Combining Mass Spectrometry with Paternò-Büchi Reaction to Determine Double-bond Positions in Lipids at the Single-cell Level.

Single cell MS (SCMS) techniques are under rapid development for molecular analysis of individual cells among heterogenous populations. Lipids are basic cellular constituents playing essential functions in energy storage and cellular signaling processes of cells. Unsaturated lipids are characterized with one or multiple carbon-carbon double (C=C) bonds, and they are critical for cell functions and human diseases. Characterizing unsaturated lipids in single cells allows for better understanding of metabolomic biomarkers and therapeutic targets of rare cells (e.g., cancer stem cells); however, these studies remain challenging. We developed a new technique using micropipette needle, in which Paterno-Buchi (PB) reactions at C=C bond can be induced, to determine lo-cations of C=C bonds in unsaturated lipids at the single-cell level. The micropipette needle is produced by combining a pulled glass capillary needle with a fused silica capillary. Cell lysis solvent and PB reagent (acetone or benzophenone) are delivered into the micropipette needle (tip size ~15 um) through a fused silica capillary. The capillary needle plays multiple functions (i.e., single cell sampling probe, cell lysis container, micro-reactor, and nano-ESI emitter) in the experiments. Both regular (no reaction) and reactive (with PB reaction) SCMS analyses of the same cell can be achieved. C=C bond locations were determined from MS scan and MS/MS of PB products assisted by Python programs. This technique can be potentially used for other reactive SCMS studies to enhance molecular analysis for broad ranges of single cells.