<p>Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for the study of protein distributions in biological tissue specimens. This has traditionally been performed using time-of-flight (TOF) systems due to their large practical mass range, high dynamic range, and high throughput of the TOF analyzer. While many proteins are detected with this technology, the unambiguous identification of these analytes remains challenging. Indirect identification strategies have been limited by insufficient mass accuracy to confidently link ion images to proteomics data. This project incorporates high mass resolving power and high mass accuracy Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) into protein IMS experiments. With sample preparation and instrument methodologies tailored for improved protein signal detection, proteins up to 17 kDa were detected with resolving powers of 75,000 and mass accuracies less than 5 ppm. Additionally, in situ digestions were utilized to investigate proteins above current observable mass ranges of the FTICR. Incubation times for enzymatic digestions were compared using IMS approaches to evaluate maximum peptide signal while minimizing delocalization.</p>
<p>In order to improve confidence in protein and peptide identifications, identification strategies that preserve some form of spatial information were employed. Initially, liquid microextractions were used to selectively interrogate regions of tissue after in situ digestion. Localization of extracted peptides provided an additional level if information to correlate back to the IMS data. To further decrease the extraction diameter, hydrogel technologies for spatially-localized protein digestion/extraction were modified. Parameters such as percent polyacrylamide used in hydrogel construction as well as concentration of trypsin with which the hydrogel is loaded were investigated to maximize the number of protein identifications from LC-MS/MS analysis of hydrogel extracts. Increased polyacrylamide concentrations led to more rigid polymers, which were more amenable to perforating hydrogels using small punch biopsies. Hydrogels were fabricated with diameters as small as 260 µm, while still providing over 600 protein identifications. These improved methods to the hydrogel process allow researchers to target smaller biological features for robust spatially-localized proteomic analyses. Integrating high mass accuracy instrumentation with regio-specific proteomics experiments allows for confident identifications of proteins, providing insight into underlying biology of heterogeneous samples.</p>
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-06072016-091330 |
| Date | 08 June 2016 |
| Creators | Rizzo, David Geoffrey |
| Contributors | John A. McLean, Michael P. Stone, Richard M. Caprioli, Kevin L. Schey |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-06072016-091330/ |
| Rights | restricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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