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Materials and Methods
Ethics
All animal procedures were conducted with prior institutional ethical approval under the requirements
of the Australian Prevention of Cruelty to Animals Act 1986 and the Code of Practice for the Care and
Use of Animals for Scientific Purposes. Prior clearance was obtained from the Animal
Experimentation Ethics Committees of Monash Medical Centre and Victorian Institutes of Animal
Science. The animals of this study were inspected by members of the Monash Medical Centre Animal
Ethics Committee.
Animals
Adult Corriedale male sheep(55kg,n=3) and female sheep(53kg, n=3) were maintained on pasture or
in feedlots under natural conditions. For intensive experimentation, the animals were housed in
individual pens with natural lighting and temperature and were fed ad libitum lucerne chaff with free
access to water. Before experiment, animals were conditioned to pen-housing and handling for 1 week.
Animals in this experiment were euthanised by overdose of 20 ml sodium pentobarbital (Lethabarb;
Virbac, Peakhurst, NSW, Australia) iv and the brains were removed, then Hypothalamus , Anterior
pituitary gland (A.P), Cerebellum (CEREB), Arcuate nucleus (ARC), Neurointermediate lobe (NIL),
Median eminence (M.E), Cerebral cortex, Spinal cord, Brain stem(B/S), and Pre-optic area(POA)
were dissected. All tissues were frozen on dry ice and stored at -80°C.
Peptide Extraction
Tissues were immediately immersed in 2N acetic acid (each 600 μl for 300mg sample) and boiled for
10 min. The samples were then homogenized by using electric homogenisers. Vortex, stand on ice
while homogenizing the rest of the samples, and once completed leave on ice for at least half an hour.
Finally, centrifuged at 6,000rpm at 4°C for 30 min. Supernatants were collected, and re-constituted in
distilled water.
Experiment 1: Mass spectrometry analysis of α-MSH in ten brain tissues
Co-immunoprecipitation (Peptide purification)
Polyclonal α-MSH-antibody (1:1000; Bachem, Bubendorf, Switzerland) was cross-linked to
Dynabeads protein A (Dynal Biotech, Olso, Norway) according to the manufacturer’s protocol.
Tissues lysates were precleared with IgG Dynabeads-protein A for 10 min and then incubated with α-
MSH-Dynabeads overnight at 4 °C, then α-MSH-immunoprecipitated complexes were washed five
times with immunoprecipitation buffer (10mM Tris/HCl, pH 7.8,1mM EDTA,150mM NaCl,1mM
NaF,0.5%NonidetP-40,0.5% glucopyranoside, 1μg/ml aprotinin, and 0.5 mM phenylmethylsul-fonyl
fluoride).Proteins were eluted by 0.1M citrate buffer (elution buffer, pH2-3 ) and then processed by
Mass Spectrometry(ABI 4700 Proteomics Analyzer ,Applied Biosystems, USA).
Peptide samples for mass spectrometry analysis
Peptide samples were purified and concentrated using a Zip Tip (Millipore) which has C18 resin fixed
at its end. The resin was rinsed according to the manufacturer’s instructions with 10 μl 0.1% TFA and
50% ACN. Peptides were eluted in 10 μl 50% ACN in 0.1 % TFA. A 0.5 μl volume of the
concentrated peptide-containing sample was mixed with a saturated solution of α-cyano-4-
hydroxycinnamic acid (0.5 μl). Each sample (0.5 μl) was spotted on the mass spectrometer sample
plate . The mass spectrometer determines the mass of the peptides and the sequence (by collisioninduced
dissociation). From the masses of the peptide fragments, sequence data were determined by
comparison with known sequences.
Experiment 2: HPLC/Enzyme immunoassay of α-MSH in ten brain tissues
α-MSH Separation
α-MSH peptides were separated by HPLC using a Symmetry C18 (5μm) column (4.6 × 150mm)
(Waters, Milford, USA). The chromatographic run was performed with an aqueous phase containing
0.1% trifluoroacetic acid (TFA) and an organic phase containing 0.085% TFA in acetonitrile(ACN):
methanol (80:20) gradient at a flow rate of 1.0 ml/min. The gradient used for separation was run 52
mins; the total run time was 72 mins .α-MSH peptides eluted between 25-30% acetonitrile: methanol.
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