The following are excerpts from Zhu et al., 2019. Please refer to this publication for more details.

Methodology

During the SCMS analysis, the redesigned T-probe was coupled to the in-home developed SCMS platform employed in our previous SCMS studies. Briefly, this platform includes an XYZ-translational stage system, two digital microscopes, and a Thermo LTQ Orbitrap XL mass spectrometer. Cells in both control and drug treatment groups were used for the SCMS experiments (detailed sample preparation procedures are described in the Supporting Information). Irinotecan is a common anticancer drug for the treatment of colon cancer that inhibits the function of Topoisomerase I, leading to DNA damage and cell apoptosis. This drug compound was selected to treat live HCT-116 colorectal cells in our experiments to demonstrate the change of cellular metabolites upon the treatment of anticancer agent. Specifically, cells were first treated using 18 mM irinotecan for 45 min, and then rinsed and detached using trypsinization. Afterwards, a droplet of cell suspension solution was placed onto a glass slide, which was attached to the XYZ-stage system controlled by a LabView software package (incremental step size = 0.1 mm). Using two digital microscopes as the visual guide, the sampling probe tip initially located above the sample plate was submerged into the solution containing cells by lifting the Z-stage. Upon selecting a target cell, the sampling probe can precisely draw the target cell with visual guidance. The XYZ-stage was then immediately lowered down to free the sampling probe tip from the culture medium and stop the suction of culture medium. Due to the complex composition of the cell culture medium that may affect the detection sensitivity, caution should be taken to minimize its amount withdrawn during cell sampling. This is particularly important for future analysis of patient cells suspended in complex biological fluids such as blood, urine, saliva, and cerebrospinal fluid (CSF). After a single cell was withdrawn, the solvent provided through the solvent-providing capillary (flowrate = 0.5 mL/min) mixed with the cell at the T-junction, and cell lysis rapidly occurred inside the nano-ESI emitter. In our SCMS analysis, an ionization voltage (~4 kV) was applied to the conductive union and transmitted throughout the solution inside the solvent-providing capillary and the nano-ESI emitter to ionize the cell lysis for MS analysis.

Instrument

Thermo LTQ Orbitrap XL mass spectrometer (Thermo Scientific, Waltham, MA, United States). Mass analyze parameters were as follows: mass resolution 60,000, +4 kV ionization voltage at positive ion mode (0.05–0.07 μA of ion current), 1 microscan, 100 ms max injection time, and automatic gain control on.

Location of experiments: University of Oklahoma, Norman, OK 73019
Cell line: HCT-116 (human colon cancer cell line)

mass_spec_metabolites.csv