Table 3.

Selected peptidomic studies in whole hypothalamus and by hypothalamic sub-region^*

Study	Animal	Extraction	Target(s) a priori?	Screen [S],Validation [V]	Map or Schematic	Major findings
Whole hypothalamus
Bures et al. 2001	Ms	dissection	N	LC-MS [S]; LC-MS/MS [S]	N	Identified 27 peptides derived from known neuropeptides as well as 25 additional peptides not known to be in the neuropeptide processing pathway; all up-regulated in carboxypeptidase E mutant mice
Svensson et al. 2003	Rt, Ms	dissection	N	nanoLC-ESI-Q-TOF-MS	N	Detected 550 endogenous peptides
Che et al. 2005	Ms	dissection	N	LC-MS/MS	N	Knockdown of carboxypeptidase E activity in two paradigms for decreasing body mass show different peptide profiles
Décaillot et al. 2006	Ms	dissection	N	Isotopic labeling & Nano-LC MS/MS	N	Detection 27 distinct peptides from hypothalamus and striatum in Cpefat/fat mice, with some showing changes in levels in mice chronically treated with morphine
Pan et al. 2006	Ms	dissection	N	Isotopic labeling & LC-ESI-MS	N	Approx. one-third of the peptides found in wild-type mice were not found in prohormone convertase KO mice
Che et al. 2007	Ms	dissection	N	Nano-LC MS/MS	N	Identified 95 peptides from samples, 64 of which were neuropeptides or other peptides derived from proteins in the secretory pathway; found OT to be preferentially abundant in hot-acid extracts over hot-water extracts
Sköld et al. 2007	Ms	dissection	N	Nano-LC MS [S]; Q-TOF LTQ MS/MS [V]	N	Identified 23 neuropeptides, hormones and potentially biologically active peptides; all were primarily up-regulated in control mouse brain relative to brains processed with longer post mortem times
Mihailova et al. 2008	Rt	dissection	N	Capillary 2-D LC/MS	N	Identified 107 peptides, 26 of which displayed differences in concentration under hypoxic stress conditions
Cai et al. 2011	Pg	dissection	N	MSPD & Nano-LC MS/MS	N	14 potential endogenous peptides were identified using MSPD extracts versus to peptides using acid extracts
Colgrave et al. 2011	Cw	dissection	N	LC-MS/MS	N	Used thermal stabilization methods to refine the yield of neuropeptides isolated from hypothalamus
Nilsson et al. 2012	Ms	dissection	N	nano-LC-ESI-LTQ MS/MS or nano-LC-ESI-LTQ-FTICR-MS/MS	N	14 peptides were significantly regulated by imipramine treatment
X. Zhang et al. 2012	Ms	dissection	N	LC-FT-MS/MS	N	Identified 367 peptides from neuropeptide precursors from hypothalamic samples.
Fouillen et al. 2013	Sw	dissection	N	LC-FT-MS/MS, SIEVE™ software-based analysis,	N	12 hypothalamic peptides were up-regulated following prolonged general anesthesia
Frese et al. 2013	Rt	dissection	N	HCD and ETD-based MS/MS [S]; LC-MS [V]	N	Identified 1,292 unique peptides from hypothalamus in rats fed on a regular diet, HFHS diet, restricted chow diet, or chocolate diet. HFHS diet produced the greatest increases in peptides as determined by label-free quantification.
Y. Gao et al. 2013	Rt	dissection	N	2D-GE, MALDI-TOF MS [S]; qRT-PCR, WB [V]	N	Identified 17 hypothalamic proteins with twofold or greater expression after electroacupuncture intervention of sciatic pain
Nakazawa et al. 2013	Rt	dissection	N	RP HPLC; Nano-LC-MS/MS [S]; WB [V]	N	Found hundreds of peptides in relaxin- and saline-treated rats; two of which exhibited signatures both in microarray experiments and peptidomic experiments: OT and CART; with OT markedly up-regulated after relaxin exposure
Schmidlin et al. 2015	Rt	dissection	N	LC-MS/MS [S]; SRM/Triple-Quad MS [V]	N	Demonstrated the feasibility of using SRM to evaluate a priori selected transitions of key neuropeptide fragments from the hypothalamus
Secher et al. 2016	Rt	dissection	N	LC-MS/MS [S]	N	Identified 14,416 peptides in 786 protein families; sorted these by LPVs to isolate 2,835 peptides derived from 356 prohormone precursors; of these, 105 LPVs were not previously described
Yang et al. 2017	Rt	dissection	N	nanoESI; nanoLC-MS/MS; MRM	N	Evaluated the efficacy of a rapid conductive sample heating system in stabilizing proteins from whole hypothalamic extracts
DeAtley et al. 2018	Cw	dissection	N	LC-MS/MS; MRM	N	Observed 143 peptides in hypothalamus of pre- and post-pubertal heifers that were assigned neuropeptide status; three of which differed between the conditions
SCH: Suprachiasmatic hypothalamic nucleus
Hatcher et al. 2008	Rt	acute tissue slice prep; μ-punch	N	HPLC, LC, SPE beads [S]; MALDI-TOF MS/MS ; LTQ FTMS [V]	N	Identified peptides released from acute slice preparations containing SCH, including after electrical stimulation of the retinohypothalamic input to the SCH; found peptide content in releasates to be stimulation-specific
J. H. Lee et al. 2010	Rt	μ-punch	N	LC-FTMS/MS	N	list of 102 endogenous peptides, including 33 that were previously unidentified; also identified novel post-translational modifications
J. E. Lee et al. 2013	Rt	μ-punch	N	LC-FTMS/MS, SIEVE™ software-based analysis	N	list of 190 endogenous peptides from 310 identified
Chiang et al. 2014	Ms	dissection	N	fractionation, LC MS/MS [S]; RT-PCR, WB, IHC [V]	N	quantified 2,112 proteins, 20% of which exhibited a time-of-day-dependent profile; found 48 proteins exhibiting circadian rhythms of expression from this time-of-day proteome
Southey et al. 2014	Rt	μ-punch	N	Spectral count, spectra index, SIEVE™ software-based analysis	N	differential peptide abundances between day and night conditions
Yang et al. 2017	Rt	dissection	N	nanoESI; nanoLC-MS/MS; MRM	N	Evaluated the efficacy of a rapid conductive sample heating system in stabilizing proteins from SCH extracts
SO: Supraoptic hypothalamic nucleus
Bora et al. 2008 [also see: Perkel 2008]	Rt	μ-punch	Y	LC/MS & tandem mass spectrometry	N	20 unique peptides identified

^*Abbreviations: 2D-GE, two-dimensional gel electrophoresis; μ-punch, micropunch; CART, cocaine- and amphetamine-related transcript; Cw, cow; ESI, Electrospray Ionization; Fs, fish; FT, Fourier transformation; FTICR, Fourier-transform Ion Cyclotron Resonance; HCD and ETD-based MS/MS, High-energy Collisional Dissociation and Electron-transfer Dissociation-based tandem mass spectrometry; HFHS, high fat and high sucrose; Hu, human; KO, knockout; LC, liquid chromatography; LPVs, longest peptide variants; LTQ, Linear Trap Quadrupole; MALDI-TOF, Matrix-Assisted Laser Desorption/Ionization-Time of Flight; MRM, multiple reaction monitoring; Ms, mouse; MS/MS, tandem mass spectrometry; MSPD, Matrix Solid-Phase Dispersion; nanoESI, nanoscale electrospray ionization; nanoLC-MS/MS, nanoscale liquid chromatography coupled to tandem mass spectrometry; OT, oxytocin; Pg, pig; qRT-PCR, quantitative real-time polymerase chain reaction; Q-TOF, quadrupole time of flight; SRM, Selected Reaction Monitoring; Triple Quad MS, triple quadrupole mass spectrometry; Rt, rat; SPE, solid-phase extraction; Sw, shrew