title: Quantitative proteomics in laser capture microdissected sleep nuclei from rat brain.
abstract: The combination of stable isotope labeling of amino acids in mammals (SILAM) and laser capture microdissection (LCM) for selective proteomic analysis of the targeted tissues holds tremendous potential for refined characterization of proteome changes within complex tissues such as the brain. The authors have applied this approach to measure changes in relative protein abundance in ventral tegmental area (VTA) of the rat brain that correlate to pharmacological perturbations. Enriched (13)C6(15)N2-lysine was introduced in vivo via diet. These animals were sacrificed during the middle of the 12-hour light period to extract isotopically "heavy" proteins, which were then used as a reference for extracts from dosed, unlabeled rats. Animals were administered an orexin peptide (Ox-B), an orexin receptor antagonist (ORA), or a mixture of both (Ox-B + ORA). All samples were obtained at same phase of the sleep cycle. Labeled-pair identification and differential quantitation provided protein identification and expression ratio data. Five proteins were found to exhibit decreased relative abundance after administration of an ORA, including α-synuclein and rat myelin basic protein. Conversely, six proteins showed increased relative abundance upon antagonist treatment, including 2',3'-cyclic nucleotide 3'-phosphodiesterase.
authors.affiliation: Department of Proteomics, Molecular Profiling and Research Informatics, Merck Research Laboratories , West Point, Pennsylvania , USA.
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Miller
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Winrow
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Spellman
authors.forename: Qinghua
authors.initials: Q
authors.lastname: Song
authors.forename: Duane R
authors.initials: DR
authors.lastname: Reiss
authors.forename: James P
authors.initials: JP
authors.lastname: Conway
authors.forename: Rhonda R
authors.initials: RR
authors.lastname: Taylor
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Coleman
authors.forename: Ronald C
authors.initials: RC
authors.lastname: Hendrickson
authors.forename: John J
authors.initials: JJ
authors.lastname: Renger
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D001835:Body Weight / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism*; D005260:Female; D047908:Intracellular Signaling Peptides and Proteins / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000737:chemistry; D008098:Litter Size / Q000187:drug effects; D008239:Lysine / Q000008:administration & dosage; D008297:Male; D009479:Neuropeptides / Q000008:administration & dosage / Q000037:antagonists & inhibitors / Q000737:chemistry; D000068797:Orexins; D010455:Peptides / Q000008:administration & dosage; D011247:Pregnancy; D060066:Protein Interaction Maps / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D040901:Proteomics; D011597:Psychomotor Performance / Q000187:drug effects / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012890:Sleep / Q000502:physiology*; D017557:Ventral Tegmental Area / Q000166:cytology*
chemical_list: D000596:Amino Acids; D047908:Intracellular Signaling Peptides and Proteins; D009479:Neuropeptides; D000068797:Orexins; D010455:Peptides; D011506:Proteins; D008239:Lysine
keywords: LC-MS; SILAC; SILAM; circadian cycle; laser capture microdissection; orexin; sleep; ventral tegmental area