Author : Rudolph K. Mignon
Publisher :
ISBN 13 : 9780438945968
Total Pages : 180 pages
Book Rating : 4.9/5 (459 download)
Book Synopsis Development of Methods for the Analysis of Proteins and Lipids in Bacteria Using Mass Spectrometry by : Rudolph K. Mignon
Download or read book Development of Methods for the Analysis of Proteins and Lipids in Bacteria Using Mass Spectrometry written by Rudolph K. Mignon and published by . This book was released on 2018 with total page 180 pages. Available in PDF, EPUB and Kindle. Book excerpt: The study of microorganisms is an important aspect in various settings including healthcare, national security, environment, and food safety. In this dissertation, various mass spectrometry (MS)-based analytical methods were developed using different instrumentation to analyze proteins and lipids in microorganisms. In the first project, high-pressure liquid chromatography (HPLC-MS) and tandem mass spectrometry (MS/MS) methods were developed to analyze the lipidome of Gemmata obscuriglobus, a unique species belonging to the planctomycetes phylum of bacteria. G. obscuriglobus shows characteristics of both prokaryotes and eukaryotes, many of which involve or are related to the lipid membrane structure and chemical composition. Using HPLC-electrospray ionization (ESI)-Orbitrap-MS/MS, seventy-six lipid species were identified spanning eight different lipid classes including glucuronosyldiacylglycerol (GlcADG), fatty acid esters of hydroxy fatty acids (FAHFA) and methylated amino acid lipids. The presence of trimethylornithine (TMO) amino acid lipids, which are unique to planctomycetes bacteria, was also confirmed in G. obscuriglobus. The variety of different amino acid lipid molecules along with phosphoethanolamine (PE) derivatives including phosphocholine (PC) is typically not observed in most bacteria including similar bacteria in the planctomycetes phylum. In addition, sterol production is a unique characteristic of G. obscuriglobus, but very little is known what functional role(s) they play. This work monitored the changes in the lipid profile between cells grown with normal sterol production and inhibited sterol production. For the detected lipids, there was not a statistically significant change in the measurements due to sterol inhibition. Although previous lipid studies have been performed, this is the first comprehensive lipidomics study of G. obscuriglobus. A different method for the analysis of biomolecules of microorganisms is matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). The use of offline HPLC-MALDI-MS/MS for bottom-up proteomics of microorganisms offers advantages in terms of cost, ease of use, and the time-decoupled nature of the separation step and the mass analysis. A method was developed to improve the capabilities of HPLC-MALDI-MS/MS in terms of protein identification in a bottom-up proteomic workflow. Enhanced protein identification was achieved by an increase in the MALDI signal intensity of the precursor peptides brought about by coating the MALDI plate with a thin film of graphite powder. Using the Escherichia coli proteome as a model microorganism, it was demonstrated that the graphite-modified MALDI plates used in an offline LC-MALDI-MS/MS bottom-up protocol led to a 50–135% increase in the number of peptide identifications, and an associated 21–105% increase in the number of proteins inferred. These improvements are achieved using a low-cost approach that is easy to implement, requires no major hardware/protocol modifications, compatible with HPLC and adds no additional analysis time. MALDI-MS is also routinely used to for differentiating/identifying bacteria down to the species level using a profile-based approach, where experimentally acquired mass spectra of bacteria are compared with those in a database. The sample preparation step in this technique is also simple, consisting of pipetting a mixture of analyte and matrix onto a metallic sample plate. However, this creates a heterogeneous crystalline morphology when dry and the inconsistences leads to variability in signal. Closely related bacteria species have subtle differences in mass spectra and high reproducibility is necessary for differentiation and/or identification. Inkjet printing has been shown to deposit liquid in an automated and controlled manner for various applications. Hence, a method was developed using a piezoelectric inkjet printer to deposit analyte/matrix in a way that reduces sample heterogeneity and increases signal reproducibility. Analysis was done using MALDI-MS and mass spectral imaging (MSI) to assess the spatial distribution and intensity of the analyte signal within a sample. The results indicated that uniform sample surfaces were observed using piezoelectric deposition in optimal conditions. Also, piezoelectric deposition of protein standards provided a reproducible signal across a sample, albeit, at a low signal intensity. But improvements in signal reproducibility were not observed for the analysis of E. coli with CHCA matrix.
