Bile acids (BA) are synthesized in the liver of mammals and plays a crucial role in regulating cholesterol homeostasis. The interaction between human host’s BA and the gut microbiome is bidirectional, where dysregulation of BA metabolism and alteration of the gut’s microbiome are associated with inflammatory bowel disease, liver disease, and metabolic disorders. The structural complexity of bile acids, including their isobaric and isomeric forms, presents significant challenges for separation using conventional LC methods.
LC-TIMS-HRMS utilizing the benefits of trapped ion mobility spectrometry (TIMS) by providing an additional dimension of separation and measuring their specific Collisional Cross Section (CCS) based on their shape and size. However, besides bile acids, the characterization of other metabolites present in biofluid could also be related to BA dysregulation. Here we present prio-PASEF, a novel timsTOF data acquisition mode that prioritizes precursor selection for predicted products while using the remaining duty cycle for untargeted data dependent acquisition. This novel MetID workflow ensures comprehensive coverage of both expected and unexpected metabolites
In this study, plasma samples and BA internal standards were extracted with methanol for LC-TIMS-HRMS analysis using both standard 4D-Metabolomics instrument parameters with dda-PASEF and prio-PASEF. The prio-PASEF was implemented using Python script and APIo on timsControl 6.1. Data were analyzed using MetaboScape 2025. Previous proof-of-principle work on a tryptic digest of NIST mAb and three known low abundant peptides, demonstrated the observation of >17000 precursors in a 13-min prio-PASEF acquisition, with improved MS2 quality compared to standard DDA. Here, we employed prio-PASEF data acquisition algorithm to prioritize the precursors of 16 BA of interest based on their RT, molecular formula and CCS values, while within the same duty cycle concurrently performing DDA for comprehensive MetID analysis. Targeted BA were also quantified based on their internal standards, ensuring precise and accurate measurement.