Journal: bioRxiv

Article Title: RISOP, a Reference-Assisted Approach for Enhanced Identification of Oxidized Phospholipids

doi: 10.64898/2026.06.01.728773

Figure Lengend Snippet: Identification of oxidized phospholipids in Fenton and H 2 O 2 -only references. ( A ) Preparation of OxPL references. KP4 cell suspensions were subjected to Fenton reaction and H 2 O 2 -only treatment for 5 different reaction time points. At the end of each reaction time point, samples were harvested for lipidomic analysis by LC-MS/MS. ( B ) OxPL identification in separate Fenton and H 2 O 2 -only references. Venn diagram showing the number of unique and commonly identified OxPLs from Fenton and H 2 O 2 -only references. ( C ) Representative Fenton-unique and H 2 O 2 -only-unique OxPLs. Top: Extracted ion chromatograms (EIC) of two representative OxPLs PC(16:0_18:3<2OH>) (left) and PS(18:0_9:0 ) (right) from Fenton references (red) and H 2 O 2 -only references (blue). Middle: Representative tandem mass spectra with diagnostic ions for identification. Each fragment ion is annotated with mass-to-charge ratio ( m/z ) and corresponding fragment identity or elemental composition. Fragments labeled in blue, purple, green, and red represent non-oxidized side chains, oxidized side chains, lipid class characteristic ions, and precursors or precursors with neutral loss (NL), respectively. Bottom: chemical structures of PC(16:0_18:3<2OH>) (left) and PS(18:0_9:0 ) (right) with isomers not specified. Annotations follow the identical color codes as in middle panel.

Article Snippet: Next, 25 μL of 10 M H 2 O 2 (Thermo Fisher Scientific, L14000.AP) and 75 μL of 333.33 mM of FeCl 2 (Sigma, 44939-50G) were added to cell suspension for a final concentration of 500 mM of H 2 O 2 and 50 mM of FeCl 2 .

Techniques: Liquid Chromatography with Mass Spectroscopy, Diagnostic Assay, Labeling

Journal: bioRxiv

Article Title: RISOP, a Reference-Assisted Approach for Enhanced Identification of Oxidized Phospholipids

doi: 10.64898/2026.06.01.728773

Figure Lengend Snippet: Reference-assisted combined analysis on OxPL references expands identification coverage and dynamic range of OxPLs. ( A ) OxPLs identified from combined analysis of Fenton and H 2 O 2 -only references. Venn diagram showing the number of unique and commonly identified OxPLs from Fenton and H 2 O 2 -only references. ( B ) Heatmap of 67 OxPLs identified in Fenton and/or H 2 O 2 references. Each row represents one OxPL, and each column represents individual replicate of the Fenton or H 2 O 2 -only references. Columns are grouped by reference type, and rows are grouped by distinct oxidative modifications. Z scores representing the relative abundance of each OxPL are calculated from normalized OxPL intensity and plotted. Grey squares with crosses indicate OxPLs that were not detected. ( C ) Principal component analysis (PCA) on log2-transformed normalized intensity of 57 common OxPLs identified in Fenton (dots in shades of red and grey) and H 2 O 2 -only (triangles in shades of blue and grey) references from untargeted lipidomics. Each data point represents one sample at a specific time point from either Fenton or H 2 O 2 -only references. ( D-E ) PCA on log2-transformed normalized intensity of 63 OxPLs identified in Fenton ( D ) and 61 OxPLs identified in H 2 O 2 -only ( E ) references from untargeted lipidomics. Each data point represents one sample at a specific time point from either Fenton references or H 2 O 2 -only references. ( F ) K-means clustering on 57 common OxPLs identified in both Fenton and H 2 O 2 -only references. K-means clustering was performed on normalized intensity of OxPLs in reference samples from different time points of Fenton or H 2 O 2 -only treatment. Number of OxPLs from Fenton or H 2 O 2 -only references in each cluster is indicated. Z scores of individual OxPLs (grey lines) are shown together with red and blue lines representing the mean Z score from Fenton and H 2 O 2 -only references, respectively. Line thickness is proportional to the number of OxPLs. Dashed line indicates a Z score of zero.

Article Snippet: Next, 25 μL of 10 M H 2 O 2 (Thermo Fisher Scientific, L14000.AP) and 75 μL of 333.33 mM of FeCl 2 (Sigma, 44939-50G) were added to cell suspension for a final concentration of 500 mM of H 2 O 2 and 50 mM of FeCl 2 .

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: RISOP, a Reference-Assisted Approach for Enhanced Identification of Oxidized Phospholipids

doi: 10.64898/2026.06.01.728773

Figure Lengend Snippet: Reference-assisted identification of sample-specific oxidized phospholipid (RISOP). ( A ) In biological samples, oxidized phospholipids (OxPLs) are often present at low abundance. Consequently, their MS spectra are often insufficiently informative, making their annotation challenging (left panel). To overcome this challenge, we experimentally generate a reference pool of oxidized lipids by 2 oxidation reactions. These references are chemically enriched for oxidized phospholipid (OxPL) species in greater number and abundance, enabling identification of low-abundance OxPLs in biological samples through retention time (RT) and mass-to-charge ratio ( m/z ) matching to the oxidized lipid references (right panel). ( B ) Workflow of RISOP. 1) Untargeted lipidomic profiling of the biological sample. Lipidomic profiles of biological samples were obtained by untargeted LC-MS/MS lipidomics. 2) Generation of chemically enriched OxPL reference pools. Oxidized lipid references were generated by subjecting a subset of the biological samples to Fenton reaction or H 2 O 2 treatment alone (H 2 O 2 -only) for different reaction times, followed by LC-MS/MS lipidomic analysis. 3) Construction of a sample-specific in silico OxPL spectral library. The endogenous non-oxidized phospholipids identified from the biological samples from step 1 were used for in silico oxidation. The in silico -generated tandem mass spectra containing diagnostic ions of each OxPL species were used to generate a sample-specific oxidized phospholipid spectral library. 4) Reference-assisted OxPL annotation. OxPL features were first detected by retention time and m/z matching between biological samples and the experimental reference pools. These features were then annotated by matching their experimental MS/MS spectra against in silico MS/MS spectra generated from the sample-specific spectral library. OxPLs identified in biological samples were reported following spectral validation to remove misannotated OxPLs.

Article Snippet: Next, 25 μL of 10 M H 2 O 2 (Thermo Fisher Scientific, L14000.AP) and 75 μL of 333.33 mM of FeCl 2 (Sigma, 44939-50G) were added to cell suspension for a final concentration of 500 mM of H 2 O 2 and 50 mM of FeCl 2 .

Techniques: Liquid Chromatography with Mass Spectroscopy, Generated, In Silico, Diagnostic Assay, Tandem Mass Spectroscopy, Biomarker Discovery

Journal: bioRxiv

Article Title: RISOP, a Reference-Assisted Approach for Enhanced Identification of Oxidized Phospholipids

doi: 10.64898/2026.06.01.728773

Figure Lengend Snippet: Non-oxidized lipidomic profiles of Fenton and H 2 O 2 -only references. ( A ) Principal component analysis (PCA) on log2-transformed normalized intensities of 715 common non-oxidized lipids identified in Fenton (dots in shades of red and grey) and H 2 O 2 -only (triangles in shades of blue and grey) references. Each data point represents one sample at a specific time point from either Fenton or H 2 O 2 -only references. ( B-C ) PCA on log2-transformed normalized intensities of 715 non-oxidized lipids identified in Fenton (B) and 739 non-oxidized lipids identified in H 2 O 2 -only (C) references. Each data point represents one sample at a specific time point from either Fenton or H 2 O 2 -only references.

Article Snippet: Next, 25 μL of 10 M H 2 O 2 (Thermo Fisher Scientific, L14000.AP) and 75 μL of 333.33 mM of FeCl 2 (Sigma, 44939-50G) were added to cell suspension for a final concentration of 500 mM of H 2 O 2 and 50 mM of FeCl 2 .

Techniques: Transformation Assay

Journal: bioRxiv

Article Title: RISOP, a Reference-Assisted Approach for Enhanced Identification of Oxidized Phospholipids

doi: 10.64898/2026.06.01.728773

Figure Lengend Snippet: Applying RISOP in studying ferroptosis-associated lipid peroxidation. ( A ) Preparation of ML210-treated cells. KP4 cells were incubated with 10 μM ML210 for six treatment time points. At the end of each incubation time point, samples were harvested for lipidomic analysis by LC-MS/MS. ( B ) Number of identified OxPLs in ML210-treated samples with or without RISOP. Stacked bar plot showing numbers of OxPLs identified in ML210-treated samples alone without RISOP (purple), and additional OxPLs identified using Fenton references (red), H 2 O 2 -only references (blue), or both Fenton and H 2 O 2 -only references combined (dark magenta) through RISOP. ( C ) Principal component analysis (PCA) on log2-transformed molar concentrations of 22 OxPLs identified in ML210-treated samples. Each data point represents one sample harvested from a given time point. ( D ) PCA on log2-transformed molar concentrations of 723 non-oxidized lipids identified in ML210-treated cells. Each data point represents one sample harvested from a given time point. ( E ) K-means clustering on 22 OxPLs identified in ML210-treated samples. K-means clustering was performed on molar concentrations of OxPLs in ML210-treated samples treated for different durations. In each cluster, number of OxPLs is indicated. Z scores of individual OxPLs (grey lines) are shown together with brown lines in different shades representing the mean Z score of each cluster. Line thickness is proportional to the number of OxPLs. Dashed line indicates a Z score of zero. ( F ) Heatmap of 22 OxPLs identified in ML210-treated samples. Each row represents one OxPL, and each column represents one biological replicate at the indicated ML210 treatment time point. Columns are grouped by treatment time and ordered from 0 to 270 min. Rows are grouped according to the K-means clusters defined in . Row-wise transformed Z scores of molar concentrations of OxPLs from each identified cluster are presented. Only OxPL isomers (indicated with letter suffixes in parentheses) that are identified in ML210-treated samples were included.

Article Snippet: Next, 25 μL of 10 M H 2 O 2 (Thermo Fisher Scientific, L14000.AP) and 75 μL of 333.33 mM of FeCl 2 (Sigma, 44939-50G) were added to cell suspension for a final concentration of 500 mM of H 2 O 2 and 50 mM of FeCl 2 .

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Transformation Assay