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This study proposed an easy-to-use method for cell identification and quantitation by ratiometric matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Two pairs of MS peaks in the molecular fingerprint of cells were selected as intracellular dual-biomarkers due to the stability and specificity of their ratio values in different types of hepatocellular cancer (HCC) cell lines. Five types of HCC cells can be thereafter differentiated based on these two pairs of intracellular peptides/proteins. Two types of HCC cells, Huh7 and LM3 were co-cultured as a model to test whether the method is feasible for cell quantitation. The results indicated that the ratiometric peak intensity of the two pair biomarkers exhibits linear relationship with the proportion of Huh7 cells. Furthermore, tumor heterogeneity was simulated by subcutaneously injecting the co-cultured cells into nude mice. The cell type and proportion in the section of grown tumor tissue can be discriminated using the ratiometric MALDI imaging approach. LC-MS/MS detection revealed that one of the biomarker pairs belongs to thymosin family, β4 and β10. The ratiometric MS spectral approach using intracellular dual-biomarkers might become a pervasive strategy for high-throughput cell identification and quantitation, which is vital in tumor heterogeneity study, clinical diagnosis and drug screening.

Ratiometric Mass Spectrometry for Cell Identification and Quantitation Using Intracellular “Dual-Biomarkers”