protein profiling of mouse lung organogenesis from embryonic day E13.5 until adulthood by gel-free two-dimensional liquid chromatography coupled to large-scale tandem mass spectrometry (MudPIT).
protein expression in Nmyc loss of function mutant with a comparison to wildtype at E18
Keywords: protein, proteomics, Mudpit, lung, development, nuclear, cytosol, mitochondria, mouse
Overall design: Mouse tissues
Mouse tissues were derived from ICR timed mated mice. Timing of conception was designated by assigning noon of the day of plug formation as day 0.5. Pregnant animals were sacrificed and uteri dissected to obtain individual embryos from which lung tissue was derived. A minimum of 0.5 grams or more lung tissue was used in all cases, which required approximately 50 pairs of lungs at E13.5, 24 at E16.5, and at later stages we homogenized lungs from a minimum of 2 litters (~24 pairs of lungs) but isolated protein extracts from an aliquot of the homogenate equivalent to 4 grams of lung tissue. Nmyc mice heterozygous for the hypomorphic allele were timed mated and litters delivered on Day 18.5. The uterus was removed and the pups dissected from the uterus. The yolk sac and amnion were carefully removed. The placental vein and artery were left intact while the pup was dried with Kim wipes, then they were cut and the pups moved under a heat lamp for warmth. The pups mouth and nose were frequently blotted to remove any mucus that was expelled. They were also gently rubbed to stimulate breathing. After 30 minutes pups that had failed to breath or had become listless were sacrificed, a tail clipping removed for genotyping as described in [1] and lungs removed and processed for protein. Geneotyping was done using the following primer pairs: for Nmyc wild type allele NmycA GGT AGT CGC GCT AGT AAG AGC and NmycB GGC GTG GGC AGC AGC TCA AAC and for Nmyc 9a hypomorphic allele NmycB and NmycC (neo) GGA GAA CCT GCG TGC AAT CC.
Tissue fractionation
The tissue fractionation was done as described [2]. Briefly, lung tissue was rinsed twice in ice cold phosphate-buffered saline and homogenized in ice cold lysis buffer, containing 250 mM sucrose, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM DDT, and 1 mM PMSF using a tight fitting Teflon pestle. The lysate was centrifuged in a bench-top centrifuge at 800 g for 15 minutes; the supernatant served as source for cytosol, mitochondria and microsomes. The pellet, which contains the nuclei was re-homogenized in lysis buffer and centrifuged as above. The nuclei were resuspended in 2 molar sucrose buffer (2M sucrose, 50 mM Tris-HCl (pH 7.4), 5 mM MgCl2, 1 mM DDT, and 1 mM PMSF) and pelleted by ultracentrifugation at 80000 g in a SW40Ti (Beckman) for 35 minutes. Mitochondria were isolated from the crude cytoplasmic fraction by bench-top centrifugation at 6000 g for 15 minutes. The mitochondrial pellet was washed and pelleted twice in lysis buffer. The cytosolic fraction was obtained after removal of the microsomal fraction by ultracentrifugation at 100000 g in a SW60Ti (Beckman) for 1 hour.
Organelle protein extraction
Nuclear proteins were extracted by resuspending the isolated nuclei in 5 volumes of 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.42 M NaCl, 0.2 mM EDTA, and 25 % glycerol for 30 minutes with gentle shaking at 4ºC. The nuclei were lysed by 10 passages through an 18-gauge needle, and debris was removed by microcentrifugation at 13000 rpm. The supernatant served as nuclear fraction. Mitochondrial proteins were isolated by incubating the mitochondria in a hypotonic lysis buffer [10 mM HEPES, pH 7.9] for 30 minutes on ice, briefly sonicated and debris pelleted at 13000 rpm for 30 minutes.
Protein digestion and MudPIT analysis
An aliquot of 150 ug of total protein from each fraction was precipitated over night with 5 volumes of ice-cold acetone followed by centrifugation at 13000 rpm for 20 minutes. The protein pellet was solubilized in 8 M urea, 50 mM Tris-HCl (pH 8.5), 1 mM DTT for 1 hour followed by carboxyamidomethylation with 5 mM iodoacetamide for 1 hour at 37ºC. The samples were then diluted to 4 M urea with 100 mM ammonium bicarbonate (pH 8.5) and digested over night with endoproteinase Lys-C at 37ºC. The next day the mixture was further diluted to 2 M urea with 50 mM ammonium bicarbonate (pH 8.5) supplemented with CaCl2 to a final concentration of 1 mM, and incubated over night with Porozyme trypsin beads at 30ºC with rotating. The resulting peptide mixture was solid phase-extracted with SPEC-Plus C18 cartridges according to the manufacture’s instruction and stored at -80ºC until further use.
Mass spectrometry
A fully automated 12-cycle, 24 hour MudPIT chromatographic procedure was set up essentially as described previously [3]. Briefly, an HPLC quaternary pump was interfaced with an LCQ DECA XP ion trap tandem mass spectrometer (ThermoFinnigan, San Jose, CA). A 100 um inner diameter fused silica capillary microcolumn (Polymicro Technologies, Phoenix, AZ) was pulled to a fine tip using a P-2000 laser puller (Sutter Instruments, Novato, CA) and packed with 10 cm of 5-um Zorbax Eclipse XDB-C18 resin (Agilent Technologies, Mississagua, Ontario, Canada) and then with 6 cm of 5-um Partisphere strong cation exchange resin (Whatman). Samples were loaded manually onto separate columns using a pressure vessel. The chromatography was carried out as described by Wolters et al. [4].
Protein identification and Validation
The SEQUEST program (a kind gift from Jimmy Eng and John Yates III) was used to search peptide spectra essentially as described previously using a minimally redundant mouse/human protein sequence database (EBI database) [5, 6]. The statistical confidence of identified proteins was validated by the use of an in-house algorithm (STATQUEST as described earlier [6].
Quantitative analysis
The profiles were clustered based on the Spearman rank correlation metric with average linkage using the freeware program Cluster 3.0 based on the original Cluster program [7]. The resulting clusters were visualized in heat-map format using Java TreeView based on the original TreeView [7]. Spectral counts were normalized essentially as described [8]. Relative protein abundance was inferred using the normalized spectral counts as a semi-quantitative metric after arcSine(H) transformation of the data.
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