Pinto, Frederico G.Giddey, Alexander D.Mohamed, NesrinAlmarri, Rauda S. B.Murtaza, MunazzaNassir, NasnaAlkhnbashi, Omer S.Uddin, Mohammed J.Soares, Nelson C.2026-01-202026-01-202025-07-24Expert Rev Proteomics. 2025 Aug;22(8):307-314. doi: 10.1080/14789450.2025.2537210. Epub 2025 Jul 241478-9450http://hdl.handle.net/10400.18/10719Background: Many of the advanced MS methods applied in proteomics such as nanoflow LC-MS with data-independent acquisition have yet to be verified and/or optimized on metabolomics applications. Research design and methods: This study evaluates the feasibility of repurposing a proteomics-optimized nanoLC-MS platform for untargeted metabolomics. Using NIST SRM 1950 reference human plasma, we compared the performance of polarity switching and separate polarity modes under DIA conditions, focusing on metabolite coverage, annotation, and response linearity. Results: We observed, in the separate polarity and switching polarity runs 669 and 353 features in (+) mode and 558 and 446 features in (-) mode, respectively. A total of 233 metabolites were annotated using the (±) separate polarities and 179 using the (±) switching polarity based on MassBank of North America (MoNA) public MS library and filtered with the Human Metabolome Database (HMDB). Both switching and separate polarity methods performed well regarding response linearities which were investigated by spiking some amino acid compounds into plasma matrix. Conclusions: The polarity switching DIA approach for metabolomics reduced sample consumption and analysis time, but led to fewer detected features and annotations compared to separate polarity runs. These findings support the use of unified nanoLC-MS platforms for integrated multi-omics analysis.Highlights: - This study evaluates the performance of polarity switching compared to separate polarity modes in nano liquid chromatography mass spectrometry (nanoLC-MS/MS) for DIA untargeted metabolomics using the NIST SRM 1950 reference human plasma. - While polarity switching offers advantages such as reduced sample consumption and faster overall analysis time, it showed however fewer detected features and annotated compounds, and lower reproducibility compared to separate polarities. - A key aspect of our study is the development of a versatile nanoLC-MS/MS method that can be employed for both high-throughput proteomics and untargeted metabolomics without the need to alter the HPLC system from nano to microflow, mobile-phases or separation column dimensions and chemistry, whereas it is currently common practice in multi-omic laboratories to change all or several of these aspects when switching between these two ‘omics applications.engUntargeted MetabolomicsData Independent AnalysisHuman PlasmaMass SpectrometrySwitching PolarityData Independent AnalysisGenómica Funcional e Estruturaljournal article10.1080/14789450.2025.25372101744-838740685892