OR 7 - µUHPLC-4D-TIMS strategy for low input lipidomics:
A case study on patient derived organoids
OR 7 - µUHPLC-4D-TIMS strategy for low input lipidomics:
A case study on patient derived organoids
The demand for low-input biological samples is rising at an exponential pace for both clinical and translational research, in this context achieving high sensitivity and throughput is essential despite the limited availability of material. Patient-derived organoids (PDOs) stand out as highly representative disease models, providing an advanced system to investigate drug responses and pathogenic mechanisms. PDOs are usually grown in bulk cultures, which provide sufficient biological material for conventional LC-MS lipidomics. The shift toward miniaturized and high-throughput formats requires substantial adaptations of the analytical strategies [1] in order to deal with small sample sizes (~10³ cells). In this study, we explored the use of a 1.0 mm I.D. UHPLC column to enhance detection sensitivity in PDOs cultivated in a 384-well format. By coupling a microbore column to trapped ion mobility spectrometry (µUHPLC-4D-TIMS) and analysing a broad lipid standard mixture, we observed pronounced improvements in terms of signal intensity, signal to noise ratio and sensitivity. In detail, we obtained an average 12-fold reduction in limits of detection (LOD) and improvements as high as 68-fold for specific compounds, exemplified by LPC 18:1. Subsequently, in order to test the applicability of the proposed approach to biological samples, we profiled colorectal cancer PDOs exposed to two chemotherapeutics under both bulk and miniaturized culture conditions. The lipidomic signatures revealed comparable drug-induced alterations between systems, particularly within phosphatidylcholines and phosphatidylethanolamines, confirming the biological reliability of the 384-well platform. If compared to the conventional narrowbore setup, the micro-4D-TIMS workflow enabled the detection of 103 additional lipid species spanning 17 subclasses, including phosphatidylserines, phosphatidylglycerols and hexosylceramides, that were undetectable in the conventional method. In summary, this work highlights how reducing column diameter constitutes a reliable and effective strategy to advance lipidomic profiling in miniaturized PDO assays, supporting sensitive and scalable applications in precision medicine.
La Gioia D,Salviati E, et al., Anal Bioanal Chem, 417, 2837–2847 (2025). https://doi.org/10.1007/s00216-024-05588-z