Global metabolic profiling of animal and human tissues via UPLC-MS

Abstract

Obtaining comprehensive, untargeted metabolic profiles for complex solid samples, e.g., animal tissues, requires sample preparation and access to information-rich analytical methodologies such as mass spectrometry (MS). Here we describe a practical two-step process for tissue samples that is based on extraction into 'aqueous' and 'organic' phases for polar and nonpolar metabolites. Separation methods such as ultraperformance liquid chromatography (UPLC) in combination with MS are needed to obtain sufficient resolution to create diagnostic metabolic profiles and identify candidate biomarkers. We provide detailed protocols for sample preparation, chromatographic procedures, multivariate analysis and metabolite identification via tandem MS (MS/MS) techniques and high-resolution MS. By using these optimized approaches, analysis of a set of samples using a 96-well plate format would take ~48 h: 1 h for system setup, 8-10 h for sample preparation, 34 h for UPLC-MS analysis and 2-3 h for preliminary/exploratory data processing, representing a robust method for untargeted metabolic screening of tissue samples.

Technical Challenges in Mass Spectrometry-Based Metabolomics

“…Recent large-scale metabolome analyses employed an experimental design using QC samples to correct a dri of the raw signal intensity during the analysis. [85][86][87] e QC samples were prepared by mixing all the sample extracts in one analysis batch or in one metabolome analysis study. e iterative analyses of the QC sample were inserted into the start, end, and between every 4-8 actual samples in batch sequences of data acquisition.…”

Hippocampal Proteomic and Metabonomic Abnormalities in Neurotransmission, Oxidative Stress, and Apoptotic Pathways in a Chronic Phencyclidine Rat Model

“…46 In brief, approximately 10mg frontal cortex and 30-50mg hippocampus tissues were weighed out into 2mL bead beater tubes and homogenized with 1.45mL of pre-chilled methanol/water (1:1) and 100 μL of 1-mm zirconium beads, using a Precellys bead beater. Homogenisation (6,5000Hz speed) cycles were 40s, followed by cooling on dry ice, and a further 40s homogenisation and cooling on dry ice.…”

“…Aqueous extracts were dried in a vacuum concentrator (Savant) for at least 180min at 45 o C. Extracts were resuspended in 120μL of methanol/water (1:1), followed by brief vortexing and sonication, and transferred into 96-well plates for analysis. Quality control (QC) samples were prepared by combining an aliquot (10μL) from each study sample to produce a representative sample -this was used for column conditioning and data quality assessment as described by Want et al 46 …”

Low invasive in vivo tissue sampling for monitoring biomarkers and drugs during surgery

“…However, these powerful instruments, and particularly mass spectrometers, require appropriate sample preparation before analysis. Several protocols describing tissue sample preparation for metabolomics, metabolic profiling, or fingerprinting have been proposed, but most of them are based on the removal of a piece of tissue, followed by labor-intensive and time-consuming homogenization, drying, and multisolvent extraction, [3][4][5] therefore limiting their onsite use. To overcome this challenge and provide a tool where direct untargeted analysis of tissue sample can be performed, matrix-assisted laser desorption/ionization (MALDI) was introduced.…”