Journal of Proteome Research a major component of the Wnt signaling pathway.9 The colon tumor cell line SW480 contains a mutation at position Q1338 in the APC coding sequence, which generates a premature stop codon.10 The remaining wild-type allele is deleted. An APC-corrected version of SW480 (SW480APC) was generated by stable transfection with wildtype APC.11 Relative to APC mutant cells (SW480Null), SW480APC cells exhibit decreased proliferation rates, decreased nuclear localization of β-catenin (CTNNB1), increased transloca- tion of epithelial cadherin (CDH1) to the plasma membrane, and enhanced cell cell adhesion.11 Thus, the SW480APC model dis- plays unique phenotypic properties that may map to underlying proteomic expression signatures. Here we employ two complementary proteomic technology platforms that provide the means to systematically characterize APC-driven proteomic diﬀerences. The ﬁrst, shotgun proteo- mics, employs liquid chromatography tandem mass spectro- metry (LC MS/MS) and provides a nondirected, global inven- tory of proteomes, together with quantitative assessments of protein abundances.12,13 The second proteomics approach is targeted analysis of individual proteins by multiple reaction monitoring (MRM) mass spectrometry intensity measurement of their constituent peptides.14,15 The data illustrate mechanisms by which APC inactivation may alter cellular functions by per- turbing the composition of cell proteomes. More generally, we demonstrate the ability of label free shotgun proteomics to assess proteomic consequences of a single gene diﬀerence. ’ EXPERIMENTAL METHODS Cell Culture and Subcellular Fractionation SW480APC and SW480Null were a kind gift from Antony Burgess (Ludwig Institute, Melbourne, Australia). Cells were grown in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and genetecin (1.5 mg/mL). Three biological replicate cultures were harvested approximately 1 week apart and these replicates were processed separately and independently through the complete analysis. Growth medium was aspirated, cells were washed once in 1 PBS and collected in 1 PBS, then centrifuged at 300 g for 5 min and the super- natant was discarded. Cell pellets were stored at 80 C until cell lysis could be carried out. For subcellular fractionation, cell pellets were resuspended in buﬀer A (10 mM HEPES, pH7.9, 10 mM KCl, 2 mM MgCl2, 0.2 mM EDTA) containing protease inhibitors (aprotinin, leupeptin) and incubated at 4 C for 20 min. IGEPAL CA-630 (Sigma-Aldrich) was added to a ﬁnal concentration of 0.5% v/v, samples were incubated at 4 C for 10 min and centrifuged at 300 g for 15 min at 4 C. The supernatant (cytosolic fraction) was collected in a separate tube. Pellets were resuspended in Buﬀer A, centrifuged at 2000 g for 15 min at 4 C and the supernatant (cytosolic fraction) was combined with supernatant from the previous step. The nuclear pellet was stored at 80 C and the cytosolic fraction was lyophilized overnight. Subsequent lyis/digestion of cytosolic lyophilates and nuclear pellets was carried out as described. Cell Lysis, Protein Digestion, and Isoelectric Focusing of Peptides Lysis of cell pellets was carried out at ambient temperature. Each biological replicate (one cell pellet from one cell line) was processed in parallel to minimize the eﬀects of systematic errors. Pellets were resuspended in 100 μL 100 mM Ammonium ARTICLE bicarbonate (AmBic) and 100 μL triﬂuoroethanol (TFE) (organic solvent) was added followed by sonication (3 20 s). Samples were incubated at 60 C and resonicated (3 20 s). Protein concentration was estimated using a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Proteins were reduced and alkylated with 40 mM tris(2-carboxyethyl)phosphine (TCEP)/ 100 mM dithiothreitol (DTT) and 50 mM iodoacetamide (IAM), respectively. Samples were diluted in 50 mM AmBic, pH 8.0 and tyrpsinized overnight at 37 C (1:50, w:w). Subsequently, pep- tides were lyophilized overnight. Peptides were desalted as described16 and separated by isoelectric focusing (IEF) using immobiline IPG strips (24 cm, pH 3.5 4.5) (GE Healthcare) as described.16,17 LC MS/MS LC MS/MS shotgun proteomic analyses were performed on LTQ XL mass spectrometer (Thermo Fisher Scientiﬁc) equip- ped with an Eksigent NanoLC AS1 autosampler and Eksigent NanoLC 1D Plus pump, Nanospray source, and Xcalibur 2.0 SR2 instrument control. Peptides were separated on a packed capil- lary tip (Polymicro Technologies, 100 mm 11 cm) with Jupiter C18 resin (5 mm, 300 Å, Phenomenex) using an in-line solid- phase extraction column (100 mm 6 cm) packed with the same C18 resin using a frit generated with liquid silicate Kasil 1.18 Mobile phase A consisted of 0.1% formic acid and mobile phase B consisted of 0.1% formic acid in 90% acetonitrile. A 90-min gradient was carried out with a 30-min washing period (100% A) to allow for solid-phase extraction and removal of any residual salts. Following the washing period, the gradient was increased to 25% B by 35 min, followed by an increase to 90% B by 50 min and held for 9 min before returning 95% A. MS/MS spectra of the peptides are acquired using data-dependent scanning in which one full MS spectrum (mass range 400 2000 m/z) is followed by ﬁve MS/MS spectra. MS/MS spectra are recorded using dynamic exclusion of previously analyzed precursors for 60 s with a repeat of 1 and a repeat duration of 1. MS/MS spectra were generated by collision-induced dissociation of the peptide ions at normalized collision energy of 35% to generate a series of b- and y-ions as major fragments. Biological samples from 3 indepen- dent cell cultures were injected in duplicate for a total of 6 replicate measurements for the SW480null and SW480APC cell lines. LC MS/MS Data Analysis MS/MS scans were transferred to mzML ﬁle format by “Scansifter”, an in-house-developed software algorithm, which reads tandem mass spectra stored as centroided peak lists from Thermo RAW ﬁles and transcodes them to universal mzML format.19 If 90% of the intensity of a tandem mass spectrum appeared at a lower m/z than the precursor ion, a single precursor charge was assumed; otherwise, the spectrum was processed under both double and triple precursor charge assumptions. The resulting mzML ﬁles were searched against the Human IPI database (v3.64) using the Myrimatch algorithm (version 1.2.11).20 The database search was conﬁgured to look for both fully tryptic and semitryptic peptides with a precursor mass/charge (m/z) tolerance of 1.25 and a fragment m/z tolerance of 0.5. Carboxamidomethylation of cysteines was included as static modiﬁcation, and oxidation of methionine as a dynamic modi- ﬁcation in the search criteria, while any number of missed cleavages was allowed. A reverse version of the Human IPI database was included in the database search to allow for the calculation of false discovery rates (FDR). The IDpicker algorithm 1185 dx.doi.org/10.1021/pr2009109 |J. Proteome Res. 2012, 11, 1184–1195