Benchmarking the Exploris 240 mass spectrometer and compound discoverer software 3.3 with yeast metabolome extracts

Authors: Cooper, Bret; Yang, Ronghui

Submitted to: Journal of Proteome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/4/2023
Publication Date: 1/23/2024
Citation: Cooper, B., Yang, R. 2024. Benchmarking the Exploris 240 mass spectrometer and compound discoverer software 3.3 with yeast metabolome extracts. Journal of Proteome Research. 14. Article e4841. https://doi.org/10.1038/s41598-024-55356-3.
DOI: https://doi.org/10.1038/s41598-024-55356-3

Interpretive Summary: The Exploris 24 mass spectrometer coupled with Compound Discoverer 3.3 software is a commercial platform designed to perform metabolomics, a scientific discipline whereby large proportions of small molecules in biological or environmental samples are identified. To test this system, USDA-ARS scientists in Beltsville, Maryland, analyzed water-soluble and organic solvent extracts from yeast, a model organism that produces a defined set of biological chemical compounds. The scientists resolved more than 5,000 different compounds and demonstrated that unique compounds could be found in each of the polar and non-polar extracts when using both positive and negative modes of electricity to charge the molecules. By performing two rounds of mass spectrometry, the scientists doubled the number of confidently identified metabolites compared to using one round of mass spectrometry. This research paper will be of interest to scientists in the government, at universities, and at private institutions who want to perform metabolomics in scientific disciplines such as medical science, environmental science, and agriculture. Examples of the types of results users may encounter are provided.

Technical Abstract: We analyzed yeast extracts with an Exploris 240 mass spectrometer, using AcquireX software for deep interrogation and Compound Discoverer 3.3 software to compare observed MS2 spectra to reference spectra from the National Institute of Standards and Technology and mzCloud. The polar fraction analyzed in negative ion mode yielded the most ions, but the polar fraction analyzed in positive ion mode yielded the most high-scoring MS2 matches. Non-polar fractions yielded the fewest ions, but these were distinct compared to the polar fractions. AcquireX increased the total number of potentially identifiable compounds by 50% through six iterations of MS2 acquisition. The union of non-redundant, high-scoring MS2 identifications from polar and non-polar extracts evaluated in positive and negative ion modes yielded 441 compounds from nearly 8,000 candidate MS2 spectra. Only 212 of these same compounds were found by parent ion mass matching to the Yeast Metabolome Database in ChemSpider. The results demonstrate the utility of MS2 spectrum library matching for compound identification and the capacity of this system for deep interrogation of a biological sample. We provide instrument and software settings, an explanation of the discovery workflow, and six examples of the types of true and false positive results users may encounter.