Method Development of Characterization of N-linked Glycoproteins in Mass Spectrometry

Protein glycosylation is one of the most important post-translational modifications, and it is involved in many biological processes, including inter-/intra cell signaling, protein recognition, and receptor binding, etc. It is estimated that 50-60% of cell-surface and secreted proteins are glycosylated. Alteration in the glycan structures on these proteins has been implicated in various disease states, such as, cancer, Alzheimer's disease, rheumatoid arthritis, and chronic obstructive pulmonary disease, etc. Thus, characterizing glycans and monitoring the changes of glycan profiles on proteins are essential to elucidate their biological significance and facilitate disease diagnosis. Mass spectrometry is a powerful tool for characterizing glycans on proteins, due to its high sensitivity, selectivity and small sample requirements for analysis. In order to elucidate a variety of glycan profiles (including both neutral and acidic glycans) on glycoproteins, several efficient MS-based approaches have been developed, and they are described herein. These approaches include an ion-pairing strategy in conjunction with ESI-MS/MS to identify the acidic functional groups (sulfate, and phosphate) in carbohydrates and glycopeptides; and a glycopeptide-based MS approach (liquid chromatography followed by MALDI-TOF/TOF) to characterize glycans on different glycoproteins that vary in the number of glycosylation sites and their corresponding glycan profiles. Aside from protein glycosylation, disulfide connectivity is another important modification present in proteins, and it plays a key role in establishing/maintaining protein structures in their biologically active forms. Therefore, determination of disulfide bond arrangement provides chemical structural information about proteins, and it may lead to insights into their functional roles. To achieve this goal of determining disulfide bonding patterns in proteins, a mass spectrometric approach using liquid chromatography followed by electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (LC/ESI-FTICR-MS) has been validated and used to determine the disulfide bond arrangement in an HIV envelope protein. This study contributes to the understanding of this protein's structure, and these findings are essential in understanding and improving the protein's immunogenicity

Magnetic nanoparticles containing labeling reagents for cell surface mapping

Cell surface proteins play an important role in understanding cell-cell communication, cell signaling pathways, cell division and molecular pathogenesis in various diseases. Commonly used biotinylation regents for cell surface mapping have shown some potential drawbacks such as crossing the cell membrane, difficult recovery of biotinylated proteins from streptavidin/avidin beads, interference from endogenous biotin and nonspecific nature of streptavidin. With aim to solve these problems, we introduced sulfo-N-hydroxysuccinimidyl (NHS) ester functionalized magnetic nanoparticles containing cleavable groups to label solvent exposed primary amine groups of proteins. Silica coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) were linked to NHS ester groups via a cleavable disulfide bond. Additionally, the superparamagnetic properties of Fe3O4@SiO2 MNPs facilitate efficient separation of the labeled peptides and removal of the detergent without any extra step of purification. In the last step, the disulfide bond between the labeled peptides and MNPs was cleaved to release the labeled peptides. The disulfide linked NHS ester modified Fe3O4@SiO2 MNPs were tested using a small peptide, and a model protein (bovine serum albumin) followed by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) of labeled peptides. In the next step, disulfide linked, NHS ester modified Fe3O4@SiO2 MNPs (150 nm) successfully labeled the solvent exposed cell surface peptides of Saccharomyces cerevisae. Electron microscopic analysis confirmed the cell surface binding of NHS ester modified Fe3O4@SiO2 MNPs. Mass spectrometric analysis revealed the presence of 30 unique proteins containing 56 peptides. Another MNPs based labeling reagent was developed to target solvent exposed carboxyl acid residues of peptides and proteins. The surface of Fe3O4@SiO2 MNPs was modified with free amine groups via a disulfide bond. Solvent exposed carboxyl groups of ACTH 4-11 and BSA were labeled by using1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. Upon cleaving the disulfide bond, labeled peptides were analyzed by LC-MS/MS. The MNPs containing labeling reagents offers specific labeling under physiological conditions and rapid magnetic separation of labeled peptides prior to mass spectrometric analysis. The ability of large Fe3O4@SiO2 MNPs to specifically attach to cell surface makes them a potential candidate to study the surface of variety of different cell types and complex proteins surrounded by lipid bilayer

SnTox3 Acts in Effector Triggered Susceptibility to Induce Disease on Wheat Carrying the Snn3 Gene

The necrotrophic fungus Stagonospora nodorum produces multiple proteinaceous host-selective toxins (HSTs) which act in effector triggered susceptibility. Here, we report the molecular cloning and functional characterization of the SnTox3-encoding gene, designated SnTox3, as well as the initial characterization of the SnTox3 protein. SnTox3 is a 693 bp intron-free gene with little obvious homology to other known genes. The predicted immature SnTox3 protein is 25.8 kDa in size. A 20 amino acid signal sequence as well as a possible pro sequence are predicted. Six cysteine residues are predicted to form disulfide bonds and are shown to be important for SnTox3 activity. Using heterologous expression in Pichia pastoris and transformation into an avirulent S. nodorum isolate, we show that SnTox3 encodes the SnTox3 protein and that SnTox3 interacts with the wheat susceptibility gene Snn3. In addition, the avirulent S. nodorum isolate transformed with SnTox3 was virulent on host lines expressing the Snn3 gene. SnTox3-disrupted mutants were deficient in the production of SnTox3 and avirulent on the Snn3 differential wheat line BG220. An analysis of genetic diversity revealed that SnTox3 is present in 60.1% of a worldwide collection of 923 isolates and occurs as eleven nucleotide haplotypes resulting in four amino acid haplotypes. The cloning of SnTox3 provides a fundamental tool for the investigation of the S. nodorum-wheat interaction, as well as vital information for the general characterization of necrotroph-plant interactions.This work was supported by USDA-ARS CRIS projects 5442-22000-043-00D and 5442-22000-030-00D

Dramatic expansion of the black widow toxin arsenal uncovered by multi-tissue transcriptomics and venom proteomics.

BackgroundAnimal venoms attract enormous interest given their potential for pharmacological discovery and understanding the evolution of natural chemistries. Next-generation transcriptomics and proteomics provide unparalleled, but underexploited, capabilities for venom characterization. We combined multi-tissue RNA-Seq with mass spectrometry and bioinformatic analyses to determine venom gland specific transcripts and venom proteins from the Western black widow spider (Latrodectus hesperus) and investigated their evolution.ResultsWe estimated expression of 97,217 L. hesperus transcripts in venom glands relative to silk and cephalothorax tissues. We identified 695 venom gland specific transcripts (VSTs), many of which BLAST and GO term analyses indicate may function as toxins or their delivery agents. ~38% of VSTs had BLAST hits, including latrotoxins, inhibitor cystine knot toxins, CRISPs, hyaluronidases, chitinase, and proteases, and 59% of VSTs had predicted protein domains. Latrotoxins are venom toxins that cause massive neurotransmitter release from vertebrate or invertebrate neurons. We discovered ≥ 20 divergent latrotoxin paralogs expressed in L. hesperus venom glands, significantly increasing this biomedically important family. Mass spectrometry of L. hesperus venom identified 49 proteins from VSTs, 24 of which BLAST to toxins. Phylogenetic analyses showed venom gland specific gene family expansions and shifts in tissue expression.ConclusionsQuantitative expression analyses comparing multiple tissues are necessary to identify venom gland specific transcripts. We present a black widow venom specific exome that uncovers a trove of diverse toxins and associated proteins, suggesting a dynamic evolutionary history. This justifies a reevaluation of the functional activities of black widow venom in light of its emerging complexity

MS/MS ANALYSIS OF IgG3 DISULFIDE BONDS AND DEVELOPMENT OF A NOVEL TOOL TO ASSESS ALGORITHMS THAT ASSIGN GLYCOPEPTIDE CID DATA

With the rapidly increasing use of proteins as biotherapeutics to treat diseases, the characterization of these large molecules using mass spectrometry has become a highly attractive field of research. A particular area of research is the identification and characterization of protein post-translational modifications. Disulfide bonds and glycosylation are among the most critical protein post-translational modifications (PTMs), as they play vital roles in maintaining the proper protein folding, structure, and functions. These two PTMs are particularly important in the development and characterization of monoclonal antibody-based drugs, which are the most prevalent protein therapeutics in the market. Among the four classes of immunoglobulins (IgG’s), the disulfide connectivity of IgG1, IgG2 and IgG4 have been effectively studied, and IgG2 and IgG4 have been shown to have disulfide bond-mediated isomers due to alternative disulfide bond connectivity. However, no studies to investigate the presence of disulfide related isomers in IgG3 have been done. In this dissertation, high resolution mass spectrometry is used map the disulfide bond connectivity in IgG3 in order to investigate the presence of disulfide-mediated isomers. The results indicate that no such isomers exist for endogenous IgG3 antibodies. The development of a novel glycoproteomics software, Glycopep Decoy Generator (Tool 1), and the generation of a large dataset of manually assigned CID spectra (Tool 2) from diverse glycopeptide compositions also are described herein. The decoy generator generates abundant decoys for any target glycopeptide composition, and when it is used along with the dataset of CID spectra, the accuracy of glycopeptide scoring algorithms can be readily determined. The tools were used to assess GlycoPep Grader, a scoring algorithm that assigns glycopeptides to CID spectra. The results indicate that GlycoPep Grader has some weaknesses in scoring spectra from fucosylated glycopeptide compositions. These weaknesses could not be easily identified without the aforementioned tools. In order to address GlycoPep Grader’s limitations, a thorough investigation of the root cause of its weaknesses is carried out, and potential updates that could improve the software are proposed

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

Mass spectrometry-based methods for identifying oxidized proteins in disease:advances and challenges

Many inflammatory diseases have an oxidative aetiology, which leads to oxidative damage to biomolecules, including proteins. It is now increasingly recognized that oxidative post-translational modifications (oxPTMs) of proteins affect cell signalling and behaviour, and can contribute to pathology. Moreover, oxidized proteins have potential as biomarkers for inflammatory diseases. Although many assays for generic protein oxidation and breakdown products of protein oxidation are available, only advanced tandem mass spectrometry approaches have the power to localize specific oxPTMs in identified proteins. While much work has been carried out using untargeted or discovery mass spectrometry approaches, identification of oxPTMs in disease has benefitted from the development of sophisticated targeted or semi-targeted scanning routines, combined with chemical labeling and enrichment approaches. Nevertheless, many potential pitfalls exist which can result in incorrect identifications. This review explains the limitations, advantages and challenges of all of these approaches to detecting oxidatively modified proteins, and provides an update on recent literature in which they have been used to detect and quantify protein oxidation in disease