An approach to the proteomic analysis of a breast cancer cell line (SKBR‐3)

Abstract

An approach to the proteomic analysis of a breast cancer cell line (SKBR-3)This report describes the profiling of proteins in a sample prepared by laser capture microdissection (LCM) from a breast cancer cell line (SKBR-3). This experimental approach serves as a model system for proteomic studies on selected tissue samples and for studies of specific cell types. The captured cells were isolated in a dehydrated and reduced state and solubilized with a denaturing buffer. After dilution the protein mixture was digested with trypsin and the resulting peptide mixture was fractionated by reversed phase HPLC (RPLC) and analyzed on an ion trap mass spectrometer. A key part of this study is the combination of the LCM process with an extraction/digestion procedure that allowed effective solubilization of a significant part of the cellular sample in a single step. The identity of the peptides was determined by tandem mass spectrometry measurements in which the resulting spectra were compared with genomic and proteomic databases and protein identifications were made. While only peptides with a high probability assignment were used, the interpretation of mass spectral fragmentation patterns were also confirmed by manual interpretation of the spectra. Also, for the more abundant proteins the initial protein assignment from the best match peptide was strengthened by the observation of additional confirmatory peptide identifications. Another selection criteria was correlation of the mass spectrometric studies with clinical and genomic studies of potential cancer markers in tumor samples. This proteomic study allowed identification of the following proteins: human receptor protein kinase HER-2 or ERBB-2 and related kinases HER-3 and HER-4, the gene products from breast cancer type I and II susceptibility genes and cytoskeletal components such as cytokeratins 8, 18 and 19. Other proteins include fibroblast growth factor receptor variants (FGFR-2&4) and T-lymphoma invasion and metastasis inducing protein 1 (TIAM1). In addition several nonreceptor protein kinases YES, FAK and JAK-1 and 3 were identified. Since the study was performed on a limited number of cells (approximately 10 000) it raises the possibility of such studies being performed on individual patient samples prepared by needle biopsy.

Competition on Nitrocellulose-immobilized Antibody Arrays

“…Here, 12 proteins have been identified as increased or decreased in expression in two breast cancer cell lines, SKBR3 versus MDA MB-231, using a microarray prepared from 72 selected antibodies. Similar data obtained by other methods confirm these results for AP-2␣ and AP-2␥ (36), ErbB2 (37,38), p53 (26), and c-Jun (37). A clear modulation of seven more proteins, including three cyclins D 1 , D 3 , and E, JNK1/2, thymidylate synthase, catalase, and 14.3.3, underlies a wider pleiotropic effect of the cancer mutation(s) in the two cell lines.…”

Profiling Core Proteomes of Human Cell Lines by One-dimensional PAGE and Liquid Chromatography-Tandem Mass Spectrometry

“…Only 30 -50 g of total protein were required as starting material compared with typical amounts of 200 -300 g of protein for 2D PAGE and 0.5-2 mg for 2D LC/LC-MS/MS experiments. As a result, this method is also applicable to the study of cellular proteomes that are of very limited availability such as samples obtained by needle biopsy or laser capture microdissection, where initial studies have reported expression proteomic analyses from as little as 1-5 g of total protein (25).…”

Proteome Analysis of Microdissected Formalin-fixed and Paraffin-embedded Tissue Specimens

“…However, the smaller quantities of samples available for microdissected populations have, to this point, restricted protein analyses to the use of only a single chromatography separation prior to tandem mass spectrometry (MS/ MS) analysis and have limited the ability to mine deeper into the tissue proteome in recent studies (Wu et al 2003;Zang et al 2004;Baker et al 2005;Crockett et al 2005;Hood et al 2005;Palmer-Toy et al 2005). Because the sizes of human tissue biopsies are becoming significantly smaller due to the advent of minimally invasive methods and early detection and treatment of lesions, a more effective discovery-based proteome technology is critically needed to enable sensitive studies of protein profiles that will have diagnostic and therapeutic relevance.…”