MS Imaging for Small Molecule, Peptide and Protein Detection in Multilayers and Bacterial Biofilms
thesisposted on 22.02.2015, 00:00 authored by Melvin B. Muttikal Thomas
A universal attribute of bacterial cells is their ability to form architecturally complex communities called biofilms, often associated with solid surfaces and typically enclosed in an extracellular polysaccharide matrix, with cells found in all possible metabolic states. This property of biofilms often results in the phenomenon of biofilm resistant to drugs and antimicrobials. Thus the study of biofilms has often tried to understand this behavior of its resistance to elimination by treatment with a variety of antimicrobial agents. When microorganisms from a biofilm are dispersed, their antimicrobial susceptibility and other properties associated with planktonic cells are usually rapidly restored. Thus there is a need for effective analytical techniques to probe samples directly from intact biofilms to understand them better. The ability of mass spectrometry (MS) imaging techniques based on secondary ion mass spectrometry (SIMS), matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser desorption postionization mass spectrometry (LDPI-MS) to probe samples directly from intact surfaces was exploited here to study chemical distributions within biofilms. A polyelectrolyte multilayer (PEM) composed of high molecular weight polysaccharides chitosan and alginate representing an ideal model system that simulates the extracellular polysaccharide matrix of biofilms was used to demonstrate depth profiling strategies using C60+ ion sputtering in conjunction with X-ray photoelectron spectroscopy. Next a comparison of SIMS and LDPI-MS protocols to probe a small molecular analyte in this PEM model was made to highlight the features of each technique with respect to their potential application in biofilm analysis. The feasibility of imaging proteomics on intact Enterococcus faecalis bacterial biofilms by MALDI-MS was also demonstrated with minimum sample preparation identifying thirteen different cytosolic and membrane proteins and spatially locating them within intact E. faecalis biofilms by MALDI-MS imaging. Finally the potential of LDPI-MS imaging for small molecule quantification was also demonstrated, indicating that 7.87 eV LDPI-MS imaging should be applicable to quantify a range of small molecular species on a variety of complex organic and biological surfaces.