Abstract
Analytical techniques with high sensitivity and selectivity are essential to the quantitative analysis of clinical samples. Liquid chromatography coupled to tandem mass spectrometry is the gold standard in clinical chemistry. However, tandem mass spectrometers come at high capital expenditure and maintenance costs. We recently showed that it is possible to generate very similar results using a much simpler single mass spectrometry detector by performing enhanced in-source fragmentation/annotation (EISA) combined with correlated ion monitoring. Here we provide a step-by-step protocol for optimizing the analytical conditions for EISA, so anyone properly trained in liquid chromatography–mass spectrometry can follow and apply this technique for any given analyte. We exemplify the approach by using 2-hydroxyglutarate (2-HG) which is a clinically relevant metabolite whose d-enantiomer is considered an ‘oncometabolite’, characteristic of cancers associated with mutated isocitrate dehydrogenases 1 or 2 (IDH1/2). We include procedures for determining quantitative robustness, and show results of these relating to the analysis of dl-2-hydroxyglutarate in cells, as well as in serum samples from patients with acute myeloid leukemia that contain the IDH1/2 mutation. This EISA–mass spectrometry protocol is a broadly applicable and low-cost approach for the quantification of small molecules that has been developed to work well for both single-quadrupole and time-of-flight mass analyzers.
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Data availability
The CIM algorithm is available at https://github.com/ricoderks/eisaCIM. Supporting data have been included in the primary research papers as well as this protocol. Further validation data are available from the authors upon request.
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Acknowledgements
This work was supported by the Program for Guangdong Introducing Innovative and Entrepreneurial Teams (2019ZT08L213), the Guangdong Provincial Key Laboratory Project (2019B121203011), Key-Area Research and Development Program of Guangdong Province (2020B1111380003), the Leiden Center for Computational Oncology (LCCO), the Spanish Ministry of Science and Innovation (PID2019-104913GB-I00 project) and the Spanish Ministry of Economy and Competitiveness (S.B.-B.’s predoctoral research contract (BES-2017-082458)). This research was partially funded by the National Institutes of Health grants R35 GM130385 (G.S.) and U01 CA235493 (G.S.). The authors are grateful to A. Llombart-Bosch (University of Valencia, Spain) for the CH2879 cell line and J.A. Block (Rush University Medical Centre, Chicago, IL, USA) for the JJ012 cell line. Authors would also like to thank Ieva Palubeckaitė and Judith Bovée (Department of Pathology, Leiden University Medical Center, the Netherlands) for providing chondrosarcoma cells and valuable discussion, Hans Dalebout and René van Zeijl (Center for Proteomics and Metabolomics, Leiden University Medical Center, the Netherlands) for their technical assistance and Peter van Balen (Department of Hematology, Leiden University Medical Center, the Netherlands) for providing the serum samples.
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Conceptualization: S.B.-B., J.X., G.S., M.Giera, E.S.-L. Methodology: S.B.-B., J.X., L.H., E.B., B.W., B.H., G.S., M.Giera, E.S.-L. Acquisition of biological material: M.W.H., S.V., E.B.A., M.Griffioen. Investigation: S.B.-B., J.X., G.S., M.Giera, E.S.-L. Funding acquisition: J.X., B.H., M.C.-P., M.L.M., E.B.A., G.S., M.Giera. Supervision: G.S., M.Giera, E.S.-L. Writing of the original draft: S.B.-B., J.X., G.S., M.Giera, E.S.-L. All authors reviewed and edited the manuscript. S.B.-B. and J.X. contributed equally as co-first authors.
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Key references using this protocol
Xue, J. et al. Anal. Chem. 92, 6051–6059 (2020): https://doi.org/10.1021/acs.analchem.0c00409
Xue, J. et al. Anal. Chem. 93, 10879–10889 (2021): https://doi.org/10.1021/acs.analchem.1c01246
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Supplementary Procedure and Figs. 1–5.
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Bernardo-Bermejo, S., Xue, J., Hoang, L. et al. Quantitative multiple fragment monitoring with enhanced in-source fragmentation/annotation mass spectrometry. Nat Protoc 18, 1296–1315 (2023). https://doi.org/10.1038/s41596-023-00803-0
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DOI: https://doi.org/10.1038/s41596-023-00803-0
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