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Proteome profile of the MCF7 cancer cell line: a mass spectrometric evaluation
Author(s) -
Sarvaiya Hetal A.,
Yoon Jung H.,
Lazar Iulia M.
Publication year - 2006
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.2677
Subject(s) - chemistry , proteome , tandem mass spectrometry , computational biology , proteomics , biomarker discovery , chromatography , mass spectrometry , isobaric labeling , tandem mass tag , electrospray ionization , cancer , biochemistry , protein mass spectrometry , quantitative proteomics , biology , gene , genetics
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, ∼60% were matched by ≥2 unique peptides. Alternatively, >99% of the proteins identified by ≥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 ≥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 ∼42 µg of sample, analyzing 16 SCX peptide fractions, and interpreting ∼55 000 MS 2 spectra. Total MS time required for analysis was 40 h. Copyright © 2006 John Wiley & Sons, Ltd.