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Clin Proteomics. 2013 Jul 4;10(1):7. doi: 10.1186/1559-0275-10-7. eCollection 2013.

Technology platform development for targeted plasma metabolites in human heart failure.

Author information

1
NHLBI Proteomics Center at UCLA, Departments of Physiology and Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA.
2
Thermo Fisher Scientific, San Jose, CA 95134, USA.
3
Ronald Reagan UCLA Medical Center, UCLA Medical Center, Los Angeles, CA 90095, USA.

Abstract

BACKGROUND:

Heart failure is a multifactorial disease associated with staggeringly high morbidity and motility. Recently, alterations of multiple metabolites have been implicated in heart failure; however, the lack of an effective technology platform to assess these metabolites has limited our understanding on how they contribute to this disease phenotype. We have successfully developed a new workflow combining specific sample preparation with tandem mass spectrometry that enables us to extract most of the targeted metabolites. 19 metabolites were chosen ascribing to their biological relevance to heart failure, including extracellular matrix remodeling, inflammation, insulin resistance, renal dysfunction, and cardioprotection against ischemic injury.

RESULTS:

In this report, we systematically engineered, optimized and refined a protocol applicable to human plasma samples; this study contributes to the methodology development with respect to deproteinization, incubation, reconstitution, and detection with mass spectrometry. The deproteinization step was optimized with 20% methanol/ethanol at a plasma:solvent ratio of 1:3. Subsequently, an incubation step was implemented which remarkably enhanced the metabolite signals and the number of metabolite peaks detected by mass spectrometry in both positive and negative modes. With respect to the step of reconstitution, 0.1% formic acid was designated as the reconstitution solvent vs. 6.5 mM ammonium bicarbonate, based on the comparable number of metabolite peaks detected in both solvents, and yet the signal detected in the former was higher. By adapting this finalized protocol, we were able to retrieve 13 out of 19 targeted metabolites from human plasma.

CONCLUSIONS:

We have successfully devised a simple albeit effective workflow for the targeted plasma metabolites relevant to human heart failure. This will be employed in tandem with high throughput liquid chromatography mass spectrometry platform to validate and characterize these potential metabolic biomarkers for diagnostic and therapeutic development of heart failure patients.

KEYWORDS:

Heart disease; Sample preparation; Targeted human plasma metabolomics

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