Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure
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| Language: | English |
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The Ohio State University / OhioLINK
2020
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| Online Access: | http://rave.ohiolink.edu/etdc/view?acc_num=osu1587409993097519 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu1587409993097519 |
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English |
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Chemistry |
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Chemistry Stiving, Alyssa Quencer Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure |
| author |
Stiving, Alyssa Quencer |
| author_facet |
Stiving, Alyssa Quencer |
| author_sort |
Stiving, Alyssa Quencer |
| title |
Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure |
| title_short |
Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure |
| title_full |
Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure |
| title_fullStr |
Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure |
| title_full_unstemmed |
Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure |
| title_sort |
development of surface-induced dissociation, ion mobility, and ultraviolet photodissociation to characterize peptide, protein, and protein complex structure |
| publisher |
The Ohio State University / OhioLINK |
| publishDate |
2020 |
| url |
http://rave.ohiolink.edu/etdc/view?acc_num=osu1587409993097519 |
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AT stivingalyssaquencer developmentofsurfaceinduceddissociationionmobilityandultravioletphotodissociationtocharacterizepeptideproteinandproteincomplexstructure |
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1719457195519639552 |
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ndltd-OhioLink-oai-etd.ohiolink.edu-osu15874099930975192021-08-03T07:14:30Z Development of Surface-Induced Dissociation, Ion Mobility, and Ultraviolet Photodissociation to Characterize Peptide, Protein, and Protein Complex Structure Stiving, Alyssa Quencer Chemistry Native mass spectrometry (MS) is gaining popularity as a useful complementary tool in the field of structural biology. The protein complexes that native MS is tasked with characterizing are growing increasingly more complex. Many biologically relevant protein complexes contain post-translational modifications, ligand binding partners, or other heterogeneity that can influence their structure and, consequently, function. Native MS techniques such as surface-induced dissociation (SID), ion mobility (IM), and ultraviolet photodissociation (UVPD) have demonstrated their ability to effectively characterize varying levels of structure. Development of these technologies and methods to study protein structural features in a more accurate, in-depth, and efficient manner is necessary to furthering the native MS and structural biology field.SID has demonstrated utility in characterizing the topology and connectivity of protein complexes in the gas phase. In Chapter 3, the development and characterization of a new, simplified SID device is accomplished using a tilted surface and an ion carpet array. This work investigates the ability to fragment peptides and protein complexes from 555 Da to 801 kDa. Furthermore, the new SID design shows the ability to achieve SID without the need to change tune settings between precursor ions of different sizes, paving the way for operation by non-expert users and, ultimately, automation.Application of ion mobility as an orthogonal dimension of separation and structural characterization within native MS experiments can help define protein structure. Charge-reducing reagents are becoming increasingly popular in native MS due to their propensity to minimize structural perturbation and ability to increase apparent resolution due to increased spacing between signals. Chapter 4 describes the development and validation of a charge-reduced protein and protein complex collision cross section (CCS) database. Because many commercial IM cells require calibration to obtain CCS values, appropriate calibrants are needed to accomplish an accurate calibration. This database establishes 19 protein calibrants under multiple charge-reducing conditions and demonstrates the need to mobility-match calibrants with the unknown protein.Chapter 5 describes investigation of the potential utility of SID as a top-down fragmentation method when complementary methods are needed to obtain protein sequence and post-translational modification (PTM) information. While the ion types and sequence coverages obtained from SID top-down of standard proteins is comparable to CID and HCD, the results demonstrate characteristic SID fragmentation behavior that can help in gaining a better understanding of SID fundamentals.The ability to identify amino acid sequences, sites of PTMs, disulfide linkages, etc. from intact proteins or protein complexes by using tandem MS methods is gaining popularity as a means of providing complementary information to higher-order structural studies. Chapter 6 describes the progress in developing an instrument platform that combines IM and 193 nm ultraviolet photodissociation (UVPD), enabling primary sequence interrogation of mobility-separated ions. This new instrument setup shows complete characterization of standard peptide systems through UVPD-only and IM-UVPD experiments.Ultimately, the work outlined within this dissertation demonstrates the development of SID, IM, and UVPD instrumentation and methods, expanding the tools available within native MS to perform more in-depth characterization of peptide, protein, and protein complex structures. 2020-09-25 English text The Ohio State University / OhioLINK http://rave.ohiolink.edu/etdc/view?acc_num=osu1587409993097519 http://rave.ohiolink.edu/etdc/view?acc_num=osu1587409993097519 unrestricted This thesis or dissertation is protected by copyright: all rights reserved. It may not be copied or redistributed beyond the terms of applicable copyright laws. |