(pH 7.8), supplemented with 1 mM CaCl2, 10 ug of trypsin and incubated for 3 hours at 37oC with shaking. The sample digests were purified with solid phase extraction using C18 columns (Discovery DSC-18, SUPELCO, 52601-U), lyophilized and resuspended in 25 mM ammonium bicarbonate pH 7.8. The peptide amounts were estimated with BCA assay. On the average the amount of purified tryptic peptides was 200 ug. To generate the 18O reference sample, 20 ug of each sample was pooled, then boiled 10 minutes, followed by immediate cooling for 10 minutes. The boiling/cooling steps were performed to inactivate trypsin (this step helps to avoid back-exchange of 18O-labeled peptides). The pooled reference was then subjected to solution-phase tryptic 18O exchange, followed by quenching of tryptic activity with formic acid. The pooled sample was then added in equal amounts with each individual sample for quantitation purposes. Characterization of the mouse liver proteome Construction of a library of proteins and tryptic peptides present in the liver is an important step for follow-up quantitation. 10 ug aliquots from all 97 strains were pooled together and subjected to LC fractionation by strong cation exchange (SCX) chromatogra- phy on a 200 mm62.1 mm Polysulfoethyl A column (PolyLC, Columbia, MD) preceded by a 10 mm62.1 mm guard column, using a flow rate of 0.2 mL/min. LC separations were performed using an Agilent 1100 series HPLC system (Agilent, Palo Alto, CA). Mobile phase solvents consisted of (A) 10 mM ammonium formate, 25% acetonitrile, pH 3.0 and (B) 500 mM ammonium formate, 25% acetonitrile, pH 6.8. Once loaded, isocratic conditions at 100% A were maintained for 10 min. Peptides were separated by using a gradient from 0–50% B over 40 min, followed by a gradient of 50–100% B over 10 min. The gradient was then held at 100% solvent B for another 10 min. Following lyophilization, all thirty fractions collected during this gradient were dissolved in 25 mM ammonium bicarbonate and stored at 280OC. Each SCX fraction was analyzed with an automated custom- built capillary HPLC system coupled online to an LTQ ion trap mass spectrometer (Thermo Fisher, San Jose, CA) by using an electrospray ionization interface. The reversed phase capillary column was prepared by slurry packing 3-mm Jupiter C18 particles (Phenomenex, Torrance, CA) into a 75 mm i.d.665 cm fused silica capillary (Polymicro Technologies, Phoenix, AZ). The mobile phase solvents consisted of (A) 0.2% acetic acid and 0.05% TFA in water and (B) 0.1% TFA in 90% acetonitrile. An exponential gradient was used for the separation, which started with 100% A, and gradually increased to 60% B over 100 min. The instrument was operated in a data-dependent mode with an m/z range of 400–2000. Ten most abundant ions from each MS scan were selected for further MS/MS analysis by using a normalized collision energy setting of 35%. Dynamic exclusion was applied to avoid repeat analyses of the same abundant precursor ion. The SEQUEST software (Thermo Fisher) was used to search the MS/MS data against the mouse International Protein Index (IPI) database (version 3.52 http://www.ebi.ac.uk/IPI). Human keratins and porcine trypsin were added into the database as expected contaminants. Trypsin cleavage specificity was required for all of the considered peptides. The following criteria were used to filter raw SEQUEST results: 1) Xcorr$1.9 and Del- taCn2$0.21 for charge state +1; 2) Xcorr$2.5 and Del- taCn2$0.26 for charge state +2; 3) Xcorr$2.8 and Del- taCn2$0.32 for charge state +3. These criteria provide the maximum number of peptide identifications not exceeding 1% false discovery rate (FDR). To estimate the FDR of peptide Global Mouse mRNA and Protein Levels Comparison identifications we searched against a reversed database as previously described [36]. Relative protein abundance quantitation Relative peptide and protein quantitation was based on ratios between intensities of natural 16O isotope containing peptides and reference peptides labeled with stable 18O isotope at the carbonyl group at the C-terminus of the peptide. To create a reference sample we pooled together 20 ug aliquots from all strains and labeled the C-termini with 18O isotopes using trypsin catalyzed exchange in the presence of heavy H218O water as described above and elsewhere [37]. Prior to the LC-MS analysis 3.75 ug aliquots from each individual sample were mixed with the same amount of 18O-labeled reference sample. The 7 ug aliquots were analyzed on a LTQ-Orbitrap mass spectrometer that was interfaced with a 75 um i.d.665 cm long LC column packed with 3 um Jupiter C18 particles (Phenom- enex). The mobile phase solvents consisted of (A) 0.2% acetic acid and 0.05% TFA in water and (B) 0.1% TFA in 90% acetonitrile. An exponential gradient was used for the separation, which started with 100% A and gradually increased to 60% B over 100 min. LC-MS datasets were analyzed by in-house software VIPER [38] that detected features in mass – elution time space and assigned them to peptides in AMT tag database as described elsewhere [39,40]. Typically an LC-MS run identifies ,3,500 16O/18O peptide pairs that co-elute with a 4.0085 Da mass difference. As we mentioned before, the relative abundances of tryptic peptides were calculated as the ratio between light and heavy isotopes. The relative abundances then were normalized with EigenMS procedure [41] to correct systematic biases that may arise for example from unequal sample loading, batch-to-batch differences in sample processing and LC column variability. Briefly, the EigenMS procedure discovers the systematic trends (so-called eigenpeptides) in the data using singular value decomposition and then removes contributions of those eigenpe- pides from each peptide. For all data analysis purposes the peptide and protein intensities were log2 transformed and zero-centered by subtracting the peptide or protein specific means taken across all the samples. Immunobloting experiments To determine the protein levels by immunobloting, liver samples were homogenized in RIPA including phosphatase and protease inhibitors (Santa Cruz Biotech sc-24948), and protein determination were done using the Biorad Dc Assay. Protein samples were boiled following addition of Laemmli loading dye, separated on Invitrogen precast gels, and transferred to PVDF membranes. Membranes were rinsed in 16 TBST (Cell signaling #9997) blocked in 5% skim milk-TBST, rinsed in TBST, and incubated with primary antibodies diluted in 3% BSA-TBST for 1 hr at 23degC or overnight at 4degC. Membranes were washed in TBST and incubated with an HRP-conjugated anti rabbit IgG KPL (#474-1516) 1/5000 in 5% skim milk-TBST. Membranes were washed again, incubated in ECL-plus, and signal detected using a Biorad Chemidoc or film. Densitometry was done using the Biorad Quantity One software. The following list of antibodies and working dilutions were used for each protein: Fasn (Cell Signaling cat #3180, 1/2000), Acyl (Cell Signaling cat #4332, 1/ 2000), Ywhae (Cell Signaling cat #9635, 1/2000), Vim (Cell Signaling cat#3932, 1/1000), Rkip (Cell Signaling cat#5291, 1/ 2000), Gapdh (Cell Signaling cat#3683, 1/5,000), Glo1 (Sigma Chemical SAB1100242, 1/20,000), GstA4 (Sigma Chemical SAB1100244, 1/20,000), AnxA5 (Sigma Chemical AV36687, 1/ 2000), Hao1 (Sigma Chemical AV42480, 1/2000), Aldh3A2 PLoS Genetics | www.plosgenetics.org 14 June 2011 | Volume 7 | Issue 6 | e1001393