Ultrahigh-Resolution Mass Spectrometry-Based Platform for Plasma Metabolomics Applied to Type 2 Diabetes Research

Abstract

Metabolomicsthe endpoint of the omics cascadeis increasingly recognized as a preferred method for understanding the ultimate responses of biological systems to stress. Flow injection electrospray (FIE) mass spectrometry (MS) has advantages for untargeted metabolic fingerprinting due to its simplicity and capability for high-throughput screening but requires a high-resolution mass spectrometer to resolve metabolite features. In this study, we developed and validated a high-throughput and highly reproducible metabolomics platform integrating FIE with ultrahigh-resolution Fourier transform ion cyclotron resonance (FTICR) MS for analysis of both polar and nonpolar metabolite features from plasma samples. FIE-FTICR MS enables high-throughput detection of hundreds of metabolite features in a single mass spectrum without a front-end separation step. Using plasma samples from genetically identical obese mice with or without type 2 diabetes (T2D), we validated the intra and intersample reproducibility of our method and its robustness for simultaneously detecting alterations in both polar and nonpolar metabolite features. Only 5 min is needed to acquire an ultra-high resolution mass spectrum in either a positive or negative ionization mode. Approximately 1000 metabolic features were reproducibly detected and annotated in each mouse plasma group. For significantly altered and highly abundant metabolite features, targeted tandem MS (MS/MS) analyses can be applied to confirm their identity. With this integrated platform, we successfully detected over 300 statistically significant metabolic features in T2D mouse plasma as compared to controls and identified new T2D biomarker candidates. This FIE-FTICR MS-based method is of high throughput and highly reproducible with great promise for metabolomics studies toward a better understanding and diagnosis of human diseases.

Systemic Metabolic Alterations Correlate with Islet-Level Prostaglandin E2 Production and Signaling Mechanisms That Predict β-Cell Dysfunction in a Mouse Model of Type 2 Diabetes

“…We performed untargeted plasma metabolomics using a workflow integrating flow injection electrospray (FIE) with ultrahigh-resolution Fourier Transform Ion Cyclotron Resonance (FTICR) mass spectrometry (MS), a platform recently established and validated for use with cryopreserved plasma samples [ 7 ] (see Supplementary Figure S1 for a summary of the workflow). Between 1200 and 2200 distinct metabolic features were detected in plasma samples from each group, with over 75% of those features being annotatable by chemical structure using MetaboScape or chemical name using the METLIN Metabolite and Chemical Entity Database ( Figure 5 A and Supplementary Table S4 ).…”

“…In our previously published work [ 7 ], the fatty acids and fatty acid conjugates family was the most highly significantly expressed for both polar and nonpolar metabolites in plasma from obese mice [ 7 ]. Eicosanoids are highly bioactive fatty acid metabolites, and a subset of essential polyunsaturated fatty acids (PUFAs) provides the substrates for their synthesis [ 17 , 18 ].…”

“…Experimental mice were singly housed in temperature- and humidity-controlled environments and maintained on a 12:12 h day/light cycle with free access to acidified water (InnoVive, San Diego, CA, USA) and one of two standard mouse chows of nearly identical macronutrient composition and energy density. A direct comparison of the nutrient content and ingredients of the Teklad global soy protein-free extruded 2920X diet (Envigo, Indianapolis, IN, USA) or the Rodent Laboratory Chow 5001 diet (Purina, Neenah, WI, USA) has been previously published [ 7 ]. Both wild-type control mice ( n = 9; 3-Purina and 6-teklad) and OB mice ( n = 17; 11-Purina and 6-teklad) were given either Purina or Teklad diets.…”

“…In the process of breeding BTBR ob mice for downstream analyses, we discovered, for an approximately 6 month period, that no 10-week-old BTBR ob mice in our investigator-accessible facility were hyperglycemic. This phenotype was partially related to changes in circulating metabolites after the brand of standard rodent chow was switched (explored in a different work [ 7 ]) but was not fully related to diet composition, as ultimately, the phenotype disappeared. Our previous publication found that the biggest differences in circulating metabolites in BTBR Ob mice was phenotype and not diet composition and that these alterations correlated directly with beta-cell function [ 7 ].…”

“…This phenotype was partially related to changes in circulating metabolites after the brand of standard rodent chow was switched (explored in a different work [ 7 ]) but was not fully related to diet composition, as ultimately, the phenotype disappeared. Our previous publication found that the biggest differences in circulating metabolites in BTBR Ob mice was phenotype and not diet composition and that these alterations correlated directly with beta-cell function [ 7 ]. Therefore, in this work, mice were grouped by phenotype, independent of diet.…”