Systematic profiling of indole-3-acetic acid biosynthesis in bacteria using LC–MS/MS

• Simultaneous determination of six intermediates in IAA biosynthesis by LC–MS/MS.
• Application in Pseudomonas putida with known IAA biosynthesis pathway.
• A simple, rapid, and reliable assay for understanding the IAA biosynthesis pathway.

Indole-3-acetic acid (IAA) is produced from tryptophan through five synthesis pathways. A comprehensive method for the quantification of IAA and biosynthesis-related intermediates in a culture medium was developed. Sample preparation was simple with protein precipitation. The analytes were separated on a superficially porous C18 silica column and detected by electrospray ionization-tandem mass spectrometry in the positive ion multiple reaction monitoring mode. The limit of detection was 0.05 μM, and the lower limits of quantification ranged from 0.05 to 2 μM. The intra-day and inter-day precision and accuracy were less than 13.96%. Ion suppression was observed, and the deuterated internal standards were used to compensate for the matrix effect. The method was applied to analyze changes in tryptophan catabolism in a culture medium of Pseudomonas putida. The proposed method is robust and suitable for the systematic profiling of IAA biosynthesis in culture supernatant.