Plamsa Proteomics

Published: 8 July 2023| Version 1 | DOI: 10.17632/7d4m8wcnrp.1
Qinqin He


The plasma metabolome from the control and CSF mice was quantified with a high throughput targeted metabolite (HM350 Metabolome). 20 μL of plasma samples and standards were added to Eppendorf vials containing 120 μL of working solution. Then, the mixtures were incubated at room temperature for 20 min to facilitate protein precipitation. After centrifugation at 18,000 g for 20 min at 4°C, 30 μL of supernatant was transferred to a 96-well plate with 20 μL derivative reagent working solution and EDC working solution. The assay plate was incubated for 60 min at 30°C on a Peltier thermocycler. 100 μL of ice-cold methanol (polar fraction) was added into each well, incubated at 1200 rpm for 5min, and centrifuged at 4000 g for 30 min at 4°C. The 100 μL of supernatant was then transferred to another microplate pre-loaded with an internal standard II working solution and the mixture was quantified using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The extract was gradient eluted from a C18 column (Waters UPLC BEH C18-2.1x100 mm, 1.7 mm) using 0.1% formic acid (solvent A) and 70% acetonitrile/30% isopropanol (solvent B). The gradient was linearly changed as follows: 0-1.0 min, 5% solvent B; 1.0-5 min 5% solvent B; 5-9 min,70% solvent B; 9-11 min, 50% solvent B; 11-13.5 min, 22% solvent B; 13.5-14.00 min, 95% solvent B, at a flow rate of 0.4 mL/min; 14.00-16.00min, 100% solvent B, the flow rate of 0.6 mL/min; 16.00-18.00min, 5% solvent B, the flow rate of 0.4 mL/min. The column was maintained at 40°C. The mass spectrometer was set for electrospray ionization (ESI) operated in ESI+/ESI- mode. The source parameters were as follows: capillary voltage, 5500 V for the positive ionization and 4500 V for the negative ionization; gas temperature, 550°C; Ion source Gas1, 60 psi; Ion source Gas2, 60 psi; CUR, 35 psi. The molecular ion and fragment for each compound were quantified based on standards.



Blood Plasma