Characterization of protein expression and folding in cell-free systems by maldi-tof mass spectrometry.
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 allow the efficient characterization of both protein identity and global degree of folding.
Jungbauer
Lisa M
LM
Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, Wisconsin 53706, USA.
Cavagnero
Silvia
S
D000595:Amino Acid Sequence; D001059:Apoproteins / Q000032:analysis / Q000737:chemistry; D002474:Cell-Free System; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000032:analysis / Q000737:chemistry; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013205:Staphylococcal Protein A / Q000032:analysis / Q000737:chemistry; D013997:Time Factors
D001059:Apoproteins; D009211:Myoglobin; D011506:Proteins; D013205:Staphylococcal Protein A; C010857:apomyoglobin; D006859:Hydrogen; D003903:Deuterium