Jonathan L. Bundy

Automated Proteomics of E. coli via Top-Down Electron-Transfer Dissociation Mass Spectrometry

Electron-transfer dissociation (ETD) has recently been introduced as a fragmentation method for peptide and protein analysis. Unlike collisionally induced dissociation (CID), fragmentation by ETD occurs randomly along the peptide backbone. With the use of the sequences determined from the protein termini and the parent protein mass, intact proteins can be unambiguously identified. Because of the fast kinetics of these reactions, top-down proteomics can be performed using ETD in a linear ion trap mass spectrometer on a chromatographic time scale. Here we demonstrate the utility of ETD in high-throughput top-down proteomics using soluble extracts of E. coli. Development of a multidimensional fractionation platform, as well as a custom algorithm and scoring scheme specifically designed for this type of data, is described. The analysis resulted in the robust identification of 322 different protein forms representing 174 proteins, comprising one of the most comprehensive data sets assembled on intact proteins to date.

Ion/ion chemistry as a top-down approach for protein analysis.

Developing methodology for analyzing complex protein mixtures in a rapid fashion is one of the most challenging problems facing analytical biochemists today. Recent advances in mass spectrometry for the analysis of intact proteins (i.e. the top-down approach) show great promise for rapid protein identification. The ion/ion chemistry approach for the detection and identification of target proteins in complex matrices, determination of fragmentation channels as a function of precursor ion charge state, and post-translational modification characterization are discussed with particular emphasis on tandem mass spectrometry of intact proteins.

Proteomic analysis reveals virus-specific Hsp25 modulation in cardiac myocytes

Viruses frequently infect the heart but clinical myocarditis is rare, suggesting that the cardiac antiviral response is uniquely effective. Indeed, the Type I interferon (IFN) response is cardiac cell-type specific and provides one integrated network of protection for the heart. Here, a proteomic approach was used to identify additional proteins that may be involved in the cardiac antiviral response. Reovirus-induced murine myocarditis reflects direct viral damage to cardiac cells and offers an excellent system for study. Primary cultures of murine cardiac myocytes were infected with myocarditic or nonmyocarditic reovirus strains, and whole cell lysates were compared by two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF/TOF) tandem mass spectrometry. Results were quantitative and reproducible and demonstrated that whole proteome changes clustered according to viral pathogenic phenotype. Moreover, the data suggest that the heat shock protein Hsp25 is modulated differentially by myocarditic and nonmyocarditic reoviruses and may play a role in the cardiac antiviral response. Members of seven virus families modulate Hsp25 or Hsp27 expression in a variety of cell types, suggesting that Hsp25 participation in the antiviral response may be widespread. However, results here provide the first evidence for a virus-induced decrease in Hsp25/27 and suggest that viruses may have evolved a mechanism to subvert this protective response, as they have for IFN.

Comprehensive defensin assay for saliva.

Defensins are highly basic cationic peptides that are important components of the innate and adaptive immune response pathways. In addition, these peptides are involved in CD8+ T cell response to HIV-1, increased pulmonary infection risk among cystic fibrosis patients, upregulated levels of HNP-5 for patients with ulcerative colitis and Crohns disease, and monitoring HNP-3 levels as a tumor classification scheme for cutaneous T cell lymphomas, and have promise in the pharmaceutical field as a new class of antibiotics. Here we present a parallel assay for the alpha (HNP1-3) and beta (HBD1-2) classes of defensins in saliva that are naturally observed in the concentration range of 1 ng/mL to 10 microg/mL. The method utilizes solid phase extraction of saliva samples combined with liquid chromatography-tandem mass spectrometry to identify and quantitate defensin targets. The approach involves limited sample manipulation and is easily amenable to automation. The saliva samples analyzed are derived from a large cohort study focused on examining the role of polymorphisms in genes of innate and adaptive immunity in modulating the response to vaccination for two gastrointestinal tract infections: typhoid and cholera. The alpha-defensin levels observed range from 1 to 10 microg/mL and correlate well with known active concentrations against a wide variety of pathogens. The observed concentration range for beta-defensins was between the detection limit and 33 ng/mL and had a sensitivity level that was comparable to immunoassay-based detection. This method is easily adapted for use in a clinical immunology setting and can be modified for other biological matrixes. This assay will facilitate examination of the production, secretion, and regulation of defensin peptides in a direct fashion to coordinate levels of these compounds with gender, age, response to vaccination, gene copy number, and oral health.

Proteomics of Pyrococcus furiosus, a hyperthermophilic archaeon refractory to traditional methods.

Pyrococcus furiosus is one of the most extensively studied hyperthermophilic archaea. Proteins from this hyperthemophile organism are extremely thermostable and are highly resistant to chemical denaturants, organic solvents and proteolytic digestion. This thermostability makes it difficult to apply traditional methods of enzymatically digesting a complex mixture of proteins, commonly a first step in peptide generation in most shotgun proteomics methods. Here, we have developed a simple shotgun proteomics approach for the global identification of the P. furiosus proteome. This methodology uses a detergent-based microwave assisted acid hydrolysis (MAAH) step coupled with an overnight trypsin digest to obtain peptides. Subsequent peptide fractionation by isoelectric focusing in immobilized pH gradients (IPG-IEF), followed by chromatographic separation with reverse phase nano-HPLC and electrospray ionization tandem mass spectrometry (ESI-MS/MS) of peptides enabled the identification of over 900 proteins representing over 44% of the proteome. In most functional classes, over 50% of the predicted proteins were identified, including a number of membrane proteins. This new sample preparation technique will enable extensive proteomics data to be obtained for this organism, thereby enabling the reconstruction of metabolic pathways and promoting a systems biology based understanding of this important extremophile.