takada ◽

Akikazu Takada

Keyword(s):

Ionic Strength ◽

Tranexamic Acid ◽

Human Plasma ◽

Human Complement ◽

Low Ionic Strength

SummaryCl, the first component of human complement, was activated to Cls by diluting human plasma in a solution of low ionic strength. The addition of lysine and its related aminoacids to plasma resulted in inhibition of Cl activation, α, ω-diaminoacids such as lysine and ornithine were most effective and ω-aminoacids such as 6 aminohexanoic acid were far less effective. Tranexamic acid which is most potent in inhibition of fibrinolysis among those aminoacids was least effective in the inhibition of Cl activation, ω-aminoacids or their analogues hardly inhibited Cls which had already been activated.

Download Full-text

Quantitation of 87 Proteins by nLC-MRM/MS in Human Plasma: Workflow for Large-Scale Analysis of Biobank Samples

Journal of Proteome Research ◽

10.1021/acs.jproteome.7b00235 ◽

2017 ◽

Vol 16 (9) ◽

pp. 3242-3254 ◽

Cited By ~ 4

Author(s):

Melinda Rezeli ◽

Karin Sjödin ◽

Henrik Lindberg ◽

Olof Gidlöf ◽

Bertil Lindahl ◽

...

Keyword(s):

Human Plasma ◽

Large Scale ◽

Scale Analysis ◽

Large Scale Analysis

Download Full-text

Single-Step Extraction Coupled with Targeted HILIC-MS/MS Approach for Comprehensive Analysis of Human Plasma Lipidome and Polar Metabolome

Metabolites ◽

10.3390/metabo10120495 ◽

2020 ◽

Vol 10 (12) ◽

pp. 495

Author(s):

Jessica Medina ◽

Vera van der Velpen ◽

Tony Teav ◽

Yann Guitton ◽

Hector Gallart-Ayala ◽

...

Keyword(s):

Human Plasma ◽

Large Scale ◽

Lipid Extraction ◽

Single Step ◽

Methyl Tert Butyl Ether ◽

Method Performance ◽

Simultaneous Extraction ◽

Lipid Species ◽

Polar Metabolites ◽

Complex Lipids

Expanding metabolome coverage to include complex lipids and polar metabolites is essential in the generation of well-founded hypotheses in biological assays. Traditionally, lipid extraction is performed by liquid-liquid extraction using either methyl-tert-butyl ether (MTBE) or chloroform, and polar metabolite extraction using methanol. Here, we evaluated the performance of single-step sample preparation methods for simultaneous extraction of the complex lipidome and polar metabolome from human plasma. The method performance was evaluated using high-coverage Hydrophilic Interaction Liquid Chromatography-ESI coupled to tandem mass spectrometry (HILIC-ESI-MS/MS) methodology targeting a panel of 1159 lipids and 374 polar metabolites. The criteria used for method evaluation comprised protein precipitation efficiency, and relative MS signal abundance and repeatability of detectable lipid and polar metabolites in human plasma. Among the tested methods, the isopropanol (IPA) and 1-butanol:methanol (BUME) mixtures were selected as the best compromises for the simultaneous extraction of complex lipids and polar metabolites, allowing for the detection of 584 lipid species and 116 polar metabolites. The extraction with IPA showed the greatest reproducibility with the highest number of lipid species detected with the coefficient of variation (CV) < 30%. Besides this difference, both IPA and BUME allowed for the high-throughput extraction and reproducible measurement of a large panel of complex lipids and polar metabolites, thus warranting their application in large-scale human population studies.

Download Full-text