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Peptidome analysis of the hypothalamus in a single mouse.

ABSTRACT: A 2-mg frozen section of hypothalamus from a single mouse was homogenized in 200 µL of denaturing solution(7 M urea, 2 M thiourea, 20 mM dithiothreitol) containing cOmplete Mini Protease Inhibitor Cocktail (Roche). Peptide extracts equivalent to 135 µg of tissue were analyzed by LC-MS/MS.

ORGANISM(S): Mus Musculus (mouse)

SUBMITTER: Yoshio Kodera

PROVIDER: PXD023475 | JPOST Repository | Sat Jan 08 00:00:00 GMT 2022

REPOSITORIES: jPOST

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			Action	DRS
	Ankgw123_001_Hy_AWK85_1_0o012ug_6ug_T50_2.raw	Raw
	Ankgw123_002_Hy_AWK85_2_0o012ug_6ug_T50_2.raw	Raw
	Ankgw123_003_Hy_AWK85_3_0o023ug_6ug_T50_2.raw	Raw
	DB%20search%20psm.csv	Csv
	protein-peptides.csv	Csv

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Project description:Four protocols were adapted and compared to enrich extracellular matrix proteins from mouse skin: 1. compartmental enrichment; 2. chemical decellularization; 3. enzymatic decellularization using phospholipase A2; 4. quantitative detergent solubility profiling (QDSP). All four protocols were started the same day, each using 35 mg of frozen back skin taken from the same initial back skin piece. The experiment was repeated three times starting from three different mice. Compartmental enrichment: skin samples were processed using the CNMCS commercial kit. Briefly, the skin tissue was homogenized in buffer C and successively incubated under agitation in buffers W, N, M and CS supplemented with the protease inhibitors from the kit. Extraction buffer C and insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. Chemical decellularization: skin samples were cut into small pieces and successively incubated in buffers containing NaCl, SDS/sodium deoxycholate, Triton X-100 and GuHCl. Extractions buffers as well as the final insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. Enzymatic decellularization: skin samples were cut into small pieces and successively incubated in buffers containing NaCl, phosopholipase A2/sodium deoxycholate. Extraction buffers as well as the insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. QDSP: skin samples were homogenized in sterile PBS and successively incubated in 3 buffers containing increasing concentrations in detergents (including SDS, sodium deoxycholate, NP-40) as previously described. Extraction buffers as well as the insoluble pellet were kept aside, flash-frozen in liquid nitrogen immediately. For comparison with the four protocols described above, total skin lysates were also prepared for two of the mice. In this case, 20 mg of mouse skin were homogenized and incubated in a lysis buffer containing SDS, β-mercaptoethanol. Samples were centrifuged and the supernatant was kept aside, flash-frozen in liquid nitrogen immediately.

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Peptidome analysis of the hypothalamus in a single mouse.

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