Skip to Main Content
 

Global Search Box

 
 
 

ETD Abstract Container

Abstract Header

Characterization of Proteins and Protein Complexes by Online Chromatographic Separations and Direct Infusion Native Mass Spectrometry

Sahasrabuddhe, Aniruddha
http://rave.ohiolink.edu/etdc/view?acc_num=osu1515171055447523

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Chemistry.
Because the functions of proteins and protein complexes are linked to their structures, structural characterization is important to understand the underlying function. Current structural biology techniques provide high-resolution structures, but have well-known challenges that limit the structural characterization of the protein complexes. These challenges inspire the development of native mass spectrometry (MS), coupled to ion mobility (IM) and integrated separations approaches, as an alternative or complementary structural biology tool. Chapter 2 of this dissertation describes the use of native MS to define the role of D32/R55 electrostatic interactions in the stability of protein dimers AV Rop, with R55 substituted by K, Q, F, L, and E. The research is a collaboration with the Magliery laboratory at OSU and applies native MS coupled to IM and surface-induced dissociation (SID). The collision cross sections (CCS) determined by IM do not differ significantly for the gas-phase dimer structures of all mutants. The gas-phase dimer stability trend determined by SID is in agreement with the expected electrostatic interactions, in contrast with relative stabilities determined by thermal and chemical denaturation in solution. Chapter 3 describes a collaboration with the computational protein design group of David Baker at the University of Washington. The goals of the three subprojects are rapid characterization of stoichiometry, oligomeric states, and inter-subunit connectivity of the designed protein complexes. Native MS, coupled to IM and SID, confirmed the computational designs of three dihedral hetero-dodecameric protein complexes. Native MS also revealed pH-dependent changes in the oligomeric states of pH-sensitive protein complexes that were developed based on the hydrogen-bond networks at the protein interface. SID showed that the gas-phase stability of the pH-induced oligomers is in agreement with the computational designs. Finally, a high-resolution native MS (Q Exactive Plus EMR Orbitrap) confirmed the interaction specificity of a set of designed asymmetric hetero-dimers. Chapter 4 describes the on-line coupling of size exclusion chromatography (SEC) and ion exchange chromatography (IEX) with a high-resolution MS (Q Exactive Plus EMR Orbitrap), with the goals of de-adducting the protein complexes and separating different oligomeric states from each other and from other contaminants. Eluting proteins are commonly monitored by on-line ultra-violet (UV) spectroscopic detection but UV detection can’t distinguish between co-eluting species. For protein mixtures, on-line SEC-MS and IEX-MS allows the detection of accurate masses (even when species co-elute), sample heterogeneity, and interaction specificity in native-like conditions. Chapter 5 describes a collaboration with the laboratory of Justin Wu at OSU. Native MS is used to determine whether an aB-chaperone protects lens protein Opj from temperature-induced aggregate formation via protein-protein interactions. Native MS coupled with SID and CID confirms the protective role of aB-chaperone in preventing Opj aggregation via protein-protein interactions. In summary, native MS can be used to characterize stability of Rop homodimers, stoichiometry and connectivity of three types of designed protein complexes, and interactions between a chaperone and a lens protein. Integration of SEC and IEX separations with native MS provide proof-of-concept measurement of accurate masses, sample heterogeneity, and interaction specificity for a small set of model hetero-dimers.
Vicki Wysocki (Advisor)
Zhengrong Wu (Committee Member)
Abraham Badu-Tawiah (Committee Member)
153 p.
Chemistry
Native mass spectrometry, Surface-induced dissociation, Protein complexes, online SEC-MS

Recommended Citations

Citations

  • Sahasrabuddhe, A. (2018). Characterization of Proteins and Protein Complexes by Online Chromatographic Separations and Direct Infusion Native Mass Spectrometry [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1515171055447523

    APA Style (7th edition)

  • Sahasrabuddhe, Aniruddha. Characterization of Proteins and Protein Complexes by Online Chromatographic Separations and Direct Infusion Native Mass Spectrometry. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1515171055447523.

    MLA Style (8th edition)

  • Sahasrabuddhe, Aniruddha. "Characterization of Proteins and Protein Complexes by Online Chromatographic Separations and Direct Infusion Native Mass Spectrometry." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1515171055447523

    Chicago Manual of Style (17th edition)

osu1515171055447523
706
© 2018, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.