Systematic, Modifying Group-Assisted Strategy Expanding Coverage of Metabolite Annotation in Liquid Chromatography–Mass Spectrometry-Based Nontargeted Metabolomics Studies

Abstract

From microbes to human beings, nontargeted metabolic profiling by liquid chromatography (LC)–mass spectrometry (MS) has been commonly used to investigate metabolic alterations. Still, a major challenge is the annotation of metabolites from thousands of detected features. The aim of our research was to go beyond coverage of metabolite annotation in common nontargeted metabolomics studies by an integrated multistep strategy applying data-dependent acquisition (DDA)-based ultrahigh-performance liquid chromatography (UHPLC)–high-resolution mass spectrometry (HRMS) analysis followed by comprehensive neutral loss matches for characteristic metabolite modifications and database searches in a successive manner. Using pooled human urine as a model sample for method establishment, we found 22% of the detected compounds having modifying structures. Major types of metabolite modifications in urine were glucuronidation (33%), sulfation (20%), and acetylation (6%). Among the 383 annotated metabolites, 100 were confirmed by standard compounds and 50 modified metabolites not present in common databases such as human metabolite database (HMDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were structurally elucidated. Practicability was tested by the investigation of urines from pregnant women diagnosed with gestational diabetes mellitus vs healthy controls. Overall, 83 differential metabolites were annotated and 67% of them were modified metabolites including five previously unreported compounds. To conclude, the systematic modifying group-assisted strategy can be taken as a useful tool to extend the number of annotated metabolites in biological and biomedical nontargeted studies.

Diagnostic Performance of Sex-Specific Modified Metabolite Patterns in Urine for Screening of Prediabetes

“…In the joint discovery cohort composed of female and male subjects, the non-parametric test revealed 1,296 significantly different metabolite features between NGT and IGT. Subsequently, after applying the MRM-Ion Pair Finder software ( 15 ) to extract modified metabolites from these potential biomarkers, 299 remained. Principal component analysis (PCA) revealed a clustering of IGT and NGT based on these 299 modified metabolite features ( Supplementary Figure 1A ).…”

“…The coverage of common metabolomics approaches was broadened applying our recently published analytical strategy that focuses on modified metabolites ( 15 ). The published approach was applied with one relevant variation.…”

“…Annotation of the modified metabolite features was performed according to our recently published strategy ( 15 ) with slight modifications of using five instead of three databases (OSI-SMMS, HMDB, Metlin, MyCompoundID, and Compound Discovery 3.0). Since Compound Discovery 3.0 database searches work only with high-resolution MS/MS spectra data from Thermo Fisher Scientific instruments for this purpose, MS/MS spectra from a QC sample were acquired on a QE-HF mass spectrometer (ESM Method).…”

“…Every tenth sample was followed by a quality control (QC) analysis of pooled urine. For modified metabolite profiling, collision energies of 15 V, 30 V, and 45 V were applied as described in detail in (15), and MS/MS fragmentation patterns of the 15 most intense ions in full scan were acquired. Curtain gas, gas 1, and gas 2 were 35, 50, and 50 psi, respectively.…”

“…(D) Only metabolites containing modifying groups were considered. The rationale to apply our novel metabolomics strategy for the untargeted profiling of modified metabolites ( 15 ) was that modified metabolites are not only less covered by common metabolomics approaches but also less represented in common metabolite databases ( 15 , 16 ), which suggests that this group of metabolites could have an added value for diagnostic biomarker patterns.…”