Protein expression varies between cells, and observing this heterogeneity helps to understand biological phenomena. We have developed a water droplet-in-oil digestion (WinO) method for single-cell proteomics. The WinO method reduces adsorption losses of proteins and peptides during sample preparation and allows parallel sample preparation in 96-well plates. However, the original WinO method requires liquid-liquid extraction during the peptide desalting step and manual sample combining after tandem mass tag labeling, which are major obstacles to automating sample preparation. The purpose of the present study was to improve the throughput of the WinO method. First, to eliminate the liquid-liquid extraction step, we used a polystyrene-divinylbenzene reversed-phase sulfonated disk for peptide desalting instead of a polystyrene-divinylbenzene reversed-phase disk. In addition, we changed the organic solvent used from highly volatile ethyl acetate to less volatile hexadecane to improve ease of use. We also used a liquid handling robot for sample combining. With this modified WinO method, the sample preparation time was reduced by three quarters, while the recovery rate remained the same as the original method. This modified WinO method enables automation of the sample preparation process and is expected to be applied to large-scale single-cell proteomics.

¹³C-Metabolic flux analysis (MFA) using mass spectrometry was applied to investigate metabolic flux distributions in undifferentiated, differentiated, and LPS-activated HL-60 myeloid leukemia cells. A series of ¹³C-tracing experiment revealed that glucose and glutamine, as well as aspartate and glutamate in the culture medium were incorporated into HL-60 cells. The metabolic model including the carbon incorporation reactions was used for the ¹³C-MFA. The estimated metabolic flux distribution showed that the level of TCA cycle fluxes was constant, while differentiated HL-60 cells decreased the level of glycolysis flux compared to that of undifferentiated HL-60 cells. LPS-activated differentiated HL-60 cells showed that increased levels of glucose uptake and pentose phosphate pathway.

In conventional ESI, electrochemical reactions occur at the interface between the emitter electrode and the solution. This study deals with “bipolar electrospray ionization,” in which positive and negative ions are generated simultaneously, with the aim of preventing electrochemical reactions in the emitter. The voltage-current and flow rate-current relationships of bipolar electrospray were found to be similar to the standard electrospray. The application of bipolar ESI to the mass spectrometry of melittin without electrochemical oxidation is demonstrated.