Mass Spectrometric Analysis of Human 40 S Ribosomal Proteins – Infectious Diseases
Many viruses use a highly structured RNA sequence, internal ribosome entry site (IRES), to initiate protein synthesis. We are interested in a comprehensive mapping of the all the proteins and the associated protein factors of the IRES bound 40S ribosome using both of the top-down and bottom-up mass spectrometry approaches, which will then facilitate the understanding of the IRES ribosome recruitment mechanism.
Figure 1. RP-HPLC separation of 40S ribosomal proteins
Figure 2. Identification of RS3 by top-down approach
After fractionation of native 40S ribosome subunit by reverse phase HPLC (Fig. 1), human 40S ribosomal proteins (XX phenotype) were identified using the top down approach (Fig. 2). All of the 40S ribosomal proteins identified by the top down approach were found to contain various posttranslational modifications, including loss of methionine, N-terminal acetylation, methylation and disulfide bonds. Using the bottom-up approach, we identified all but one 40S ribosomal proteins.
Figure 3. Pull-down experiment was used to extract the IRES bound human 40S ribosome subunit.
For the IRES pull-down 40S ribosome subunit (Fig. 3), nucleolin and various eIF3 subunits were found, which were not present in native 40S subunit and thus may be involved in IRES mediated translation. Experiments are ongoing to identify various post-translational modifications.
Enzyme Mechanisms and Kinetics
We are using ESI-FTICR MS to investigate protein-ligand noncovalent complexes. Finely controlled instrument conditions allow these ions to be transferred into the gas phase without dissociation. Proteins are mixed with their ligands in solution and the intact noncovalent complexes can be observed in the gas phase for protein-substrate, protein-inhibitor and intact multimeric protein complexes. Moreover, once these desolvated molecules are in the gas phase, important information about these complexes such as molecular weight, binding stoichiometry and relative binding strength can be determined using mass spectrometry. In particular, we are interested in the NodH sulfotransferase (NodST) from Rhizobium meliloti, which belongs to the GlcNAc6OST carbohydrate sulfotransferase family. NodST complexed with its substrate, PAPS and inhibitor, PAP were observed in the mass spectrum. Relative binding constants determined from the gas phase are close to those determined from solution. In addition, peptide mapping was used to identify the covalent sulfate enzyme intermediate formed via the random hybrid ping-pong mechanism for this enzyme.
Research is ongoing which involves using our newly developed screening assay (IEMS assay) for the analysis and identification of combinatorial libraries of inhibitors before and after incubation with immobilized enzyme. Mass spectrometric analysis of a combinatorial library of compounds which were both active and inactive towards various sulfotransferases indicates that we can identify the enzyme inhibitor compounds when eluted through a column of immobilized enzyme. Our methodology shows that inhibition is evidenced by a depletion or complete absence of ion signal in the mass spectrum obtained after reaction with the enzyme.
A variety of immobilized enzymes with combinatorial libraries exceeding 250 compounds/enzyme are currently being screened. Different immobilization techniques including reductive amination on agarose gel, metal chelation, etc, are being investigated. In general these procedures involve formation of an isourea, a diazo linkage, a peptide bond, or an alkylation reaction. Previous work in our lab has focused on immobilizing enzymes on agarose and our results indicate that approximately 90% of the activity for each enzyme studied is retained after immobilization. The unique and advantageous facets of analyzing for inhibition using our proposed method is that very small quantities of inhibitor (low pmol) can be screened in a relatively short time frame with a large number of potential inhibitors.
A new method has been developed that allows for the determination of kinetic parameters, such as Km, Vmax, and kcat, for various enzymatic systems. The mass spectrometric method allows for study of enzymatic reactions that do not involve chromophoric changes. Using this method, the kinetic constants have been measured for a series of sulfotransferases and phosphotransferases. This novel mass spectrometric technique has been further adapted to determine the inhibition pattern of enzyme inhibitors, measure their inhibition constants, and investigate the catalytic mechanism of bi-substrate enzymatic systems. A hybrid random Ping-Pong mechanism has been determined for NodH sulfotransferase using the ESI-MS kinetic assay, which was confirmed by the sulfated enzyme intermediate identification using ESI-FTICR-MS.
We have also developed a novel strategy for rapid determination of enzyme substrate specificity using one reaction system containing multiple competing substrates. In this multiplex ESI-MS assay method, the electrospray ionization mass spectrometry (ESI-MS) technique was used for simultaneous quantification of multiple products and a steady-state kinetics model was established for efficient specificity constant calculation. In our lab, the reaction specificity of NodST for four chitooligosaccharide acceptor substrates of different chain length (chitobiose, chitotriose, chitotetraose, and chitopentaose) was determined by both individual kinetic measurements and the new multiplex ESI-MS assay. The results obtained from the two methods were compared and consistency was found. The multiplex ESI-MS assay is an accurate and valid method for substrate specificity evaluation, in which multiplex substrates can be evaluated in one assay.
Using a novel ion/molecule reaction strategy, a mutase system whose substrate and product are positional isomers (as shown in the figure below) has also been successfully studied. The mass spectrometry results are identical to those obtained using traditional UV assays and are more accurate and precise.
- Immobilize low concentrations of different classes of enzymes with high efficiency and with retention of enzyme activity for the purpose of screening large numbers of inhibitor libraries (ligands) using the IEMS method.
- Identify possible inhibitors showing at least 40% inhibition and apply MS and MS/MS as needed to ensure correct combinatorial synthesis and identify synthetic byproducts.
- Calculate Km, Vmax, and kcat as needed, of both immobilized and soluble enzymes using our MS method.
- Determine if inhibition is competitive or non-competitive.
- Calculate Kiís of all possible inhibitors.
- Obtain mechanistic information of therapeutic interesting enzymes to direct combinatorial inhibitor synthesis and drug design.
- Perform efficient substrate specificity study of interesting enzymes using multiplex ESI-MS assay, in order to further understand the enzyme’s catalytic function, which will also contribute to inhibitor design and library synthesis.
- Investigate the enzyme/substrate and enzyme/inhibitor complexes by mass spectrometry.
Structural Characterization of Lipoarabinomannans and Arabinogalactans from Mycobacterium tuberculosis and Mycobacterium smegmatis
Mycobacterium tuberculosis
Mycobacterium tuberculosis, the intracellular pathogen causative agent of pulmonary tuberculosis, is the leading infectious cause of death in the world.1 Approximately 2 billion people are infected with Mycobacterium tuberculosis making it a serious worldwide problem in which ~ 8 million new active cases and 2-3 million deaths occur every year. Following decades of decline of occurrence, in the last 20 years tuberculosis has seen resurgence in industrialized countries. This resurgence can be directly related to the persistence of drug-resistant strains as well as to the human immunodeficiency virus (HIV). Consequently, the factors that lead to virulence of tuberculosis are of great interest. Yet, despite considerable effort towards understanding pathogenic species, the mechanisms by which they avoid destruction and flourish in cells are not well defined.
The
unique cell wall of M. tuberculosis forms a waxy envelope and has been established as an important factor leading to bacterial virulence and survival. As a consequence, effective antibiotics often target the biosynthetic pathways that lead to formation of components of the cell wall. Two of the principle components of the cell wall of mycobacteria are the lipoarabinomannans (LAM) and mycolyl arabinogalactans (AG). LAM and AGs are complex lipopolysaccharides that are characterized by a polysaccharide backbone that consists of a mannan or galactan backbone, respectively, and branched arabinan chains. While much is known concerning the general composition of LAM and AGs from various mycobacteria there is a great need to correlate their structure with their biological functions. Furthermore, it is becoming increasing apparent that subtle differences in structure may shape the host reponse to virulent and avirulent species.
The Leary lab is interested in developing analytical methods to define the structures of LAM and AGs and subsequently correlating the structures with their function. Currently, we utilize Fourier-transform ion cyclotron resonance (FT-ICR), MALDI-time-of-flight, and quadrupole ion trap mass spectrometry, coupled with multiple stages of collision-induced dissociation to analyze the structure of lipoarabinomannans isolated from M. smegmatis and M. tuberculosis. Utilizing as little as 10 ug of LAM, mild acid hydrolysis experiments and enzymatic digestion are used to facilitate MS characterization of small segments of the LAM. FT-ICR mass spectrometry offers high resolution and mass accuracy to unambiguously identify the molecular formula of analytes. Structural characterization of LAM and AG is achieved in a QIT mass spectrometer by collision-induced dissociation (CID) experiments. CID of lithiated arabinose trimer standards yields ions that could be used to differentiate between arabinose trimers that are a(1-›2), a(1-›5) and a(1-›5), a(1-›5) linked. We are actively utilizing these analytical methods to probe the structure of LAM and AG from mycobacteria grown under a variety of environmental conditions and in the presence of antibiotics.
Structural characterization of glycosaminoglycans using mass spectrometry
The
cellular surface is copiously covered with carbohydrates modifying proteins and lipids in the plasma membrane. These oligosaccharides mediate a variety of functions in the body through modulation of interactions between cells and with extracellular matrix components. However, the role of oligosaccharide structure as regards function has been minimally studied due to the complexity of these biomolecules and the limitations of the analytical methods used to study them. One of our objectives, in the Leary lab, is to develop methods for carbohydrate structural elucidation using mass spectrometry.
Heparin
and heparan sulfate glycosaminoglycans (GAGs), which are involved in the regulation of many patho(physiological) processes, consist of a variably sulfated repeating disaccharide unit. In fact, the high degree of sulfation of heparin gives it the highest charge density of any known biological macromolecule, further complicating its analysis. Traditional methods for carbohydrate sequencing rely on using exoglycosidases for removal of non-reducing terminal monosaccharides one at a time, in conjunction with fluorescent labeling and high resolution polyacrylamide gel electrophoresis or LC/CE separation, which can be quite laborious and time-consuming. More recently, mass spectrometry, and specifically MSn is becoming an established tool for the analysis of carbohydrates, such as these GAGs.
MSn analysis of HS7-3, isolated from heparinase III digestion of HS. [a] MS2 430.13-?, [c] Structure of HS7-3 with major product ions indicated.
Our approach to the sequencing of heparin/HS oligosaccharides utilizes a combination of MSn analysis of the saccharide, and disaccharide composition analysis using an established MS method after complete enzymatic degradation by a mixture of heparinases.1 For MSn analysis, isolation and activation of the molecular ion with a high charge state, yields a spectrum consisting of product ions generated mainly through various glycosidic and cross-ring cleavages, with minimal loss of sulfate. The collision-induced dissociation of the oligosaccharide produces a series of B- and Y-ions, as well as distinctive 0,2A- and X-ions from the reducing and non-reducing ends, respectively.
With the help of HOST (Heparin Oligosaccharide Sequencing Tool), a new EXCEL-based software application in development, the data generated from both MS experiments can be integrated and analyzed to provide sequence information in much the same way that peptide mapping is currently done from MS/MS data. This methodology is currently being applied to various heparin oligosaccharides isolated from partial enzymatic digests of heparin/HS, as well as to heparin fragments that are found to be ligands that bind to and influence the function of different protein targets.
