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Sample GSM335832

Query DataSets for GSM335832

Status

Public on Jun 30, 2009

Title

Mouse E17.5 placental labyrinth

Sample type

protein

Source name

E17.5 placental labyrinth

Organism

Mus musculus

Characteristics

Tissue: placental labyrinth
Strain: C57Bl/6J
Stage: E17.5

Extracted molecule

protein

Extraction protocol

EXTRACTION: Placentas from naturally-mated crosses of C57Bl/6J mice on embryonic day 17.5 were cut into thick slices. A scalpel blade was used to isolate the blood-red labyrinth tissue from the more poorly vascularized, and hence paler, spongiotrophoblast tissue, and to remove the superficial chorionic vasculature. From each litter, a quarter of the collected labyrinth tissues were set aside for RNA extraction and microarray analysis and ¾ for cellular fractionation and proteomic analysis, as recently described (Kislinger et al., 2006).

SEPARATION: The preparation of tissue organelle fractions was previously described (Kislinger et al., 2006). Briefly, tissue was quickly excised, collected and washed several times in ice-cold phosphate-buffered saline to remove blood, and homogenized in ice-cold lysis buffer (250 mM sucrose, 50 mM HEPES (pH 7.4), 25 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol (DTT), and 1 mM phenylmethylsulfonyl fluoride (PMSF) using a tight fitting glass pestle homogenizer. All subsequent steps were performed at 4C or on ice. The lysate was centrifuged in a benchtop centrifuge at 800g for 10 min, with the supernatant serving as the source of cytosol, mitochondria and microsomes. The nuclear pellet was re-homogenized in lysis buffer, recentrifuged, solubilized in 5 ml of lysis buffer, and over-layered onto 4 ml of 0.9 M sucrose cushion buffer and centrifuged at 1000g for 10 min in a swing-bucket rotor. The pellet was then re-solubilized in 5 ml of 1.9 M sucrose cushion buffer (1.9 M sucrose, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM DTT, and 1 mM PMSF). After over-laying onto 4 ml of 1.9 M cushion buffer, the nuclei were pelleted by ultracentrifugation at 150,000g for 1 h (Beckman SW 40Ti rotor). The mitochondria were isolated from the crude cytoplasm by centrifugation at 8,000g for 15 min, with the resulting pellet washed twice in lysis buffer. Mixed membranes were isolated by centrifugation of the post-mitochondrial cytoplasmic fraction at 150,000g for 1 h (Beckman SW 40Ti rotor), while the supernatant served as the cytosolic fraction. Nuclear proteins were extracted by re-suspending the nuclei in 5 volumes of nuclear extraction (NE) buffer (25 mM HEPES, pH 7.4, 400 mM NaCl), followed by 30 min incubation with gentle shaking at 4ÂºC. The nuclei were then lysed by 10 passages through an 18-gauge needle. The debris was removed by microcentrifugation at 20,000g for 30 min and the supernatant saved (NUC1 fraction). The pellet was resuspended in 5 volumes NET buffer (NE with addition of 1% Triton-X-100), gently shaken for 30 minutes 4C, and the nuclei lysed by 10 passages through an 18-gauge needle. Debris was removed by microcentrifugation at 20,000g for 30 minutes, with the supernatant saved (NUC2 fraction). Mitochondrial proteins were isolated by incubating the isolated mitochondria in hypotonic lysis buffer (10 mM HEPES, pH 7.4, 1 mM DTT, 1 mM PMSF) for 30 min on ice. The suspension was briefly sonicated, shaken at 4ÂºC for 1 h, and debris removed by microcentrifugation at 20,000g for 30 min. The supernatant was saved (MITO fraction). Membrane proteins were extracted by re-suspending the microsomes in 5 volumes of NET buffer. The suspension was incubated with gentle shaking for 1 h. Insoluble membranes were removed by microcentrifugation at 20,000g for 30 min, while the supernatant was saved (MEMB fraction).

DIGESTION: An aliquot of 150 micrograms of protein from each sample was precipitated overnight at -20C with 5-volumes of ice-cold acetone, followed by centrifugation at 20,000 g for 15 min. The protein pellet was solubilized in 8M urea, 2 mM DTT, 50 mM Tris-HCl, pH 8.5 at 37C for 1 hour, followed by carboxyamidomethylation with 10mM iodoacetamide for 1 hour at 37C in the dark. The samples were then diluted with 100 mM Tris-HCl, pH 8.5 to ~1.5 M urea. Calcium chloride was added to a final concentration of 1mM and digested with a 1:25 molar ratio of recombinant, proteomics grade trypsin (Roche Diagnostics, Laval, QC) at 37C overnight. The resulting peptide mixtures were solid phase-extracted with Varian OMIX cartridges (Mississauga, ON, Canada) according to the manufacturers instructions and stored at -80C until further use.

SET-UP: A fully automated 9-cycle, 18-h MudPIT procedure was set up similar as previously described (Kislinger et al., 2006; Washburn et al., 2001). A quaternary HPLC-pump was interfaced with a LTQ linear ion-trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) equipped with a nano-electrospray source (Proxeon Biosystems, Odense, Denmark). A 100 micrometer inner diameter fused silica capillary (InnovaQuartz, Phoenix, AZ) was pulled to a fine tip using a P-2000 laser puller (Sutter Instruments, Novato, CA) and packed with ~ 7cm of JupiterTM 4 micron Proteo 90Ã… C12 reverse phase resin (Phenomenex, Torrance, CA), followed by ~ 5cm of Luna 5 micron SCX 100… strong cation exchange resin (Phenomenex, Torrance, CA). Samples were loaded manually on separate columns using an in-house pressure vessel. As peptides eluted from the microcapillary columns, they were electrosprayed directly into the MS. A distal 2.3 kV spray voltage was applied to the microsplitter tee (Proxeon Biosystems). The MS operated in a cycle of one full-scan mass spectrum (400-1400 m/z), followed by 6 data-dependent MS/MS spectra at 35% normalized collision energy, which was continuously repeated throughout the entire MudPIT separation. The MS functions and the HPLC solvent gradients were controlled by the Xcalibur data system (Thermo Fisher Scientific, San Jose, CA).

Description

The placental labyrinth was microdissected away from the spongio trophoblast layer

Data processing

Raw files were converted to m/zXML using ReAdW and searched by X!Tandem against a mouse (v3.28) or human (v3.28) IPI (International Protein Index; http://www.ebi.ac.uk/IPI) protein sequence database. To estimate and minimize our false positive rate the protein sequence database also contained every IPI protein sequence in its reversed amino acid orientation (target-decoy strategy) (Gortzak-Uzan et al., 2008; Sodek et al., 2008). A conservative false discovery rate was set to 0.5% on the peptide level, as recently described (Gortzak-Uzan et al., 2008; Sodek et al., 2008). Only fully tryptic peptides matching these criteria were accepted to generate the final list of identified proteins. We only accepted proteins identified either with two unique peptides or one unique, unambigious peptide found in greater that or equal to 2 MudPIT runs per analyzed organelle fraction. To minimize protein inference, we developed a database grouping scheme and only report proteins with substantial peptide information, as recently reported (Gortzak-Uzan et al., 2008; Sodek et al., 2008).

Submission date

Oct 21, 2008

Last update date

Jun 30, 2009

Contact name

Brian Joseph Cox

E-mail(s)

b.cox@utoronto.ca

Organization name

University of Toronto

Department

Physiology