The diel vertically migrating copepod, Pleuromamma xiphias, migrates approximately 600 m daily, up and down in the water column. At the surface, the copepods feed on plankton at night, and then descend to the depths during the day, likely to escape visual predators. Both the migratory behavior and the physiological pathways that are up- and down-regulated throughout the migration are likely under at least partial circadian clock control. This experiment compares the protein abundances of select physiological processes from copepods collected in situ with copepods incubated in the dark to remove the influence of exogenous stimuli on physiology.

Individual female copepods (P. xiphias) were collected throughout a diel cycle in the waters off Bermuda. Some of these copepods were incubated in the dark for >24 hours to distill the physiological signal of their circadian rhythm. Both copepods collected in situ and those in the circadian incubation were analyzed using targeted proteomics mass spectrometry on an TSQ Altis over 2 injections to cover 2 transition lists. A pre-column (2.5 cm) and analytical column (27 cm) were packed in-house with Dr. Maisch C18 3 µm beads. Each copepod was analyzed individually to capture a range of biological variation in physiological response. For each of the time series time points (5,6,7,8,9), 4 copepods were analyzed with SRM; for each of the circadian experiment time points (1,2,3,4,6), 3 copepods were analyzed. To create 25 µl of sample to be injected on the MS/MS, 20 µl of sample (20 µg) was added to 1.25 µl PRTC (25 fmol) and 3.75 µl of water. Peptides were eluted from the column using an acidified (formic acid, 0.1% v/v) water-acetonitrile gradient (4.8-40% acetonitrile over 25 minutes). The mass spectrometry method was built off of the transition lists exported from Skyline. Cycle time was 1.2 s, chromatographic peak width was 15.0 s, data mode was centroid, Q1 resolution was 0.7 FWHM and Q3 resolution was 1.2 FWHM.

Individual Pleuromamma xiphias (female)