Characterization of protein expression and folding in cell-free systems by maldi-tof mass spectrometry

Analytical Chemistry
Lisa M Jungbauer, Silvia Cavagnero

Abstract

Recent advances in basic research, medicine, and biotechnology provide great motivation for the development of analytical tools to probe the behavior of target biomolecules in complex biological environments. Cell-free transcription-translation systems are an attractive medium for such studies, because they mimic several biochemical features of living cells, yet they are much more amenable to manipulation and spectroscopic analysis. However, few methods are currently available to characterize target proteins in cell-free systems. We have employed matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry for the detection and characterization of two cell-free expressed model proteins, cold shock protein A and apomyoglobin (apoMb) in cell-free systems. We exploited a combination of multiple selective isotope-labeling patterns for the identification of both full-length proteins and their in situ-generated proteolytic fragments. MALDI-TOF mass spectrometry-detected hydrogen/deuterium exchange, performed directly in the cell-free medium, allowed the assessment of apoMb's global degree of folding. The above methods are straightforward in that they do not require high levels of protein expression and all...Continue Reading

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