Developmental Research Pilot Project

Daniel Martin, MD, Institute for Systems Biology

"Isotopically Heavy Protein Standards to Calculate Absolute Protein Concentration"

Abstract

In this proposal we utilize advances in mass spectrometry based proteomics to develop a generalizable method for determining the absolute concentration of a specific protein in a cell or in a biofluid. We will produce an identical isotopically Heavy Protein Standard (HPS) for addition to a complex biological sample followed by high efficiency protein purification to capture both the HPS and the endogenous protein while excluding background proteins. We will co-purify the endogenous target and HPS at the protein level using either polyacrylamide gel electrophoresis (PAGE) or the combination of preparative isoelectric focusing followed by PAGE. Excised gel bands containing the co-purified heavy spiked protein and endogenous target will be subjected to in-gel digest followed by quantitative multiple reaction monitoring mass spectrometry. This strategy provides a high degree of purification while preserving all potentially informative peptides for the quantitative analysis. We will determine the intracellular protein concentration of the androgen receptor, TMPRSS2-ERG, and NKX3.1 in whole cell lysates from prostate cancer cells. We will also measure the concentrations of the secreted proteins PSA, jagged 1 ectodomain, and IGFBP-2 in serum from mice xenografted with prostate cancer cells to determine our ability to quantify potential cancer biomarkers in a complex "biofluid". This proposal addresses a critical deficiency in protein measurement in molecular biology and translational research; by using an innovative combination of newly developed technologies, our proposed method could alter the existing paradigm of protein measurement in both the clinical and laboratory setting.