Acetaldehyde reacts with a fluorescent nitric oxide probe harboring an o-phenylenediamine structure that interferes with fluorometry.

Nitric oxide (NO) is a ubiquitous signaling molecule, and thus a variety of methods have been developed for its detection and quantification. Fluorometric analyses using a fluorescent NO probe harboring an o-phenylenediamine (OPD) structure are widely used for NO analyses in various organisms, including yeast. Here, we discovered that an NO-independent fluorophore (UNK436) was generated from a fluorescent NO probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM), which has an OPD structure, in yeast cells. The molecules responsible for this undesirable fluorescence and their reaction mechanisms were analyzed. Our mass spectrometric analysis showed that two carbon atoms from glucose were incorporated into UNK436. Subsequent analyses indicated that a non-proteinous small compound leads to the synthesis of UNK436 through an oxidative reaction. Furthermore, our LC/MS/MS analysis of the reaction mixture of DAF-FM with acetaldehyde in combination with stable isotope labeling demonstrated that acetaldehyde reacts with DAF-FM oxidatively, generating UNK436. Another NO probe with an OPD structure, diaminorhodamine-4M, reacted with acetaldehyde in the same way to emit fluorescence. Based on our findings, we recommend that in researches using OPD-based fluorescent NO probes, alternative analyses also be performed to identify the reaction products of the probes with NO to avoid false-positives.