A Transferable Workflow for Confident Annotation of Polar Analytes in 4D-Metabolomics

ABSTRACT: Introduction High-confidence annotation of polar metabolites remains a central challenge in non-targeted metabolomics. While derivatization improves chromatographic retention and ionization of derivatized compounds, it changes the molecules, making annotation using conventional annotation workflows difficult. Trapped ion mobility spectrometry (TIMS) provides collisional cross section (CCS) values as an orthogonal, instrument-agnostic molecular descriptor, but CCS reference data for derivatized metabolites is scarce. Objectives We present a four-dimensional annotation strategy integrating 3-nitrophenylhydrazine 3-NPH derivatization with orthogonal retention time (RT), CCS, accurate mass, isotopic pattern, and MS/MS information acquired by LC-TIMS-HRMS, providing a large-scale experimental CCS reference dataset for 3-NPH-derivatized metabolites. Methods Experimentally measured TIMS CCS values were acquired for 3-NPH derivatives on a timsTOF Pro2. Combined with standardized RTs from the biocrates MxQuant kit workflow, these values form a curated 4D target list for automated annotation that is instrument independent and transferable. Coverage was further extended via an in silico derivatization workflow scoring candidate structures against experimental MS/MS and CCS data. Results CCS values were transferred directly to the timsMetabo platform with inter-instrument agreement consistent with published benchmarks (<2 % ΔCCS). Applied to time-resolved brown adipocyte extracts following norepinephrine stimulation, the workflow resolved temporal trajectories across fatty acids, organic acids, and amino acid derivatives matching previous reports. Conclusion Anchoring 3-NPH metabolite annotation to experimentally measured TIMS CCS values and standardized biocrates kit retention times substantially reduces annotation ambiguity, allowing confident profiling of polar analytes in complex biological systems.

INSTRUMENT(S): Liquid Chromatography MS - negative - reverse-phase

PROVIDER: MTBLS16057 | MetaboLights | 2026-10-19

REPOSITORIES: MetaboLights