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. 2026 Feb 17;98(6):4571-4584.
doi: 10.1021/acs.analchem.5c05677. Epub 2026 Feb 5.

MS/MS Mass Spectrometry Filtering Tree for Bile Acid Regio- and Stereoisomer Annotation

Ipsita Mohanty  1 , Shipei Xing  1 , Vanessa Castillo  1 , Julius Agongo  1 , Abubaker Patan  1 , Yasin El Abiead  1 , Helena Mannochio-Russo  1 , Wilhan D Gonçalves Nunes  1 , Jasmine Zemlin  1 , Itzhak Mizrahi  2   3 , Dionicio Siegel  1 , Mingxun Wang  4 , Lee R Hagey  5 , Pieter C Dorrestein  1   6   7   8
Affiliations

MS/MS Mass Spectrometry Filtering Tree for Bile Acid Regio- and Stereoisomer Annotation

Ipsita Mohanty et al. Anal Chem. .

Abstract

Bile acids are essential steroids with a wide range of biological roles, including the regulation of immunity, nutrient absorption, insulin signaling, appetite, and body temperature. However, due to similarities in their MS/MS spectra, spectral matching with reference MS/MS libraries generally fails to differentiate between isomers. This study introduces a proof-of-concept workflow that uses a mass spectrometry query language filtering tree to distinguish isomeric bile acids in untargeted LC-MS/MS data by leveraging intensity ratios of ions that are close to one another in the MS/MS spectrum. It can be retrospectively applied to existing LC-MS/MS data in data repositories. The filtering tree concept provides the opportunity to annotate and distinguish previously unknown bile acid isomers across LC-MS/MS data sets. To facilitate the ease of applying these filters to LC-MS/MS data sets, we developed a web-based application that simplifies the stepwise filtering tree workflow, removing the need for coding expertise. Here, we apply the multistep filtering application to a representative public data set, which revealed distinct patterns of bile acids associated with different diet types across diverse mammalian species. We further identified the previously uncharacterized bile acid deoxycholyl-N-acetyl-putrescine, which was elevated in carnivores.

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Figures

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MS/MS fragmentation of bile acid isomers. (a) Structure of bile acids highlighting mono-, di-, and trihydroxylated steroid cores, with experimentally observed potential hydroxylation sites on the steroid core indicated by red stars. (b) MS/MS fragmentation spectra of the regioisomers, taurochenodeoxycholic (TCDCA) acid and taurodeoxycholic acid (TDCA), illustrating a low-intensity mass region containing ions unique to each isomer. (c) Enlarged view of the MS/MS fragmentation spectra for taurochenodeoxycholic and taurodeoxycholic acids, emphasizing the ion pair used to calculate relative intensity ratios for differentiating these isomers.
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Development of MS/MS fragmentation-based MassQL filtering tree. Sequential MS/MS fragmentation-based filtering tree designed to classify regio- and stereoisomers of dihydroxylated bile acids. Structures at each filtering step are shown, with terminal bins color-coded for clarity.
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Workflow of MS/MS fragmentation-based MassQL filtering tree. Schematic diagram of the workflow for using the multistep MassQL filters for distinguishing different bile acid isomers.
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Filtering of bile acid isomers in an untargeted LC-MS/MS data set. Representative example for using the MassQL filters for identifying bile acid isomers. (a) Overview of study design. Fecal samples from herbivores (n = 3), omnivores (n = 23), and carnivores (n = 13) were analyzed by LC-MS/MS and processed using MZmine4 and FBMN on GNPS2. Stepwise MassQL filtering refined candidate bile acids from 543 features (Step 1) to 96 (Steps 2 and 3) and 49 stereo and regiochemistry assigned MS/MS spectra (Step 4). Differential abundance of bile acids between herbivores/omnivores and carnivores is illustrated in the volcano plots (b) at Step 1 and (c) Step 4 of the dihydroxy filtering tree. The annotation of a previously uncharacterized bile acid was refined from (OH)2-N-acetyl-putrescine to 3,12α-N-acetyl-putrescine.
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Structural elucidation of 3,12α-N-acetyl-putrescine using MassQL filtering and experimental validation. (a) Output from the multistep MassQL app for the candidate bile acids shown as a Sankey plot. The branch with the progressive filtering down from (OH)2-N-acetyl-putrescine to 3,12α-N-acetyl-putrescine is highlighted. The pipeline identified 3,12α-(OH)2 stereochemistry, while showing the potential ambiguity at C3 (α/β).The isomer assignment of the bile acid core was experimentally validated by (b) retention time matching of the synthetic standard of deoxycholyl-N-acetyl-putrescine, biological sample of tiger feces, and spiked sample, confirming coelution. (c) MS/MS spectral comparison of standard with biological extract yielded a cosine similarity of 0.9801. (d) The final confirmed structure, deoxycholyl-N-acetyl-putrescine (3α,12α-(OH)2), is shown with regio-and stereochemical assignments.
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