Measurement of tryptophan metabolites

Heads from 14-day old flies were dissected and immediately frozen in liquid nitrogen and kept at −80°C until processing. We collected at least four independent replicates (30 heads/sample) per genotype, sex, and treatment group. Fly heads were homogenized by sonication in 300 μL of ultrapure water containing 0.01% BHT (butylated hydroxytoluene). Fly homogenates and calibration standards were prepared in ultrapure water containing 0.01% BHT (100 μL) with the appropriate amount of tryptophan (TRYP), kynurenic acid (KYNA), kynurenine (KYN) and 3-hydroxykynurenine (3-HK) to achieve concentrations ranging from 0.5-1,000 ng/mL homogenate. Additional QCs were prepared in control homogenate to ensure accuracy of extraction from the sample matrix. Standards, QCs and samples were treated with deuterated internal standards (1 μg/mL TRYP-d5, KYNA-d5, KYN-d4, and 3-HK ¹³C₂¹⁵N) and 10 μl of 20% perchloric acid for protein precipitation. After centrifugation for 5 minutes at 21000xg, the supernatant was transferred to autosampler vials and analyzed in positive ion mode by LC/MS/MS consisting of a Shimadzu system (Columbia MD) equipped with LC20-AD dual HLPC pumps, an SIL20-AC HT autosampler, and a DGU-20A2 in-line degasser.

Mass calibration, data acquisition, and quantitation were performed using Applied Biosystem Analyst 1.6.2 software (Applied Biosystems, Foster City, CA). We used a Phenomenex Luna Omega Polar C18, 100 X 2 mm 5 μm particle column to separate the tryptophan metabolites and the internal standards from the homogenate matrix. The mobile phase was delivered at a flow rate of 400 μL/min using a gradient elution profile consisting of DI water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). A gradient elution profile was used in which mobile phase B was held at 3% for 2.5 minutes, then increased to 90% over 2.5 minutes, held at 90% for 1 minute, returned to 3%, and equilibrated for 5 minutes. The analytes were detected using multiple reaction monitoring for the following transitions: TRYP (m/z 205.0→188.0), KYNA (m/z 190.1→144.0), KYN (m/z 209.2→192.0), 3-HK (m/z 225.0→110.0). The internal standard transitions were as follows: TRYP-d5 (m/z 210.0→192.0), KYNA -d5 (m/z 195.1→149.0), KYN-d4 (m/z 213.0→196.0), 3-HK ¹³C₂¹⁵N (m/z 228.0→110.0).