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(A, B) Confocal imaging of bifunctional fatty acid analogues ( cl-SA , cl-2MeSA , and cl-10MeSA ) in HeLa cells after 4 hour incubation and subsequent SPAAC labeling with BODIPY-BCN (CO-1 ). Images were taken with co-treatment of oleic acid to induce lipid droplet formation. (A) Co-staining with LipidSpot 610 (1/1000 dilution) <t>and</t> <t>ER-Tracker</t> Red (1 μM). (B) Co-staining with CellMask Plasma Membrane Deep Red (2 μg/mL) and BioTracker 405 Blue Mitochondria (100 nM). (C–J) Representative MS/MS spectra of modified glycerophospholipids incorporating azido-functionalized fatty acids and branched analogues. (C, D) MS/MS spectra of modified PC species. (E, F) MS/MS spectra of modified PE species. (G, H) MS/MS spectra of modified PI species. (I, J) MS/MS spectra of PS species. Diagnostic fragment ions are highlighted to support lipid class identification and incorporation of modified acyl chains.
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(A, B) Confocal imaging of bifunctional fatty acid analogues ( cl-SA , cl-2MeSA , and cl-10MeSA ) in HeLa cells after 4 hour incubation and subsequent SPAAC labeling with BODIPY-BCN (CO-1 ). Images were taken with co-treatment of oleic acid to induce lipid droplet formation. (A) Co-staining with LipidSpot 610 (1/1000 dilution) <t>and</t> <t>ER-Tracker</t> Red (1 μM). (B) Co-staining with CellMask Plasma Membrane Deep Red (2 μg/mL) and BioTracker 405 Blue Mitochondria (100 nM). (C–J) Representative MS/MS spectra of modified glycerophospholipids incorporating azido-functionalized fatty acids and branched analogues. (C, D) MS/MS spectra of modified PC species. (E, F) MS/MS spectra of modified PE species. (G, H) MS/MS spectra of modified PI species. (I, J) MS/MS spectra of PS species. Diagnostic fragment ions are highlighted to support lipid class identification and incorporation of modified acyl chains.
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(A, B) Confocal imaging of bifunctional fatty acid analogues ( cl-SA , cl-2MeSA , and cl-10MeSA ) in HeLa cells after 4 hour incubation and subsequent SPAAC labeling with BODIPY-BCN (CO-1 ). Images were taken with co-treatment of oleic acid to induce lipid droplet formation. (A) Co-staining with LipidSpot 610 (1/1000 dilution) <t>and</t> <t>ER-Tracker</t> Red (1 μM). (B) Co-staining with CellMask Plasma Membrane Deep Red (2 μg/mL) and BioTracker 405 Blue Mitochondria (100 nM). (C–J) Representative MS/MS spectra of modified glycerophospholipids incorporating azido-functionalized fatty acids and branched analogues. (C, D) MS/MS spectra of modified PC species. (E, F) MS/MS spectra of modified PE species. (G, H) MS/MS spectra of modified PI species. (I, J) MS/MS spectra of PS species. Diagnostic fragment ions are highlighted to support lipid class identification and incorporation of modified acyl chains.
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(A, B) Confocal imaging of bifunctional fatty acid analogues ( cl-SA , cl-2MeSA , and cl-10MeSA ) in HeLa cells after 4 hour incubation and subsequent SPAAC labeling with BODIPY-BCN (CO-1 ). Images were taken with co-treatment of oleic acid to induce lipid droplet formation. (A) Co-staining with LipidSpot 610 (1/1000 dilution) and ER-Tracker Red (1 μM). (B) Co-staining with CellMask Plasma Membrane Deep Red (2 μg/mL) and BioTracker 405 Blue Mitochondria (100 nM). (C–J) Representative MS/MS spectra of modified glycerophospholipids incorporating azido-functionalized fatty acids and branched analogues. (C, D) MS/MS spectra of modified PC species. (E, F) MS/MS spectra of modified PE species. (G, H) MS/MS spectra of modified PI species. (I, J) MS/MS spectra of PS species. Diagnostic fragment ions are highlighted to support lipid class identification and incorporation of modified acyl chains.

Journal: bioRxiv

Article Title: Selective Editing and Functionalization of the Mammalian Lipidome

doi: 10.64898/2026.04.24.720406

Figure Lengend Snippet: (A, B) Confocal imaging of bifunctional fatty acid analogues ( cl-SA , cl-2MeSA , and cl-10MeSA ) in HeLa cells after 4 hour incubation and subsequent SPAAC labeling with BODIPY-BCN (CO-1 ). Images were taken with co-treatment of oleic acid to induce lipid droplet formation. (A) Co-staining with LipidSpot 610 (1/1000 dilution) and ER-Tracker Red (1 μM). (B) Co-staining with CellMask Plasma Membrane Deep Red (2 μg/mL) and BioTracker 405 Blue Mitochondria (100 nM). (C–J) Representative MS/MS spectra of modified glycerophospholipids incorporating azido-functionalized fatty acids and branched analogues. (C, D) MS/MS spectra of modified PC species. (E, F) MS/MS spectra of modified PE species. (G, H) MS/MS spectra of modified PI species. (I, J) MS/MS spectra of PS species. Diagnostic fragment ions are highlighted to support lipid class identification and incorporation of modified acyl chains.

Article Snippet: Media is then removed and replaced with 200 μL of 1 mg/mL Hoechst-33342 in phenol red-free DMEM media (10% FBS, 1% P/S) for 20 min or, for co-staining experiments, the organelle tracking dyes were used at the following concentration diluted in phenol red-free DMEM media (without FBS or P/S) and incubated for 10 min, BioTracker 405 Blue Mitochondria Dye (Millipore Sigma #SCT135, 100 nM) and CellMaskTM Plasma Membrane Deep Red (Thermo Fisher C10046, 2 μg/mL) or ER-Tracker red (MCE #HY-D1431, 2 μM) and LipidSpotTM 610 (Biotium #70069-T, 1/1000 dilution).

Techniques: Imaging, Analogues, Incubation, Labeling, Staining, Clinical Proteomics, Membrane, Tandem Mass Spectroscopy, Modification, Diagnostic Assay