| Metadata |
| datasetIdentifier | PASS00550 |
| datasetType | SRM |
| submitter | Christopher Colangelo <christopher.colangelo@yale.edu> |
| submitter_organization | Yale University |
| lab_head_full_name | Christopher Colangelo |
| lab_head_email | christopher.colangelo@yale.edu |
| lab_head_organization | Yale University |
| lab_head_country | United States |
| datasetTag | Mouse_PSD |
| datasetTitle | Extended MRM/SRM experiment for 112 Mouse/Rat Brian Proteins |
| publicReleaseDate | 2014-12-31 00:00:00 |
| finalizedDate | 2014-08-19 08:55:47 |
| summary | Synaptosomes from the hippocampus of 28-day old male mice exposed to BDS and control conditions were used to examine the effects of BDS on synaptic composition using a LC-MRM mouse/rat postsynaptic density assay. |
| contributors | Wei L, Hao J, Lacher RK, Abbott T, Chung L, Colangelo CM, Kaffman, A. |
| publication | Wei L, Hao J, Lacher RK, Abbott T, Chung L, Colangelo CM, Kaffman, A. Early life stress causes a global decrease in synaptosomal proteomic content in the developing mouse hippocampus. Proteomics. Submitted
|
| growth | BALB/cByj mice (Stock # 001026, Jackson Laboratories) were housed in standard Plexiglas cages and kept on a standard 12:12 hour light-dark cycle (lights on at 7:00AM), constant temperature and humidity (22 ºC and 50%), and food provided ad libitum. The Institutional Animal Care and Use Committee (IACUC) at Yale University approved all studies. |
| treatment | In brief, visibly pregnant dams were placed individually in maternity cages with 750cc of corncub bedding but no nesting material. At birth (PND=0), pups were culled to 6-8 pups per litter, and litters were randomly assigned to either brief daily separation (BDS) or control condition. The separation procedure occurred daily from PND1-21 and was done from 11:00-11:40AM. During each BDS session, the dam was removed from the home cage and placed in a holding cage covered with fresh corncob bedding, and provided with food and water. Pups were transferred individually into a new cage covered with clean corncob bedding and placed at different corners of a 20.3 X 27.9 cm standard cage. Cages were left undisturbed for 15 min at ambient temperature in the vivarium (22C° ±2C°) after which the pups were individually transferred back to their home cage, followed by the return of the dam. On PND 22, pups were weighed and housed in groups of 3-5 littermates of the same sex in cages with 500cc of corncub bedding but no nesting material. On PND28, mice were processed to harvest synaptosomes from the hippocampus. |
| extraction | PND28 offspring were rapidly decapitated and the right hippocampus was dissected and placed in a 2ml dounce homogenizer (Kontes Glass Co) containing 500uL of ice cold buffer A [HEPES 4 mM pH 7.4, 320 mM sucrose, 1mM DTT, and protease inhibitor cocktail (Cat# P2714, Sigma)]. Hippocampal tissue was homogenized using 10 slow strokes. The homogenate was transferred into a cold Eppendorf tube and centrifuged for 5min at 1000g at 4°C to remove nuclei and cell debris (P1 fraction). The supernatant was transferred to a new Eppendorf tube and centrifuged for 20min, 15,000g, at 4°C. The supernatant containing soluble proteins was transferred to a new Eppendorf tube (S2) and snap frozen in liquid nitrogen. The remaining pellet (P2 fraction), containing the crude synaptosomes was snap frozen in liquid nitrogen, and stored at -80 °C for later use in proteomic studies and/or western blots. |
| separation | |
| digestion | Samples were treated with 4.1 mM DTT to reduce cysteines, digested with trypsin (1:15 ratio) for 15 h at 37°C, and quenched with 12 ul 20% TFA. Peptides were then desalted, dried, and dissolved in 20ul 3:8 v/v 70 % FA/0.1% TFA. Total peptide concentration was adjusted to 0.2 ug/ul using 0.1% TFA. Stable isotope peptide standards (SIS) were added to the unlabeled peptides at a final concentration of 100 fmol/ul. |
| acquisition | For multiple reaction monitoring (MRM), 1ug (5uL) of peptide/SIS mixture was loaded onto a 180 um x 20 mm 5 um Symmetry C18 nanoACQUITY trapping column with 2% ACN/0.1% FA at 15 ul per min for 3 min. After trapping, a 2-40%, 60 min linear ACN/0.1% FA gradient, was run at flow rate of 500 nl/min with a 75 um x 150 mm 1.7 um BEH130 C18 nanoAcquity column. Data were acquired with 1371 transitions/sample at peak windows of 5 min, and a cycle time of 2.5 sec. |
| informatics | Transition peaks were quantitated using Multiquant™ 2.1 software (research version) and the SignalFinder™ 2 algorithm. |
| instruments | AB SCIEX 5500 QTRAP |
| species | Mouse |
| massModifications | static: C+57.021464, variable: K+8.014199, R+10.008269 |
