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Structural analysis of proteins by covalent labeling and mass spectrometric detection

Yuping Zhou, University of Massachusetts Amherst

Abstract

Covalent labeling and mass spectrometry are seeing increased use together as a way to obtain insight into the 3-dimensional structure of proteins and protein complexes. Several amino acid specific (e.g., diethylpyrocarbonate) and non-specific (e.g., hydroxyl radicals) labeling reagents are available for this purpose. Diethylpyrocarbonate (DEPC) is a promising labeling reagent because it can potentially probe up to 30% of the residues in the average protein and gives only one reaction product, thereby facilitating mass spectrometric analysis. It was recently reported, though, that DEPC modifications are labile for some amino acids. ^ This dissertation focuses on the improvement of diethylpyrocarbonate (DEPC)-based covalent labeling for increased protein structural resolution. The number of DEPC modified residues and, thus, protein structural information, can be significantly increased by decreasing the time between the covalent labeling reaction and the mass spectrometric analysis. This is most effectively accomplished using short (e.g., 2 h) proteolytic digestions with enzymes such as immobilized chymotrypsin or Glu-C rather than using methods (e.g., microwave or ultrasonic irradiation) that accelerate proteolysis in other ways. Besides, Cys residues that form disulfide bonds appear to be modified by DEPC as well. We demonstrate that disulfide linked Cys residues are not actually reactive with DEPC but, instead, once reduced, free Cys residues can capture a carbethoxy group from other modified amino acids via a solution-phase reaction that can occur during the protein digestion step. This "scrambling" of carbethoxy groups decreases the amount of modification observed at other residues and can potentially provide incorrect protein structural information. Fortunately, label scrambling can be completely avoided by alkylating the free thiols after disulfide reduction. ^ We also developed novel methods as indicators of protein structural integrity using isotopically encoded labeling reagent Tandem Mass Tags. Because most covalent labels are relatively large, steps must be taken to ensure the structural integrity of the modified protein during the labeling reactions so that correct structural information can be obtained. Measuring labeling kinetics is a reliable way to ensure that a given labeling reagent does not perturb a protein's structure, but obtaining such kinetic information is time and sample intensive because it requires multiple liquid chromatography (LC)–MS experiments. Here we present a new strategy that uses isotopically encoded labeling reagents to measure labeling kinetics in a single LC–MS experiment. We illustrate this new strategy by labeling solvent-exposed lysine residues with commercially available tandem mass tags. After tandem MS experiments, these tags allow the simultaneous identification of modified sites and determination of the reaction rates at each site in a way that is just as reliable as experiments that involve multiple LC-MS measurements. ^ This improved technique is then applied to study the structural change of proteins that undergo forced degradation. Because covalent labeling combined with MS can be used to monitor conformational changes of proteins, it can also be applied to study structural changes of proteins after exposure to degradation conditions. β2m was used as a model protein to examine the potential of this method. Forced degradation studies using thermal and oxidative conditions were carried out. Covalent modification patterns of the protein show clear differences under these degradation conditions as compared to the native protein. The general trend that we found was that less modification was observed for both thermal and oxidative condition, indicating that aggregation could occur at higher temperatures or in the presence of hydrogen peroxide. Size exclusion chromatography confirmed that there was dimer formation during the forced degradation conditions, which is consistent with the covalent labeling data. This method could be applied to other systems to study degradation caused structural changes of proteins as it is a fast and relatively simple approach.^

Subject Area

Biochemistry

Recommended Citation

Zhou, Yuping, "Structural analysis of proteins by covalent labeling and mass spectrometric detection" (2014). Doctoral Dissertations Available from Proquest. AAI3615467.
http://scholarworks.umass.edu/dissertations/AAI3615467

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