Improved workflow for mass spectrometry–based metabolomics analysis of the heart

Abstract

MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS–based metabolomics workflow that uses insoluble protein–derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.

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“…Following heating, samples were transferred to v-shaped glass chromatography vials and 80 μl of MTSFA + 1% TMCS (Thermo Scientific) was added. Samples were then heated for 60 min at 60 °C, allowed to cool to room temperature, and then analyzed via GCMS with parameters as previously described [ 28 ]. Briefly, a GC temperature gradient of 130 °C was held for 4 min, rising at 6 °C/min to 243 °C, rising at 60 °C/min to 280 °C and held for 2 min.…”

Oral Gavage Delivery of Stable Isotope Tracer for In Vivo Metabolomics

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