Seasonally relevant changes in UV-A alter neurohormone levels and behavior via cOpsin1
Publication pending in Nature Ecology & Evolution
- Organism: Platynereis dumerilii
- Instrument: Q Exactive HF-X
Targeted MS, PRM, quantification, neurohormones
Many animals exhibit strong seasonal rhythms in their physiology and behavior, but the sensors conveying the environmental input are still unknown. In our study we provide detailed light measurements from the natural habitat of the marine bristle worm Platynereis dumerilii, showing that the ratios between different seasons differ more for day-time UV-A light than for longer wavelength. Mimicking seasonal UV-A differences, without photoperiodic changes, significantly decreased locomotor activities in non-mature worms and equaled the reduction occurring with short photoperiod. This UV-A dependent modulation of locomotion requires cOpsin1, likely signaled via Gi-signalling. cOpsin1 mutants also show a strong reduction of rate limiting enzymes for monogenic amine synthesis, as well as regulation of several neurohormones, including NPY, PDF and Vasotocin, as determined via parallel-reaction monitoring in this dataset. Together, these data provide strong functional evidence that cOpsin1 functions as a seasonal light receptor and suggest that daylengths, as well as UV-A levels signal annual time to animals.
PRM assays were set up based on a DDA measurement of PDF, NPY-1, NPY-4, GnRH-1 and Vasotocin synthetic peptides, with Lys-C protease specificity and amidated C-termini. The acquired spectra were used to prepare a spectral library in Proteome Discoverer (version 2.3) using MS Amanda (Dorfer et al., 2014) and Percolator (Käll, Canterbury, Weston, Noble, & MacCoss, 2007) with the following search parameters: database of target peptides and common contaminants, 10 ppm MS1 and 20 ppm MS2 mass tolerance, carbamidomethylation of Cys and amidated peptide c-termini as fixed and oxidation of Met as variable modifications, 5% FDR on PSM level. PRM assay generation was performed using Skyline software (version: daily 188.8.131.52) (MacLean et al., 2010). Samples were spiked with 100 fmol Pierce Peptide Retention Time Calibration Mixture (PRTC, Thermo-Fisher) to monitor the chromatographic and nano-spray stability across the PRM measurements of all samples. A wild-type sample spiked with the synthetic peptides served as positive control and retention time reference.
Peptides were separated on an Ultimate 3000 RSLC nano-flow chromatography system (Thermo-Fisher), using a pre-column for sample loading (Acclaim PepMap C18, 2 cm × 0.1 mm, 5 μm, Thermo-Fisher), and a C18 analytical column (Acclaim PepMap C18, 50 cm × 0.75 mm, 2 μm, Thermo-Fisher), applying a segmented linear gradient from 2% to 45% solvent B (80% acetonitrile, 0.1% formic acid; solvent A 0.1% formic acid) at a flow rate of 230 nL/min over 60 minutes. Eluting peptides were analyzed on a Q Exactive HF-X Orbitrap mass spectrometer (Thermo-Fisher), which was coupled to the column with a customized nano-spray EASY-Spray ion-source (Thermo-Fisher) using coated emitter tips (New Objective). For parallel reaction monitoring (PRM) data acquisition of c-opsin1+/+ and c-opsin1Δ8/Δ8 samples we operated the instrument with the following MS parameters: survey scan with 60k resolution, AGC 1E6, 50 ms IT, over a range of 400 to 1300 m/z, PRM scan with 60k resolution, AGC 2E5, 500 ms IT, isolation window of 0.7 m/z with 0.2 m/z offset, and NCE of 27%.
Data analysis, manual validation of all peptides and their transitions (based on retention time, relative ion intensities, and mass accuracy) was performed in Skyline. All peptides were quantified across all samples, except GnRH-1, which was not consistently detected and therefore removed from further analysis. The most intense non-interfering transitions were selected and their peak areas were summed up for each target (total peak area). Intensities of unmodified and oxidized (Met) peptide species were summed up for quantification of the neuropeptides. To correct for varying peptide amounts across replicates and instrument stability over the measurements, the peptide intensities were normalized based on the relative total MS1 ion current, which was extracted using the MaxQuant software (version 184.108.40.206) (Tyanova, Temu, & Cox, 2016). MaxQuant was run with default settings and the msScans table was used to extract the total ion current for each sample.
Platynereis heads (3heads/BR; n=10BRs for LD16:8; n=12BRs for LD12:12) were sampled from both c-opsin1+/+ and c-opsin1Δ8/Δ8 worms after 5 days of entrainment to respective photoperiod under broad spectrum white light containing UVA wavelength. LD 12:12 experiment entrained by white light without UVA (n=12BRs) was achieved by installing UVAR filters. The heads were first rinsed in natural sea water (NSW) and collected in 200ul of ice-cold acidified methanol (methanol/water/acetic acid – 90/9/1 – v/v/v) to extract peptides 75. The collected heads were homogenized by sonicating three times for 30sec on ice and the supernatant was collected in a fresh collection tube. This procedure was repeated twice by adding 200ul of ice-cold acidified methanol to the remaining pellet each time for all samples to dissociate remaining pellets and the resultant supernatants in each step were collected in same collection tube resulting in about 600ul of total volume.
Synthetic PDF, NPY-1, NPY-4, GnRH-1 and Vasotocin peptides were dissolved in 50% acetonitrile at a final concentration of 5 mM and mixed in a 40:10:2:2:1 ratio to obtain similar signal strength in the mass spectrometer, as determined by a scouting run. To avoid disulfide bond formation 1 uL (10 pmol) of the peptide mixture was dissolved in 45 uL ammonium bicarbonate (ABC), reduced with 10 mM dithiothreitol (DTT) for 30 min at room temperature, alkylated with 20 mM iodoacetamide (IAA) for 30 min at room temperature in the dark and the remaining IAA was quenched by adding 5 mM DTT.
The extracts were concentrated by vacuum centrifugation, freeze-dried and resuspended in 8 M urea, 50 mM ABC. Reduction and alkylation of disulfide bonds was performed as described for the synthetic peptides. Samples were diluted with 50 mM ABC to 4 M urea and peptides were digested with 500 ng Lys-C (Wako) at 25°C for 4 hours. After stopping the digestion by addition of 0.5% trifluoroacetic acid (TFA), the peptides were desalted using C18 stagetips prior to LC-MS (Rappsilber, Mann, & Ishihama, 2007).
Created on 6/22/20, 6:32 AM