384 well ground steel target

Frozen cell suspensions were thawed on ice and diluted with ddH₂O to desired concentrations. For dried droplet application equal volume amounts of a cell suspension and matrix solution were mixed together and left on ice for 10 min. To avoid the problem of non-specific interactions of cellular proteins with the pipette surface each pipette tip was saturated with matrix-analyte mixture (by pipetting the mixture three times) prior to application. Then 1–1.5 µL of the resulting mixture was spotted on 384-well ground steel target (Bruker Daltonics) in n technical replicates (n=3, 4, 16 or 24) and air dried. For double layer application first 1–1.5 µL of SA in acetonitrile (1 mg/mL of ACN) was applied to 384-well ground steel plate and air dried. Then it was followed by dried droplet application of matrix-analyte mixture as described above.

Live cells in PBS media were diluted with PBS to desired concentrations. The samples were kept on ice in between dilution steps and sample preparation. Equal volume amounts of a cell suspension and matrix solution were mixed together and left on ice for 10 min. To avoid the problem of non-specific interactions of cellular proteins with the pipette surface each pipette tip was saturated with matrix-analyte mixture (by pipetting the mixture three times) prior to application. Then 1.5 µL of the resulting mixture was spotted on 384-well ground steel target (Bruker Daltonics) in triplicates and air dried. Dried droplet applications that were washed with water to remove PBS salts were prepared following all the steps described above. Then 1.5 μL of ice-cold water was gently applied to the top of a dry crystalline preparation and briefly removed with Kimwipe trying not to disturb the sample integrity. After washing step crystalline samples were dried at room temperature.

Frozen MOE and OVCAR8 cell suspensions at 1×10⁴ cells/μL were thawed on ice and diluted with cold ddH₂O to 2,500 cells/μL. Then MOE and OVCAR8 suspensions were mixed to obtain a range of two-component cell line populations containing 50 to 10% (with 10% increment) or 9 to 1% (with 1% increment) of OVCAR8 cancer cells in the mixture. All cell suspensions were kept on ice during dilution and mixing steps. For dried droplet application equal volume amounts of a cell suspension and SA20 matrix solution were mixed together and left on ice for 10 min. To avoid the problem of non-specific interactions of cellular proteins with the pipette surface each pipette tip was saturated with matrix-analyte mixture (by pipetting the mixture three times) prior to application. Then 1.5 µL of the resulting mixture was spotted on 384-well ground steel target (Bruker Daltonics) in 16 or 24 replicates per population and air dried.