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Michael B Goshe

Publications and source records attributed to Michael B Goshe.

17 recordsLinked to original sources
PubMed2006

Collision-induced dissociative chemical cross-linking reagents and methodology: Applications to protein structural characterization using tandem mass spectrometry analysis.

Chemical cross-linking combined with mass spectrometry is a viable approach to study the low-resolution structure of protein and protein complexes. However, unambiguous identification of the residues involved in a cross-link remains analytically challenging. To enable a more effective analysis across various MS platforms, we have developed a novel set of collision-induced dissociative cross-linking reagents and methodology for chemical cross-linking experiments using tandem mass spectrometry (CID-CXL-MS/MS). These reagents incorporate a single gas-phase cleavable bond within their linker region that can be selectively fragmented within the in-source region of the mass spectrometer, enabling independent MS/MS analysis for each peptide. Initial design concepts were characterized using a synthesized cross-linked peptide complex. Following verification and subsequent optimization of cross-linked peptide complex dissociation, our reagents were applied to homodimeric glutathione S-transferase and monomeric bovine serum albumin. Cross-linked residues identified by our CID-CXL-MS/MS method were in agreement with published crystal structures and previous cross-linking studies using conventional approaches. Common LC/MS/MS acquisition approaches such as data-dependent acquisition experiments using ion trap mass spectrometers and product ion spectral analysis using SEQUEST were shown to be compatible with our CID-CXL-MS/MS reagents, obviating the requirement for high resolution and high mass accuracy measurements to identify both intra- and interpeptide cross-links.

Amino Acid Sequence↗
PubMed2006

A mass spectrometry-based proteomic approach to study Marek's Disease Virus gene expression.

Marek's Disease Virus (MDV) is an avian herpesvirus that causes a lymphoproliferative disorder in chickens. MDV transitions between a lytic phase in which new viruses are produced and a latent phase in which the virus lays dormant. The mechanism controlling this lytic-to-latent switch remains unclear. To better understand the lytic phase of MDV infection, a mass spectrometry-based strategy was developed to identify viral proteins and to qualitatively examine their abundance in lytically infected chicken embryo fibroblast (CEF) cells. A combination of strong cation exchange chromatography (SCXC) and microcapillary reversed-phase liquid chromatography-tandem mass spectrometry (murpLC/MS/MS) was used to resolve peptides from tryptic digests of MDV-infected CEF cell lysates. Peptides were identified by searching the tandem mass spectra against a protein database containing both MDV proteins and all currently available Gallus gallus proteins using the SEQUEST algorithm. A total of 427 MDV peptides, corresponding to 82 unique proteins, were identified, with 56 of them detected with at least two unique peptides. Overall, nearly 80% of all putative MDV proteins expressed in infected CEF cells were identified. We anticipate that this approach will be a viable method for determining how viral and host proteome changes occurring in Marek's Disease pathogenesis regulate the switch between the lytic and latent phases of the MDV life cycle.

Animals↗
PubMed2006

Characterizing phosphoproteins and phosphoproteomes using mass spectrometry.

The reversible phosphorylation of proteins plays a major role in many vital cellular processes by modulating protein function and transmitting signals within cellular pathways and networks. Because phosphorylation is dynamic and the sites of modification cannot be predicted by an organism's genome, proteomic measurements are required to identify sites of and changes in the phosphorylation state of proteins. The low stoichiometry of phosphorylation sites that accompany the multifarious nature of protein phosphorylation in biological systems continues to challenge the dynamic range of present mass spectrometry (MS) technologies and proteomic measurements, despite the preponderance of research and analytical methods devoted to this area. This review addresses some of the strategies and limitations involving the use of MS to map and quantify changes in protein phosphorylation sites for samples that range from a single protein to an entire proteome, and presents several compelling reasons as to why comprehensive phosphorylation site analysis has proven to be so elusive without a hypothesis-driven experimental approach to elicit more meaningful and confident results.

Chromatography, Liquid↗
PubMed2005

Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker.

Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S-transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.

Amino Acid Sequence↗
PubMed2005

Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase.

Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.

Arabidopsis↗
PubMed2004

Evaluation of the DNA binding tendencies of the transition state regulator AbrB.

Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors. One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity. Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes. MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets. MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner. UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes. General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed. It is likely that these parameters contribute to the differential binding proclivities of AbrB. In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner.

Bacillus subtilis↗
PubMed2003

Phosphoprotein isotope-coded solid-phase tag approach for enrichment and quantitative analysis of phosphopeptides from complex mixtures.

Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.

Amino Acid Sequence↗
PubMed2003

Initial implementation of external accumulation liquid chromatography/electrospray ionization Fourier transform ion cyclotron resonance with automated gain control.

Capillary liquid chromatography (LC) separation coupled with external accumulation Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has recently been demonstrated to have significant potential for proteomics research. Accumulation of an excessive space charge external to the FTICR cell ion trap has been shown to result in increased mass measurement error, undesirable ion discrimination and/or fragmentation, potentially causing misrepresentation or incorrect assignments of lower abundance peptides in the acquired mass spectra. In this work we report on the capability of data-dependent adjustment of ion accumulation times in the course of LC separations, further referred to as automated gain control (AGC). Three different AGC approaches were evaluated based on the number of putative peptides from a tryptic digest of four casein proteins detected in the course of LC/FTICR separations. When compared with the conventional technique, AGC was found to increase, up to a factor of 3, the total number of peptides identified.

Automation↗
PubMed2003

Stable isotope-coded proteomic mass spectrometry.

Developing the ability to quantify changes in protein abundance between cells subjected to a variety of physiological and environmental conditions is an extremely active area of proteome research. Although advances in chromatography, mass spectrometry instrumentation, and bioinformatics have contributed to producing a viable method for comparative proteome-wide analyses, the highest precision of quantitation is based, in part, upon improved methods for chemical and metabolic stable isotope labeling of proteins and peptides. The ability to quantify differences in protein expression and post-translational modifications using stable isotope labeling has been achieved, but insights into the biochemical mechanisms that will contribute to the development of new biotechnologies have yet to be realized.

Affinity Labels↗
PubMed2002

Myxoma virus immunomodulatory protein M156R is a structural mimic of eukaryotic translation initiation factor eIF2alpha.

Phosphorylation of the translation initiation factor eIF2 on Ser51 of its alpha subunit is a key event for regulation of protein synthesis in all eukaryotes. M156R, the product of the myxoma virus M156R open reading frame, has sequence similarity to eIF2alpha as well as to a family of viral proteins that bind to the interferon-induced protein kinase PKR and inhibit phosphorylation of eIF2alpha. In this study, we demonstrate that, like eIF2alpha. M156R is an efficient substrate for phosphorylation by PKR and can compete with eIF2alpha. To gain insights into the substrate specificity of the eIF2alpha kinases, we have determined the nuclear magnetic resonance (NMR) structure of M156R, the first structure of a myxoma virus protein. The fold consists of a five-stranded antiparallel beta-barrel with two of the strands connected by a loop and an alpha-helix. The similarity between M156R and the beta-barrel structure in the N terminus of eIF2alpha suggests that the viral homologs mimic eIF2alpha structure in order to compete for binding to PKR. A homology-modeled structure of the well-studied vaccinia virus K3L was generated on the basis of alignment with M156R. Comparison of the structures of the K3L model, M156R, and human eIF2alpha indicated that residues important for binding to PKR are located at conserved positions on the surface of the beta-barrel and in the mobile loop, identifying the putative PKR recognition motif.

Amino Acid Sequence↗
PubMed2002

Low-energy collision-induced dissociation fragmentation analysis of cysteinyl-modified peptides.

The development of methods to chemically modify and isolate cysteinyl-residue-containing peptides (Cys-peptides) for LC-MS/MS analysis has generated considerable interest in the field of proteomics. Methods using isotope-coded affinity tags (ICAT) and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (iodoacetyl-PEO-biotin) employ similar Cys-modifying reagents that contain a thiolate-specific biotin group to modify and isolate Cys-containing peptides in conjunction with immobilized avidin. For these strategies to be effective on a proteome-wide level, the presence of the ICAT or acetyl-PEO-biotin tag should not interfere with the efficiency of induced dissociation in MS/MS experiments or with the identification of the modified Cys-peptides by automated database searching algorithms. We have compared the collision-induced dissociation (CID) fragmentation patterns of peptides labeled with iodoacetyl-PEO-biotin and the ICAT reagent to those of the unmodified peptides. CID of Cys-peptides modified with either reagent resulted in the formation of ions attributed to the modified Cys-peptides as well as those unique to the labeling reagent. As demonstrated by analyzing acetyl-PEO-biotin labeled peptides from ribonuclease A and the ICAT-labeled proteome of Deinococcus radiodurans, the presence of these label-specific product ions provides a useful identifier to discern whether a peptide has been modified with the Cys-specific reagent, especially when a number of peptides analyzed using these methods do not contain a modified Cys residue, and to differentiate identical Cys-peptides labeled with either ICAT-d0 or ICAT-d8.

Animals↗
PubMed2002

Phosphoprotein isotope-coded affinity tags: application to the enrichment and identification of low-abundance phosphoproteins.

The use of a phosphoprotein isotope-coded affinity tag (PhIAT), which employs differential isotopic labeling and biotinylation, has been shown capable of enriching and identifying mixtures of low-abundance phosphopeptides. A denatured solution of beta-casein was labeled using the PhIAT method, and after proteolytic digestion, the labeled peptides were isolated using immobilized avidin. The recovered peptides were separated by capillary reversed-phase liquid chromatography and identified by tandem mass spectrometry. PhIAT-labeled peptides corresponding to known O-phosphorylated peptides from beta-casein were identified along with the phosphorylated peptides from alphas1-casein and alphas2-casein, known low-level (<5%) contaminants of commercially available beta-casein. All of the casein-phosphorylated residues identified by the present PhIAT approach correspond to previously documented sites of phosphorylation. The results illustrate the efficacy of the PhIAT-labeling strategy to not only enrich mixtures for phosphopeptides but also, more importantly, permit the detection and identification of low-level phosphopeptides. In addition, the differences in the phosphorylation state could be determined between phosphopeptides in comparative samples by stoichiometric conversion using the light and heavy isotopic versions of the PhIAT reagents. Overall, our results exemplify the application of the PhIAT approach and demonstrate its utility for proteome-wide phosphoprotein identification and quantitation.

Affinity Labels↗
PubMed2002

The postgenomic age: characterization of proteomes.

Global analysis of biological systems is becoming increasingly feasible as technologies that facilitate genome-wide analyses of gene expression are developed. Proteomics is the global analysis of expressed proteins (including posttranslational modifications) and seeks to establish the relationship between genome sequence, expressed proteins, protein-protein interactions, and cell and tissue phenotype. While the relative abundance of transcripts can be quantified using gene expression microarrays, the identification and quantitation of expressed proteins is more challenging. Nevertheless, the potential payoff for global protein analyses is immense because identification of distinctive protein signatures associated with cell function may provide novel therapeutic targets, molecular markers of disease, and increased understanding of determinants of cell phenotype. The challenges and promises of applications of established and emerging proteome strategies to detect and quantify differentially expressed proteins in culture cells are discussed.

Animals↗
PubMedDate not supplied

Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry.

An increasing number of proteomic strategies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures.

Amino Acid Sequence↗
PubMedDate not supplied

Affinity labeling of highly hydrophobic integral membrane proteins for proteome-wide analysis.

The ability to identify and quantitate integral membrane proteins is an analytical challenge for mass spectrometry-based proteomics. The use of surfactants to solubilize and facilitate derivatization of these proteins can suppress peptide ionization and interfere with chromatographic separations during microcapillary reversed-phase liquid chromatography-electrospray-tandem mass spectrometry. To circumvent the use of surfactants and increase proteome coverage, an affinity labeling method has been developed to target highly hydrophobic integral membrane proteins using organic-assisted extraction and solubilization followed by cysteinyl-specific labeling using biotinylation reagents. As demonstrated on the membrane subproteome of Deinococcus radiodurans, specific and quantitative labeling of integral membrane proteins was achieved using a 60% methanol-aqueous buffer system and (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine as the cysteinyl-alkylating reagent. From a total of 220 unique Cys-labeled peptides, 89 proteins were identified, of which 40 were integral membrane proteins containing from one to nine mapped transmembrane domains with a maximum positive GRAVY of 1.08. The protocol described can be used with other stable isotope labeling reagents (e.g., ICAT) to enable comparative measurements to be made on differentially expressed hydrophobic membrane proteins from various organisms (e.g., pathogenic bacteria) and cell types and provide a viable method for comparative proteome-wide analyses.

Affinity Labels↗
PubMedDate not supplied

Global analysis of the membrane subproteome of Pseudomonas aeruginosa using liquid chromatography-tandem mass spectrometry.

Pseudomonas aeruginosa is one of the most significant opportunistic bacterial pathogens in humans causing infections and premature death in patients with cystic fibrosis, AIDS, severe burns, organ transplants, or cancer. Liquid chromatography coupled online with tandem mass spectrometry was used for the large-scale proteomic analysis of the P. aeruginosa membrane subproteome. Concomitantly, an affinity labeling technique, using iodoacetyl-PEO biotin to tag cysteinyl-containing proteins, permitted the enrichment and detection of lower abundance membrane proteins. The application of these approaches resulted in the identification of 786 proteins. A total of 333 proteins (42%) had a minimum of one transmembrane domain (ranging from 1 to14) and 195 proteins were classified as hydrophobic based on their positive GRAVY values (ranging from 0.01 to 1.32). Key integral inner and outer membrane proteins involved in adaptation and antibiotic resistance were conclusively identified, including the detection of 53% of all predicted opr-type porins (outer integral membrane proteins) and all the components of the mexA-mexB-oprM transmembrane protein complex. This work represents one of the most comprehensive proteomic analyses of the membrane subproteome of P. aeruginosa and for prokaryotes in general.

Affinity Labels↗