Proteome profile of the MCF7 cancer cell line: a mass spectrometric evaluation

Abstract

The development of novel proteomic technologies that will enable the discovery of disease specific biomarkers is essential in the clinical setting to facilitate early diagnosis and increase survivability rates. We are reporting a shotgun two-dimensional (2D) strong cationic exchange/reversed-phase liquid chromatography/electrospray ionization tandem mass spectrometry (SCX/RPLC/ESI-MS/MS) protocol for the analysis of proteomic constituents in cancerous cells. The MCF7 breast cancer cell line was chosen as a model system. A series of optimization steps were performed to improve the LC/MS experimental setup, sample preparation, data acquisition and database search protocols, and a data filtering strategy was developed to enable confident identification of a large number of proteins and potential biomarkers. This research has resulted in the identification of >2000 proteins using multiple filtering and p-value sorting. Approximately 1600-1900 proteins had p < 0.001, and, of these, approximately 60% were matched by >or=2 unique peptides. Alternatively, >99% of the proteins identified by >or=2 unique peptides had p < 0.001. When searching the data against a reversed database of proteins, the rate of false positive identifications was 0.1% at the peptide level and 0.4% at the protein level. The typical reproducibility in detecting overlapping proteins across replicate runs exceeded 90% for proteins matched by >or=2 unique peptides. According to their biological function, approximately 200 proteins were involved in cancer-relevant cellular processes, and over 25 proteins were previously described in the literature as putative cancer biomarkers, as they were found to be differentially expressed between normal and cancerous cell states. Among these, biomarkers such PCNA, cathepsin D, E-cadherin, 14-3-3-sigma, antigen Ki-67, TP53RK, and calreticulin were identified. These data were generated by subjecting to MS analysis approximately 42 microg of sample, analyzing 16 SCX peptide fractions, and interpreting approximately 55,000 MS2 spectra. Total MS time required for analysis was 40 h.

Differential protein expression analysis using stable isotope labeling and PQD linear ion trap MS technology

“…In a previous work, we generated a full proteome profile of the MCF-7 cell line using 2DLC-MS/MS technology [35]. The study resulted in the identification of ϳ2000 proteins (P Ͻ 0.001), of which 200 were correlated with cellular processes relevant to cancer.…”

“…For initial optimization studies, MCF-7 breast cancer cells were cultured according to a procedure described in detail elsewhere [35]. Briefly, the cells were grown to 70% confluence in EMEM containing 10% FBS and 10 g/mL bovine insulin (maintenance medium), at 37°C, in a humidified atmosphere of 5% CO 2 .…”

“…Cells were thawed and lysed following a procedure described in previous work [35]. The cell lysis solution was prepared from 1 mL RIPA buffer (500 mM TrisHCl pH 7.4, 1.5 M NaCl, 10% NP-40, 2.5% deoxycholic acid, 10 mM EDTA), 100 L protease inhibitor cocktail (104 mM AEBSF, 0.08 mM aprotinin, 2 mM leupeptin, 4 mM bestatin, 1.5 mM pepstatin A, 1.4 mM E-64), 100 L NaF (ϳ100 mM) and 50 L Na 3 VO 4 (ϳ200 mM) as phosphatase inhibitors, and 8.75 mL of ice cold water.…”

“…Coupling of the LC system to the LTQ was accomplished via an on-column/no-split injection set up described in detail elsewhere [35] MS data were acquired using data-dependent acquisition conditions: each MS event was followed by zoom/MS 2 scans on the five top-most intense peaks; zoom scan width was Ϯ5 m/z; dynamic exclusion was enabled at repeat count 1, repeat duration 30 s, exclusion list size 200, exclusion duration 60 s, and exclusion mass width Ϯ 1.5 m/z; PQD parameters were set at isolation width 3 m/z, normalized collision energy 35%, activation Q 0.7, and activation time (T) 0.1 ms; the threshold for MS/MS acquisition was set to 100 counts. Protein identification was performed with the Bioworks 3.3 software (Thermo Electron Corp., San Jose, CA) using a minimally redundant human protein database downloaded from the ExPASy/SwissProt website on January 22, 2007 (37,690 entries, including 12 bovine proteins that were used for spiking the sample) [36].…”

“…Thus, the number of identified proteins can change from one chromatographic run to another (typical variations were ϳ5%-10%). We have shown, however, that if the stringency of the data filtering parameters is high, and if only proteins that are matched by Ն2 unique peptides are considered for comparison, the reproducibility between consecutive runs can be as high as ϳ90%-98% [35].…”