AN ABSTRACT OF THE DISSERTATION OF Mohamed Zaneer Segu Mohideen, for the Doctor of Philosophy degree in Chemistry, presented on November 3 2008, at Southern Illinois University Carbondale. TITLE: TARGET MODIFICATION FOR ENHANCED PERFORMANCE MATRIX ASSISTED LASER DESORPTION IONIZATION (MALDI) MASS SPECTROMETRY MAJOR PROFESSOR: Dr. Gary R Kinsel MALDI MS, a powerful tool for the analysis of biomolecules, has undergone major advancement in instrumentation to yield improvements in robustness, sensitivity and throughput since its invention. Despite these developments in instrumentation, the performance of MALDI is in question when it comes to the analysis of complex protein/peptide mixtures. For these types of mixtures the performance of MALDI can be improved by either simplifying the sample complexity, modifying the sample preparation approach to increase the ionization efficiency of mixture components or seeking further enhancements to instrument performance. In this work these improvements are pursued through modifications to the MALDI target itself. In the MALDI analysis of high MW proteins a primary limitation is thought to be related to inefficient desorption of these compounds as proteins are expected to experience relatively stronger interaction with the MALDI target surface. This insight led to investigations of the use of various sublayers, deposited directly on the MALDI target, as a means to improve high molecular weight protein MALDI ion signals. In the first approach the protein / matrix mixture is applied on a laser desorbable polyaromatic hydrocarbon layer which serves as a barrier to protein surface binding interactions. These sublayers are also shown to be useful for on probe sample purification from salts that are known to interfere with MALDI performance. In the second approach the sublayer is formed from bovine serum albumin, a protein that is known to have strong binding affinity for surfaces and is also expected to form a barrier to protein surface binding interactions. Enhancements in MALDI performance and reductions in the limit of detection for proteins on these albumin precoated probes clearly demonstrate the influence of surface-protein interaction in the analysis of these species by MALDI MS. In further studies, methods to improve on-MALDI-target approaches to the simplification of sample complexity are investigated. These on-target separation approaches have been previously developed and shown to be successful for reducing sample complexity in the Kinsel Research Group. However, one significant limitation to this separation approach is the limited surface binding capacity of the MALDI probe. This limitation led to theoretical and experimental studies of methods to improve the surface protein binding capacity. Studies performed show that the surface binding capacity can be improved significantly through attachment of gold beads and through physical / chemical roughening of the target surface. Both approaches are shown to yield higher performance MALDI probes with lowered limits of detection for deposited / affinity captured proteins.

abstract: Protein-surface interactions, no matter structured or unstructured, are important in both biological and man-made systems. Unstructured interactions are more difficult to study with conventional techniques due to the lack of a specific binding structure. In this dissertation, a novel approach is employed to study the unstructured interactions between proteins and heterogonous surfaces, by looking at a large number of different binding partners at surfaces and using the binding information to understand the chemistry of binding. In this regard, surface-bound peptide arrays are used as a model for the study. Specifically, in Chapter 2, the effects of charge, hydrophobicity and length of surface-bound peptides on binding affinity for specific globular proteins (&beta-galactosidase and &alpha1-antitrypsin) and relative binding of different proteins were examined with LC Sciences peptide array platform. While the general charge and hydrophobicity of the peptides are certainly important, more surprising is that &beta-galactosidase affinity for the surface does not simply increase with the length of the peptide. Another interesting observation that leads to the next part of the study is that even very short surface-bound peptides can have both strong and selective interactions with proteins. Hence, in Chapter 3, selected tetrapeptide sequences with known binding characteristics to &beta-galactosidase are used as building blocks to create longer sequences to see if the binding function can be added together. The conclusion is that while adding two component sequences together can either greatly increase or decrease overall binding and specificity, the contribution to the binding affinity and specificity of the individual binding components is strongly dependent on their position in the peptide. Finally, in Chapter 4, another array platform is utilized to overcome the limitations associated with LC Sciences. It is found that effects of peptide sequence properties on IgG binding with HealthTell array are quiet similar to what was observed with &beta-galactosidase on LC Science array surface. In summary, the approach presented in this dissertation can provide binding information for both structured and unstructured interactions taking place at complex surfaces and has the potential to help develop surfaces covered with specific short peptide sequences with relatively specific protein interaction profiles. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2014