Time-Resolved cAMP Level Determination in Frog Retina Samples Using LC-MS/MS.

The phototransduction cascade allows photoreceptors to detect light across a wide range of intensities without saturation, with cGMP serving as the second messenger and calcium feedback as the key regulatory mechanism. While experimental evidence suggests that cAMP may also play a role in modulating this cascade, such regulation would necessitate rapid changes in cAMP levels on a timescale of seconds. However, data on the dynamics of intracellular cAMP changes in photoreceptors remain scarce, primarily due to the limitations of conventional fluorescence-based methods in this specialized sensory system. To address this gap, we developed a methodology combining rapid cryofixation of retinal samples following light stimulation with the isolation of outer segment preparations. The rapid cryofixation setup comprises six computer-controlled sections, each with a high-speed stepper motor-driven lever that rapidly moves the specimen in a 180° arc within ~80 ms to press it against a liquid nitrogen-cooled copper cylinder for fixation. Using highly sensitive metabolomics techniques, we measured cAMP levels in these samples. This approach enables the investigation of rapid cAMP dynamics and its potential regulatory role in phototransduction, providing a foundation for understanding the interplay between cAMP and PKA signaling in photoreceptor function. Key features • The protocol provides ms time resolution in retina outer segment sampling in response to light stimulus with cryofixation, conserving proteome and metabolome response features. • The protocol allows direct cAMP quantification with an average level of 11.4 ± 0.5 pmol/mg of protein in the dark.