Endogenous peptide discovery of the rat circadian clock: a focused study of the suprachiasmatic nucleus by ultrahigh performance tandem mass spectrometry

Abstract

Understanding how a small brain region, the suprachiasmatic nucleus (SCN), can synchronize the body's circadian rhythms is an ongoing research area. This important time-keeping system requires a complex suite of peptide hormones and transmitters that remain incompletely characterized. Here, capillary liquid chromatography and FTMS have been coupled with tailored software for the analysis of endogenous peptides present in the SCN of the rat brain. After ex vivo processing of brain slices, peptide extraction, identification, and characterization from tandem FTMS data with <5-ppm mass accuracy produced a hyperconfident list of 102 endogenous peptides, including 33 previously unidentified peptides, and 12 peptides that were post-translationally modified with amidation, phosphorylation, pyroglutamylation, or acetylation. This characterization of endogenous peptides from the SCN will aid in understanding the molecular mechanisms that mediate rhythmic behaviors in mammals.

Fig. 1.

FTMS and FTMS/MS data allow identification of peptides present in SCN sample with high confidence. The peptides derived from VIP (A) and PACAP (B) were identified with one b-ion and seven y-ions and with eight b-ions and eight y-ions, respectively. VIP is known to be present in SCN core neurons, and PACAP is synthesized within the retinal ganglion cells that innervate the SCN.

Fig. 2.

Identification of co-eluted truncated cerebellin and cerebellin. The two peptides derived from cerebellin-1 precursor were detected in the same FTMS scan (A) and fragmented by a data-dependent MS/MS acquisition strategy and identified as SGSAKVAFSAIRSTN (B) and SGSAKVAFSAIRSTNH (C), respectively.

Fig. 3.

Identification of manserin with E-value of 6 × 10⁻⁵⁰ (A) and phosphorylated manserin with E-value of 4 × 10⁻²³ (B) by tailored software, ProSightPC. The FTMS/MS spectrum of phosphorylated manserin exhibited the fragment ion generated by neutral loss of H₃PO₄ as the most prominent signal, which is a typical fragmentation pattern of Ser(P)/Thr(P) phosphopeptides by CID.

Fig. 4.

Multiplexed identification from high resolution FTMS/MS mass spectrum. The two isotopic distributions corresponding to 1744.964 and 2623.345 Da (A) are seen in the isolation window for m/z 875.79 and generate the chimeric FTMS/MS spectrum (B). The tailored software, ProSightPC, produces the two peptides derived from Rhombex-40 and pro-SAAS precursors, respectively (C).