Quantitative peptidomics for discovery of circadian-related peptides from the rat suprachiasmatic nucleus

Abstract

In mammals the suprachiasmatic nucleus (SCN), the master circadian clock, is sensitive to light input via the optic chiasm and synchronizes many daily biological rhythms. Here we explore variations in the expression levels of neuropeptides present in the SCN of rats using a label-free quantification approach that is based on integrating peak intensities between daytime, Zeitgeber time (ZT) 6, and nighttime, ZT 18. From nine analyses comparing the levels between these two time points, 10 endogenous peptides derived from eight prohormones exhibited significant differences in their expression levels (adjusted p-value <0.05). Of these, seven peptides derived from six prohormones, including GRP, PACAP, and CART, exhibited ≥ 30% increases at ZT 18, and the VGRPEWWMDYQ peptide derived from proenkephalin A showed a >50% increase at nighttime. Several endogenous peptides showing statistically significant changes in this study have not been previously reported to alter their levels as a function of time of day, nor have they been implicated in prior functional SCN studies. This information on peptide expression changes serves as a resource for discovering unknown peptide regulators that affect circadian rhythms in the SCN.

Figure 1

FTMS with high accuracy and ion trap MS/MS data allow the identification of endogenous peptides present in the SCN. The peptides derived from (A) proSAAS and (B) secretogranin 1 were identified with 14 b-ion and 15 y-ions, and 13 b-ions and 11 y-ions, respectively.

Figure 2

Venn Diagrams showing the distribution of a total of 310 peptides identified from ZT 6 and ZT 18 in which each measurement time consists of nine nanoLC-MS/MS runs. The (A) combined, (B) prohormone-derived, and (C) non-prohormone protein-derived numbers of peptides identified at each time point. The tables list the identified peptides from (D) known prohormones and (E) non-prohormone proteins that exhibited statistically significant changes (FDR-adjusted p<0.05).

Figure 3

Results of the SIEVE analysis of VGRPEWWMDYQ peptide (AA 219-229) derived from proenkephalin A showing significant changes between ZT 6 and ZT 18. The integrated intensities of VGRPEWWMDYQ peptide from two frames exhibited a 1.7-fold increase (FDR-adjusted p-value = 0.00027) in levels at ZT 18.