The following are excerpts from Zhu et al., 2020. Please refer to this publication for more details.

Methodology

Fabrication of the Micropipette Needle. The micropipette needle (tip size ≈ 15 um) was pulled from a glass capillary tube (size: 0.8 × 90 mm2, Kimble Chase Life Science and Research Products, Rockwood, TN) using a pipet puller (KOPF, Tujunga, CA). UV epoxy (Prime-Dent, Chicago, IL) was used to connect the micropipette needle to a fused silica capillary (OD: 150 μm, ID: 75 μm, Polymicro Technologies, Phoenix, AZ). A syringe was connected to the fused silica capillary via a conductive union (IDEX Health & Science LLC, Oak Harbor, WA).

Sampling device

The Micropipette Needle was produced for sampling. The micropipette needle (tip size ≈ 15 um) was pulled from a glass capillary tube (size: 0.8 × 90 mm2, Kimble Chase Life Science and Research Products, Rockwood, TN) using a pipet puller (KOPF, Tujunga, CA). UV epoxy (Prime-Dent, Chicago, IL) was used to connect the micropipette needle to a fused silica capillary (OD: 150 μm, ID: 75 μm, Polymicro Technologies, Phoenix, AZ). A syringe was connected to the fused silica capillary via a conductive union (IDEX Health & Science LLC, Oak Harbor, WA). Using an Eppendorf cell manipulation system and a syringe pump, a target cell was sucked into the glass micropipette (flow rate 10 μL/min) containing prefilled acetone or benzophenone solution. Additional solution was drawn into the micropipette needle to ensure cell lysis. The syringe pump was turned on to deliver (flow rate 0.2 μL/min) the single cell lysate toward the nano-ESI emitter. Both the regular (no UV irradiation) and the reactive (after UV irradiation) single cell MS experiments can be conducted for the same single cell. Specifically, after accomplishing data acquisition of the regular SCMS experiment, the ionization energy was turned off and the syringe pump was paused. The UV lamp (BHK, Ontario, CA) was then turned on to generate UV radiation and initiate PB reactions between the reagents and unsaturated cellular lipids. After 15 min of reaction, the UV light was turned off, and then

the reactive single cell MS experiment was started by turning on the ionization voltage and resuming the syringe pump. Products from the PB reactions were analyzed using both MS scan (to obtain accurate m/z values of all ions) and tandem MS (MS/MS) analysis (to acquire fragments of selected ions).

Instrument

Thermo LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, United States). Mass analyze parameters were as follows: mass resolution 60,000, 1 microscan, 100 ms max injection time, and automatic gain control on. A DC ionization voltage (+4 kV in the positive ion mode or −4 kV in the negative ion mode) was applied on a conductive union and transmitted through the solvent to induce ionization of cell lysis at the tip of the micropipette for MS analysis.

Location of experiments: University of Oklahoma, Norman, OK 73019

Cell line: HCT-116 (human colon cancer cell line)

Reactions: Paternò-Büchi (PB) between unsaturated lipids (contain carbon-carbon double bonds) and acetone or benzophenone

mass_spec_double_bonds.csv