Nitric oxide (NO) plays important roles in cell functions, including neuronal signaling, immune response, and cancer progression. Recent studies suggest that the functions of NO are related to their intracellular concentrations. Cell heterogeneity has been reported in nearly all biological systems, and it is commonly regarded as a major challenge for numerous human diseases such as cancer. Thus, quantification of NO concentrations in individual cells present in heterogeneous populations of cells can substantially improve our understanding of the functions and mechanisms of NO in biological systems. NO is a small molecule with short lifetime, and it is very challenging to directly detect it from live cells using mass spectrometry (MS). A recent study indicates that NO can rapidly (&lt;1 s) and completely (~100% efficiency) react with amlodipine (AML) to produce dehydro amlodipine (DAM), which can be readily detected by MS. In our proposed studies, we will design a new single cell MS technique, allowing for NO detection and quantification in single cells. Briefly, we selected HCT116 colorectal cancer cell line as the model system and cultured using glass chips containing microwells. We used doxorubicin to stimulate NO production in cells, and treated cells by AML to produce DAM. We selected single cells in individual wells and then used the Single-probe SCMS technique to analyze cells. The produced DAM were detected and quantified using internal standard.

Development of real-time reactive single cell mass spectrometry methods to quantify nitric oxide (NO) inside live single cells

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