As the site of the master biological clock, the SCN controls circadian rhythms. Multiple peptides in the SCN act as critical synchronizers and mediators of the SCN. Changes in peptide expression within the SCN across the 24-h day appear to mediate time-of-day-specific circadian functions. While antibody-based approaches have been previously used with success for measuring changes in peptide expression in the SCN,39–42
these methods only provide information on pre-selected peptides and can lack sufficient specificity to identify an exact peptide form due to cross-reactions with longer peptides or peptides containing distinct PTMs. MS-based quantification offers the benefit of being able to examine changes in the expression of multiple peptides without preselecting the peptides, and without prior knowledge of their structure.
Using high resolution FTMS, we expanded our previous identification of rat SCN endogenous peptides at ZT 615
to include nighttime, ZT 18. In comparison with the previous SCN study, 63 peptides, including APVSTGAGGGTVLAKMYP (AA 24-41) derived from GRP, were identified in both our previous and current SCN peptidomic studies. We found another 26 peptides, including the truncated VIP (HSDAVFTDNYTRL (AA 125-137)), that were identified in the previous SCN peptidomic analysis and also in the current SCN mass spectral data by use of manual validation based on the precursor peptide masses within 10 ppm of mass accuracy (data not shown). We surmise that most of these peptides either had molecular weights of ≥3 kDa, and, thus, did not generate sufficient fragment ions for confident identification in the ion trap MS/MS scans, or may not have been subjected to CID fragmentation due to low abundances.
In this study, we performed a label-free quantitative peptidomic analysis of rat SCN tissue samples collected at daytime (ZT 6) and nighttime (ZT 18), respectively. The daytime and nighttime samples contained distinct peptides and so factors arising from these differences such as distinct co-eluting analytes may have affected the quantitative accuracy (for example, from differences in ion suppression). However, ion suppression from complex mixtures is reduced by using nanoliter volume/minute flow rates and increased separation gradient lengths.43–45
Therefore in the current study, peptides were analyzed using a 300 nL/min flow rate and separated over 100 min of the gradient. In addition, as seen in , approximately 75% of the identified peptides were found in both the ZT 6 and ZT 18 samples, suggesting that the composition of the samples generated from ZT 6 and ZT 18 are similar. Based on these two factors, ion suppression effects were minimized.
Integrated mass spectrometric peak intensities, acquired from high resolution FTMS and CID ion trap data, were analyzed between circadian time points. While substance P derived from protachykininn 1 is known to play a role in the generation and entrainment of circadian rhythmicity,46, 47
we observed that substance P peptide exhibited about a 1.5-fold increase in its peptide level at ZT 18. In addition, many of the peptides derived from prohormones that are known to be involved in circadian functions of the SCN exhibited significant changes between daytime and nighttime. For example, truncated forms of GRP (AA 24-41, 1.4-fold) and VIP (AA 125-137, 1.6-fold) were found to significantly increase at ZT 18 compared to ZT 6. Full length GRP (AA 24-52) and VIP (AA 125-152) peptides are known to have phase-shifting properties in the SCN.48–52
According to a previous study, GRP and VIP mRNA levels in mice exhibited circadian rhythms, with the highest levels during the middle of subjective night.7
In an enzyme immunoassay-based study in rats, VIP immunoreactivity increased over the course of the dark period, whereas GRP immunoreactivity increased during the light period.42
Our observation that the elevated expression of the truncated form of VIP is significantly higher at ZT 18 than ZT 6 correlates with previous studies using antibodies against full-length VIP.7, 42
This may imply that the truncated form of VIP could work as a bioactive peptide. In the case of the truncated form of GRP, in this study we found the level was higher at nighttime. This could indicate that the truncated form and full-length form of GRP may have different modes of action for SCN circadian functions, or may indicate that the immunoreactivity studies were not set up to distinguish such closely related peptides. The truncated peptide is predicted by our general mammalian53
prohormone cleavage models. While the truncated GRP peptide could be generated from the sample collection and handling process rather than endogenous intracellular processing, our approaches were designed to minimize such effects. Lastly, the full length form of GRP may not have been detected by our current LC-MS analysis platform because it was present below the MS detection limit. A previously unidentified short form of PACAP-related peptide (AA 111-128; GMGENLAAAAVDDRAPLT), generated by cleavage of PACAP-related peptide at a monobasic Arg site, had a significant 1.3-fold increase at ZT 18 compared to ZT 6; a similar peptide has been detected via MS in other animal models.55
PACAP is known to transmit photic signals to the SCN via the retinohypothalamic tract for SCN functions.56–58
However, discrepancies between photic resetting in the SCN from mouse PACAP knockout models, with and without the additional deletion of the PACAP-related peptide, have been noted.59, 60
PACAP 38 (AA 131-168) peptide levels have been reported to be low during the light period and high during the dark period,40
which correlates with our observation of the altered level for the truncated form of PACAP-related peptide between daytime and nighttime. In addition to the endogenous peptides that are well known to be involved in SCN circadian rhythmicity, the peptides derived from proenkephalin A, CART, prothyroliberin, and secretogranin 2 that exhibited ≥30% increases in their peptide levels at nighttime have not been previously implicated in SCN circadian functional studies. Proenkephalin A-derived peptide, VGRPEWWMDYQ (AA 219-229), exhibited a significant increase of more than 1.5-fold at ZT 18 compared to ZT 6. It was reported that the proenkephalin gene interacts with cAMP response-element binding protein (CREB), which is a key regulatory transcription factor influencing timing and entrainment of the Drosophila
In our previous analysis,62
VGRPEWWMDYQ (AA 219-229) was observed as one of the released peptides from the SCN stimulated via the optic tract; however, the functional roles of the proenkephalin-A peptides in SCN circadian functions have not been elucidated yet, and deserve further study. Two of the three CART-derived peptides in exhibited a ≥30% higher level at ZT 18. While we first showed that CART-derived peptides are present in the SCN in our previous peptidomic analysis,15
these peptides have not been yet reported to be related to SCN function. CART is involved in appetite and drug addiction.63
CART-derived peptides exhibit a diurnal rhythm in several brain regions, including the hypothalamus, depending on food intake; peptides are lower in the morning compared to evening hours under normal feeding regimens.64
There are few studies of the direct involvement of prothyroliberin-derived peptide in SCN circadian regulation. Thyrotropin-releasing hormone (TRH) derived from prothyroliberin is known to be expressed in the SCN65
and has a diurnal rhythm in the rat hypothalamus.66
While secretogranin 2 gene is reported to be highly expressed in the SCN,67
it was first reported in our previous study15
that several endogenous peptides derived from secretogranin 2 were actually present in the SCN. As yet, there are no reports on the involvement of secretogranin 2-derived peptides in SCN circadian functions.15, 67
While most of the peptides derived from prohormones in the present study exhibited higher levels at nighttime over daytime, two peptides (listed in supplemental Table S3
), VGYDLVPEPKIIDA (AA 77-87) derived from cytochrome c oxidase subunit 5A, and vimentin, IKTVETRDGQVINETSQHHDDLE (AA 444-466), exhibited significant increases of more than 30% at daytime compared to nighttime. Although cytochrome c oxidase-derived peptide has not been previously reported in the direct involvement of SCN circadian functions, it was found that metabolic activity in the SCN is known to be higher during the daytime than at night, and that mitochondrial cytochrome c oxidase activity was higher during the light period.68–70
In the case of vimentin, there are no reports that it is directly involved in SCN regulation.