Oct 22, 2024
Version 3
Quantitative targeted metabolomics for ASO mouse model using Biocrates Q500 Platform V.3
- 1Duke School of Medicine
Protocol Citation: Siamak MahmoudianDehkordi 2024. Quantitative targeted metabolomics for ASO mouse model using Biocrates Q500 Platform. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5pwyg47/v3Version created by Catherine Oikonomou
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 20, 2024
Last Modified: October 22, 2024
Protocol Integer ID: 110267
Funders Acknowledgement:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
This protocol describes the use of the biocrates MxP‱ Quant 500 kit (biocrates life sciences AG, Innsbruck, Austria), a commercially available targeted metabolomics kit, to assess levels of 630 metabolites across 26 biochemical classes in different tissues collected from mice. All samples are processed through the metabolomics phenotyping services platform of biocrates life sciences.
Sample Preparation
Sample Preparation
Thaw previously-collected, frozen samples On ice
For plasma samples, use directly for analysis
For tissue samples:
Resuspend in 3 µL ethanol/phosphate buffer [x85 % (v/v) HPLC-grade ethanol in 0.01 M phosphate buffer] per 1 mg wet weight
Sonicate samples
Vortex samples
Homogenize samples with ceramic beads in a
Equipment
Precellys
NAME
Homogenizer
TYPE
Bertin
BRAND
P000669-PR240-A
SKU
LINK
3 times 00:00:30 each at 5,800 rpm with a 00:00:30 pause in between
1m
Centrifuge sample 10000 x g, 4°C, 00:05:00 and transfer supernatant to fresh tube On ice
Note
for measurement of some metabolites, we find it necessary to dilute some tissue samples before centrifugation. For instance, we dilute duodenum samples 1:5 in ethanol/phosphate buffer
5m
For intestinal content samples:
Resuspend in ethanol/phosphate buffer, vortexing thoroughly until dissolved
Ultrasonicate in a chilled bath for 00:05:00
5m
Centrifuge and transfer supernatant to fresh tube On ice
To analyze highly concentrated bile acids, prepare an additional 1:1,000 dilution
Quantification of metabolite concentrations
Quantification of metabolite concentrations
Add 10 µL of each sample to the 96-well sample plate (containing inserts impregnated with internal standards) of the biocrates MxP‱ Quant 500 kit (biocrates life science ag, Innsbruck, Austria) following manufacturer's instructions
Perform measurement by FIA-MS/MS and/or LC-MS/MS
Note
Exact methods depend on sample and analyte type. For lipids (e.g, acylcarnitines, glycerophospholipids, sphingolipids, triglycerides) and hexoses, we use flow injection analysis-tandem MS (FIA-MS/MS) using a 5500 QTRAP‱ instrument (AB Sciex, Darmstadt, Germany) with an electrospray ionization (ESI) source for plasma and tissue samples, and a Xevo TQ-S (Waters, Vienna, Austria) instrument with an ESI source for gut content samples. For small molecules, we use liquid chromatography-tandem MS (LC-MS/MS), also using a 5500 QTRAP‱ instrument for all samples. For gut tissue and content samples we also use LC-MS/MS on a Xevo TQ-S instrument.