Abstract
The emergence of acquired drug resistance results from multiple compensatory mechanisms acting to prevent cell death. Simultaneous monitoring of proteins involved in drug resistance is a major challenge for both elucidation of the underlying biology and development of candidate biomarkers for assessment of personalized cancer therapy. Here, we have utilized an integrated analytical platform based on SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring mass spectrometry, a versatile and powerful tool for targeted quantification of proteins in complex matrices, to evaluate a well-characterized model system of melphalan resistance in multiple myeloma (MM). Quantitative assays were developed to measure protein expression related to signaling events and biological processes relevant to melphalan resistance in multiple myeloma, specifically: nuclear factor-κB subunits, members of the Bcl-2 family of apoptosis-regulating proteins, and Fanconi Anemia DNA repair components. SDS-PAGE protein fractionation prior to liquid chromatography coupled to multiple reaction monitoring methods were developed for quantification of these selected target proteins in amounts of material compatible with direct translation to clinical specimens (i.e. less than 50,000 cells). As proof of principle, both relative and absolute quantification were performed on cell line models of MM to compare protein expression before and after drug treatment in naïve cells and in drug resistant cells; these liquid chromatography-multiple reaction monitoring results are compared with existing literature and Western blots. The initial stage of a systems biology platform for examining drug resistance in MM has been implemented in cell line models and has been translated to MM cells isolated from a patient. The ultimate application of this platform could assist in clinical decision-making for individualized patient treatment. Although these specific assays have been developed to monitor MM, these techniques are expected to have broad applicability in cancer and other types of disease.
Highlights
From the ‡Molecular Oncology, §Experimental Therapeutics, ¶Translational Research Laboratory, ʈBiomedical Informatics, H
Rationale for Selection of SDS-PAGE Coupled with LCMRM—Immunoprecipitation and LC-ESI-MS/MS can guide the selection of target peptides and transitions; LC-ESI-multiple reaction monitoring (MRM) screening of proteins fractionated by SDSPAGE has been demonstrated to be effective for developing quantitative assays for signaling proteins and applying them to study cancer biology [45]
A GeLC-MRM based platform for assessment of protein expression related to NFB survival signaling, apoptosis, and the Fanconi Anemia DNA damage-response has been developed using models for acquired drug resistance (ADR) to melphalan in two MM cell lines (RPMI-8226 and U266), which have different underlying oncogenic biology
Summary
Reagents—All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) at the highest purity, unless otherwise specified. Antibodies directed against Bcl-xL (2764), Bad (9268), Bim (4582), and Bid (2002) were purchased from Cell Signaling Technology (Boston, MA). Synthesis of Isopeptide Standards for Monoubiquitinated FANCD2 and FANCI—To develop standards for the tryptic peptides containing the sites of ubiquitination, the isopeptides (branched peptides with glycyl-glycine moieties connected to the epsilon amino group of a lysine via amide bonds) were synthesized with FMOC-Lys(Boc)-Wang resin or FMOC-Arg(Pbf)-Wang resin at the 25 mol scale (Tribute, Protein Technologies). Aliquots of each cell lysate containing 50 g of protein (corresponding to the extract from 250,000 cells) were denatured by boiling in loading buffer and separated on 4 –12% gels (Criterion XT, Bio-Rad, Hercules, CA) for 80 min at 150 V. One sixth of each digest was analyzed by LC-MRM in each technical
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