Quantitative Profiling of Polar Cationic Metabolites in Human Cerebrospinal Fluid by Reversed-Phase Nanoliquid Chromatography/Mass Spectrometry

Citations Over TimeTop 10% of 2009 papers

Abstract

Reversed-phase (RP) nanoliquid chromatography (LC)/mass spectrometry (MS) is widely used for proteome analysis, but hydrophilic metabolites are poorly retained on RP columns. We describe here the development and application of an efficient, robust, and quantitative nano-LC/MS method for cationic metabolome analysis in the positive ionization mode without any derivatization of analytes. Various stationary phases for nano-LC, coating of the internal wall of the capillary column, and various mobile phases were evaluated in terms of separation and peak shapes for 33 hydrophilic metabolites, including nonderivatized amino acids. Polar cationic compounds were strongly bound to mixed-functional RP with cation exchange mode resin, and the best separation was obtained with hydrophilic internal wall coating and a two-step trifluoroacetic acid (TFA) gradient in methanol as the mobile phase. Simple, but optimized, sample processing and the use of a high content of methanol allowed robust nano-LC/MS analysis. Our developed method was applied for biomarker discovery in Alzheimer's disease (AD). Several hundred peaks were detected from 10 microL of cerebrospinal fluid (CSF). In a principal component analysis (PCA) plot using peak intensities without normalization, peak separation depended on the experimental date, not disease state. Therefore, constant amounts of two stable isotope-labeled amino acids, Val and Lys, were added as internal standards (ISs) to each sample before processing. These ISs were eluted in different gradient slopes in the two-step gradient, and the normalized peak ratios using the corresponding ISs gave a unique group of PCA scores which could distinguish AD CSF samples from age-matched control CSF samples.

Related Papers

• → Chemical derivatization in LC-MS-based metabolomics study(2020)169 cited
• → Determination of vitamin D metabolites in various biological samples through an improved chemical derivatization assisted liquid chromatography-tandem mass spectrometry approach(2023)4 cited
• → Monophasic Variant of Salmonella Typhimurium Infection Affects the Serum Metabolome in Swine(2023)3 cited
• → First Insights into the Urinary Metabolome of Captive Giraffes by Proton Nuclear Magnetic Resonance Spectroscopy(2020)9 cited
• → Insights into the Metabolome of the Cyanobacterium Leibleinia gracilis from the Lagoon of Tahiti and First Inspection of Its Variability(2020)8 cited