MS/MS Analysis and Automated Tool Development for Protein Post-Translational Modifications

Protein post-translational modifications (PTMs) are important for a variety of reasons. PTMs confer the final protein product and biological functionality onto a nascent protein chain. Two common PTMs are glycosylation and disulfide bond formation. Both glycosylation and disulfide bond formation contribute to a variety of biological processes, including protein folding and stabilization. Mass spectrometry (MS) has shown to be an essential technique to study PTMs, especially when tandem mass spectrometry (MS/MS) experiments are performed. In the characterization of PTMs using MS/MS, different fragmentation techniques are often used. Regardless of the dissociation method that is employed, MS/MS data interpretation is a tedious and lengthy process. To render this analysis more efficient, the use of automated tools is necessary. In this work, collision induced dissociation (CID) MS/MS experiments were carried out in order to create a set of fragmentation rules applicable to any N-linked glycopeptide. These rules were then used to develop an algorithm to power publicly available software that accurately determines glycopeptide composition from MS/MS data. This program greatly reduces the time it takes researchers to manually assign the identity of an N-linked glycopeptide to an acquired CID spectrum. In addition, electron transfer dissociation (ETD) experiments were performed in order to devise a computational approach that works to determine precursor charge state directly from MS/MS data of peptides containing disulfide bonds. Lastly, alternate fragmentation patterns found to be detected in glycopeptides containing labile monosaccharide residues such as sialic acid are discussed. These patterns, along with other trends noticed after extensive analysis of N-linked glycopeptide CID data, were then used to propose future updates to the GPG analysis tool

Applications of ion mobility spectrometry, collision-induced dissociation and electron activated dissociation tandem mass spectrometry to structural analysis of proteins, glycoproteins and glycans

This dissertation mainly focuses on analytical method development for characterization of proteins, glycoproteins and glycans using the recently developed ion mobility spectrometry (IMS) techniques and various electron activated dissociation (ExD) tandem mass spectrometry methods. IMS and ExD have become important techniques in structure analysis of biomolecules. IMS is a gas-phase separation method orthogonal to liquid chromatography (LC) fractionation. ExD is capable of producing a large number of structurally informative fragment ions for elucidation of structural details, complementary to collision-induced dissociation (CID). We first applied the selected accumulation-trapped IMS (SA-TIMS)-electronic excitation dissociation (EED) method to analyze various mixtures of glycan isomers. Glycan linkage isomers with linear or branched structure were successfully separated and subsequently identified. Theoretical modeling was also performed to gain a better understanding of isomer separation. The calculated collisional cross section (CCS) values match well with the experimentally measured ones, and suggested that the choice of metal charge carrier and charge state is critical for successful IMS separation of isomeric glycans. In addition, a SA-TIMS-electron capture dissociation (ECD) approach was employed to study gas-phase protein conformation, as the ECD fragmentation pattern is influenced by both the charge distribution and the presence of various non-covalent interactions. We demonstrated that different conformations of protein ions in a single charge state could produce distinct fragmentation pattern, presumably because of their differences in tertiary structures and/or proton locations. The second part describes characterization of glycoproteins using LC-hot ECD. To improve the cleavage coverage of glycopeptides, hot ECD, a fragmentation method utilizing the irradiation of high-energy electrons, was optimized for both middle-down and bottom-up analyses of glycopeptides, including peptides with multiple glycosylation sites. Hot ECD was shown to be an effective fragmentation technique for sequencing of glycopeptides, even for ions in lower charge states. In addition, the online LC-hot ECD approach was applied to characterize extensively modified glycoproteins from biological sources in which all glycosylation sites could be unambiguously determined. This study expands the applications of IMS, CID and ExD to structural analysis of various biomolecules, and explores the analytical potential of combining them for investigation of complex biological systems, in particular, enzyme mechanisms

Electron Capture Dissociation Mass Spectrometry of Metallo-Supramolecular Complexes

The electron capture dissociation (ECD) of metallo-supramolecular dinuclear triple-stranded helicate Fe2L3 4 ions was determined by Fourier transform ion cyclotron resonance mass spectrometry. Initial electron capture by the di-iron(II) triple helicate ions produces dinuclear double-stranded complexes analogous to those seen in solution with the monocationic metal centers CuI or AgI. The gas-phase fragmentation behavior [ECD, collision-induced dissociation (CID), and infrared multiphoton dissociation (IRMPD)] of the di-iron double-stranded complexes, (i.e., MS3 of the ECD product) was compared with the ECD, CID, and IRMPD of the CuI and AgI complexes generated from solution. The results suggest that iron-bound dimers may be of the formFeI 2L2 2 and that ECD by metallo-complexes allows access, in the gas phase,to oxidation states and coordination chemistry that cannot be accessed in solution

Charge Transfer Dissociation Mass Spectrometry of Biomolecules

Recent advances in many biological disciplines are closely related to the development and application of new mass spectrometry techniques. The investigation of gas-phase ion activation techniques is one of the active research fields. Although researchers have developed a variety of ion activation techniques, they all suffer from certain intrinsic limitations---either limited in the types of fragment ions, or limited by the inefficiency with low charge-state precursor ions. Most of the ion activation techniques are not commercially available, and are at their developing stages.;As an attempt to explore new possibilities of fragmenting a gas-phase ion, charge transfer dissociation (CTD) was developed by the Jackson group. CTD is not only workable with low charge state precursor ions (1+ and 2+), but also is workable with highly charged precursor ions (4+, 5+, and 6+). For peptide analysis, CTD produces extensive backbone fragment ions, including a/x, b/y, and c/z ions. Additionally, CTD generates characteristic amino acid side-chain losses, which can complement the sequence information from backbone fragments. An interesting phenomenon of CTD reaction is that the type of predominant fragment ions shifts from a/x to c/z as the precursor charge state increases from 1+ to 3+, or more.;For intact insulin analysis, CTD enables the oxidation through a one-electron (dominant) or two-electron (minor) oxidation pathway, which increased the charge state of the intact protein by 1 or 2, respectively. Direct CTD produces a few fragment ions outside the loop defined by disulfide linkages together with charge-increased/charge-decreased species. The MS3-level CID fragmentation of the charge-increased species shows the capability of breaking disulfide linkages, thus provides enhanced structural information. Making use of the ability of being workable with 1+ precursor ions, CTD was employed to fragment phospholipids with various degree of unsaturation. CTD extensively fragments the C-C single bonds within lipid acyl chain, and provides information regarding C=C double bond location. For lipids with various head groups, CTD shows the capability of fragmenting the acyl chains to some extent, but the efficiencies are not suitable for on-line HPLC experiments at this time. CTD was also applied to structural characterization of a methylated linear oligosaccharide, generating both extensive between-ring and cross-ring cleavages. Given the similarity in radical nature between CTD and ETD, CTD was integrated into a HDX workflow to probe the gas-phase conformation of ubiquitin. CTD shows comparable performance to ETD, which demonstrated the potential of CTD in pinpointing the secondary structural elements of gas-phase proteins/peptides.;In addition to the exploration to CTD technique, some efforts were devoted to examine the fragmentation behavior of radical cations generated from metastable atom-activated dissociation (MAD) reactions. ESI-generated protonated, sodiated and potassiated POPC ions were firstly subjected to MAD reactions, and then the resulting [POPC]+• ions were further mass-selected and subjected to MS3-level CID reactions respectively. The resulting mass spectra are almost identical---independent of the first generation adducting species. Moreover, the MS3 CID experiment produced extensive fragmentation along the lipid acyl chain, providing valuable structural information associated with C=C double bond positioning

Expanding the Applications of Ion Mobility Spectrometry and Mass Spectrometry in Integrative \u27Omics Analyses

Over the past few decades, biomolecular analyses ranging from the study of complex mixtures to protein structural interrogation have increased significantly. These studies range from small molecule separations[1, 2] to observing structural trends in large proteins and protein sub-complexes.[3, 4] Traditionally, the use of liquid chromatography mass spectrometry (LC-MS), electrophoresis and nuclear magnetic resonance (NMR) spectroscopy have been at the forefront of these respective studies. Because complex mixtures can contain a variety of components over a wide dynamic range and proteins and their complexes can contain a diverse array of structures, few analytical techniques are capable of providing information across all experimental areas (e.g. small molecule mixtures to large individual proteins). In contrast, the use of Ion Mobility Spectrometry-Mass Spectrometry (IMS-MS) has emerged as a powerful tool for measuring ion(s) structural heterogeneity. While IMS-MS is a relatively newer method, workflows are becoming more common as the commercialization of IMS instruments has created a larger user base. Such workflows now include metabolomic,[1, 5, 6] lipidomic,[7] proteomics and protein structural analyses[8, 9]. Taken collectively, these areas encompass the field of \u27omics\u27 analysis. While each field has its respective difficulties, IMS-MS is well poised to enhance and even expand the repertoire of analytical platforms for omics analyses.;Much of the current bottlenecks in traditional techniques suffer from an inability to sample measureable species rapidly in a reproducible manner over a wide dynamic range. For example, Anderson and coworkers have proposed that the plasma proteome includes 106--107 species that span a concentration range of 1011.[10] In many cases, IMS has shown improved resolution of isomeric species compared to either LC or Gas Chromatography (GC) analyses.[11, 12] The utility of IMS-MS in profiling is largely attributed to its rapid ability to resolve low-abundance species from spectral regions containing high-abundance species, thereby increasing measurement sensitivity, dynamic range and peak capacity.[13--17] Additionally, IMS is capable of separating isobaric species that cannot be resolved by MS alone. In \u27omic profiling directed toward biomarker discovery, it is imperative to identify compounds of interest. The identification is complicated by compound diversity (class and structural variation).;Traditionally, as well as all commercially available, IMS-MS instruments use Time-of-Flight (ToF) mass analyzers for determining an ion\u27s mass-to-charge ratio (m/z). The obvious advantage is the ability to nest the m/z measurement (micros) within the drift measurement (ms). This creates an orthogonal separation where many m/z measurements are made during the drift separation. Although this combination creates a rapid, multidimensional analysis, ToF mass analyzers are not capable of multistage tandem mass spectrometry (MSn) or nonergodic dissociation methods such as electron transfer dissociation (ETD). These MS fragmentation methods are often used as standalone techniques in applications ranging from small molecule identification within complex mixtures to identifying high order structure in proteins using Hydrogen Deuterium exchange (HDX) MS. To this end, new applications of IMS-MS that leverage the use of ion trapping MS are useful for supplementing these limitations of ToF analyzers. Trapping mass analyzers add the capability to perform ion-neutral or ion-ion reactions on drift-selected ions. In such experiments, fragment ions are generated and are structurally useful in identifying and quantifying individual components or those that compose protein structures or post translational modifications (PTMs). To date, very few, if any, experiments have attempted to combine the unique capabilities of IMS-MS with MSn or ETD-MS for uncovering ion structural information or heterogeneity. As will be shown in the coming chapters, coupling IMS to trapping mass analyzers expands the capabilities into new areas of \u27omics analysis and enhances the information that can be obtained from either technique alone

Degrees of freedom effect on fragmentation in tandem mass spectrometry of singly charged supramolecular aggregates of sodium sulfonates

The characteristic collision energy (CCE) to obtain 50% fragmentation of positively and negatively single charged non-covalent clusters has been measured. CCE was found to increase linearly with the degrees of freedom (DoF) of the precursor ion, analogously to that observed for synthetic polymers. This suggests that fragmentation behavior (e.g. energy randomization) in covalent molecules and clusters are similar. Analysis of the slope of CCE with molecular size (DoF) indicates that activation energy of fragmentation of these clusters (loss of a monomer unit) is similar to that of the lowest energy fragmentation of protonated leucine-enkephalin. Positively and negatively charged aggregates behave similarly, but the slope of the CCE vs DoF plot is steeper for positive ions, suggesting that these are more stable than their negative counterparts