LC-MS/MS Iron-Binding Metabolomics (Data Analysis).

Feature finding and ion identity networking were performed using an in-house modified version of MZmine2.37 (68), corr.17.7 available at https://github.com/robinschmid/mzmine2/releases. Feature tables, MS/MS spectra files (mgf), and ion identity networking results were exported, uploaded to the MassIVE repository, and submitted to GNPS (35) for feature-based molecular networking analysis.

MS/MS spectra were converted to .mzML files using MSconvert (ProteoWizard) (69). All raw and processed data are publicly available at ftp://massive.ucsd.edu/MSV000086287/. MS1 feature extraction and MS/MS pairing were performed with MZmine 2.37 (68) corr17.7_kai_merge2. An intensity threshold of 1E5 for MS1 spectra and of 1E3 for MS/MS spectra was used. MS1 ADAP chromatogram building was performed within a 10 ppm mass window and a minimum peak intensity of 3E5 was set. Extracted Ion Chromatograms (XICs) were deconvoluted using baseline cutoff with m/z range for MS2 pairing of 0.01 and RT range for MS2 scan pairing of 0.2. After chromatographic deconvolution, MS1 features linked to MS/MS spectra within 0.01 m/z mass and 0.2 min retention time windows. Isotope peaks were grouped and features from different samples were aligned with 10 ppm mass tolerance and 0.2 min retention time tolerance. MS1 peak lists were joined using an m/z tolerance of 10 ppm and retention time tolerance of 0.1 min; alignment was performed by placing a weight of 75 on m/z and 25 on retention time. Correlation of coeluting features was performed with the metaCorrelate module; retention time tolerance of 0.1, minimum height of 1E5 and noise level of 1E5 were used. A correlation of 0.85 was set as the threshold for the min feature shape corr., and feature height correlation was not used. The following adducts were searched: [M + H⁺]⁺, [M + Na⁺]⁺, [M + K⁺]⁺, [M + NH]²⁺, [M + Fe³⁺ - 2H⁺]⁺, with an m/z tolerance of 10 ppm, a maximum charge of 2, and maximum molecules/cluster of 2.

Peak areas and feature correlation pairs were exported as .csv files and the corresponding consensus MS/MS spectra were exported as an .mgf file. For spectral networking and spectrum library matching, all files were uploaded to the feature-based molecular networking workflow on GNPS (35) (https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp). For spectrum library matching and spectral networking, the minimum cosine score to define spectral similarity was set to 0.7. The Precursor and Fragment Ion Mass Tolerances were set to 0.01 Da and Minimum Matched Fragment Ions to 4, Minimum Cluster Size to 1 (MS Cluster off). When analog search was performed, the maximum mass difference was set to 100 Da. The GNPS job can be accessed at https://gnps.ucsd.edu/ProteoSAFe/status.jsp?task=76115ecaad9c46e9aaa30413d0d435eb. Molecular networks were visualized with Cytoscape 3.7.1 (70).

The XCalibur Quant Browser (Thermo) was used to obtain integrated peak areas of both apo- and iron-bound adducts. The XCalibur processing method integrates exact mass +/− 10 ppm, utilizing ICIS Peak Detection with 7 smoothing points, a baseline window of 40, area noise factor of 5, and peak noise factor of 6. Statistical significance between integrated peak areas was calculated using an unpaired t test.