Quantitative Profiling of Drug-Associated Proteomic Alterations by Combined 2-Nitrobenzenesulfenyl Chloride (NBS) Isotope Labeling and 2DE/MS Identification

Abstract

The identification of drug-responsive biomarkers in complex protein mixtures is an important goal of quantitative proteomics. Here, we describe a novel approach for identifying such drug-induced protein alterations, which combines 2-nitrobenzenesulfenyl chloride (NBS) tryptophan labeling with two-dimensional gel electrophoresis (2DE)/mass spectrometry (MS). Lysates from drug-treated and control samples are labeled with light or heavy NBS moiety and separated on a common 2DE gel, and protein alterations are identified by MS through the differential intensity of paired NBS peptide peaks. Using NBS/2DE/MS, we profiled the proteomic alterations induced by tamoxifen (TAM) in the estrogen receptor (ER) positive MCF-7 breast cancer cell line. Of 88 protein spots that significantly changed upon TAM treatment, 44 spots representing 23 distinct protein species were successfully identified with NBS-paired peptides. Of these 23 TAM-altered proteins, 16 (70%) have not been previously associated with TAM or ER activity. We found the NBS labeling procedure to be both technically and biologically reproducible, and the NBS/2DE/MS alterations exhibited good concordance with conventional 2DE differential protein quantitation, with discrepancies largely due to the comigration of distinct proteins in the regular 2DE gels. To validate the NBS/2DE/MS results, we used immunoblotting to confirm GRP78, CK19, and PA2G4 as bona fide TAM-regulated proteins. Furthermore, we demonstrate that PA2G4 expression can serve as a novel prognostic factor for disease-free survival in two independent breast cancer patient cohorts. To our knowledge, this is the first report describing the proteomic changes in breast cancer cells induced by TAM, the most commonly used selective estrogen receptor modulator (SERM). Our results indicate that NBS/2DE/MS may represent a more reliable approach for cellular protein quantitation than conventional 2DE approaches.

Expression and Role of the ErbB3-Binding Protein 1 in Acute Myelogenous Leukemic Cells

“…For example, p48 Ebp1 interacts with nuclear PKB/ Akt to prevent apoptotic cell death in neuronal cells (8). High expression of Ebp1 in breast cancers predicts a poor clinical outcome, whereas tamoxifen treatment of MCF-7 breast cancer cells markedly decreases both the transcription and protein expression of p48 Ebp1 (26). It has also been shown recently that the phosphorylation of p48 Ebp1 by CDK2 is required for its tumorigenic function (6).…”

The importance of protein profiling in the diagnosis and treatment of hematologic malignancies

“…In addition to the current advanced techniques for the assessment of cellular proteins, this area is still open to development. For instance, 2-nitrobenzenesulfenyl chloride (NBS) isotope labeling coupled with 2-DE and mass spectrometry was shown to yield more reliable results than the conventional methods of analyzing protein levels [109].…”

Negative Regulation of p53 by the Long Isoform of ErbB3 Binding Protein Ebp1 in Brain Tumors

“…Furthermore, tamoxifen treatment of MCF-7 breast cancer cells dramatically decreases p48 Ebp1 transcription and, thus, protein levels. Breast cancer patients that express high level of PA2G4 have poor clinical outcomes, suggesting Ebp1 may promote aggressive tumor behavior (6).…”