%0 Journal Article
%~ PubMed
%A Paulech, Jana
%A Solis, Nestor
%A Cordwell, Stuart J
%T Characterization of reaction conditions providing rapid and specific cysteine alkylation for peptide-based mass spectrometry.
%B Biochimica et Biophysica Acta
%D 2013
%C Netherlands
%I Elsevier BV
%V 1834
%N 1
%P 372-379
%@ 0006-3002
%X Alkylation converts Cys thiols to thioethers and prevents unwanted side reactions, thus facilitating mass spectrometric identification of Cys-containing peptides. Alkylation occurs preferentially at Cys due to its high nucleophilicity, however reactions at other such sites are possible. N-ethylmaleimide (NEM) shows rapid reaction kinetics with Cys and careful definition of reaction conditions results in little reactivity at other sites. Analysis of a protein standard alkylated under differing reaction conditions (pH, NEM concentrations and reaction times) was performed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and selected reaction monitoring (SRM) of NEM-modified and unmodified peptide pairs. Mis-alkylation sites at primary and secondary amines were identified and limited to one equivalent of NEM. No evidence for hydroxyl or thioether alkylation was observed. Improved specificity was achieved by restricting the pH below neutral, NEM concentration below 10mM and/or reaction time to below 5min. Maximal removal of Cys activity was observed in tissue homogenates at 40mM NEM within 1min, dependent upon efficient protein denaturation. SRM assays identified peptide-specific levels of mis-alkylation, indicating that NEM-modified to unmodified ratios did not exceed 10%, with the exception of Cys alkylation that proceeded to 100%, and some Lys residues that resulted in tryptic missed cleavages. High reactivity was observed for His residues considering their relatively low abundance. These data indicate that rapid and specific Cys alkylation is possible with NEM under relatively mild conditions, with more abrasive conditions leading to increased non-specific alkylation without appreciable benefit for MS-based proteomics.
%Z FOR Codes: 60101


%0 Journal Article
%~ PubMed
%A Henningham, Anna
%A Chiarot, Emiliano
%A Gillen, Christine M
%A Cole, Jason N
%A Rohde, Manfred
%A Fulde, Marcus
%A Ramachandran, Vidiya
%A Cork, Amanda J
%A Hartas, Jon
%A Magor, Graham
%A Djordjevic, Steven P
%A Cordwell, Stuart J
%A Kobe, Bostjan
%A Sriprakash, Kabada S
%A Nizet, Victor
%A Chhatwal, G S
%A Margarit, Immaculada Y R
%A Batzloff, Michael R
%A Walker, Mark J
%T Conserved anchorless surface proteins as group A streptococcal vaccine candidates.
%B Journal of Molecular Medicine
%D 2012
%C Germany
%I Springer
%V 90
%N 10
%P 1197-1207
%@ 1432-1440
%X Streptococcus pyogenes (group A Streptococcus (GAS)) causes ?700 million human infections each year, resulting in over 500,000 deaths. The development of a commercial GAS vaccine is hampered by the occurrence of many unique GAS serotypes, antigenic variation within the same serotype, differences in serotype geographical distribution, and the production of antibodies cross-reactive with human tissue that may lead to autoimmune disease. Several independent studies have documented a number of GAS cell wall-associated or secreted metabolic enzymes that contain neither N-terminal leader sequences nor C-terminal cell wall anchors. Here, we applied a proteomic analysis of serotype M1T1 GAS cell wall extracts for the purpose of vaccine development. This approach catalogued several anchorless proteins and identified two protective vaccine candidates, arginine deiminase and trigger factor. These surface-exposed enzymes are expressed across multiple GAS serotypes exhibiting ?99% amino acid sequence identity. Vaccine safety concerns are alleviated by the observation that these vaccine candidates lack human homologs, while sera from human populations suffering repeated GAS infections and high levels of autoimmune complications do not recognize these enzymes. Our study demonstrates anchorless cell surface antigens as promising vaccine candidates for the prevention of GAS disease.
%Z FOR Codes: 60502 60114 110309


%0 Journal Article
%~ PubMed
%A Iwashkiw, Jeremy A
%A Seper, Andrea
%A Weber, Brent S
%A Scott, Nichollas E
%A Vinogradov, Evgeny
%A Stratilo, Chad
%A Reiz, Bela
%A Cordwell, Stuart J
%A Whittal, Randy
%A Schild, Stefan
%A Feldman, Mario F
%T Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation.
%B PLoS Pathogens
%D 2012
%C United States
%I Public Library of Science
%V 8
%N 6
%P e1002758
%@ 1553-7374
%X Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening "superbugs" for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.
%Z FOR Codes: 60501 60109 100499


%0 Journal Article
%~ PubMed
%A Scott, Nichollas E
%A Nothaft, Harald
%A Edwards, Alistair V G
%A Labbate, Maurizio
%A Djordjevic, Steven P
%A Larsen, Martin R
%A Szymanski, Christine M
%A Cordwell, Stuart J
%T Modification of the Campylobacter jejuni N-linked glycan by EptC-mediated addition of phosphoethanolamine.
%B Journal of Biological Chemistry
%D 2012
%C United States
%I American Society for Biochemistry and Molecular Bi
%V 287
%N 35
%P 29384-29396
%@ 1083-351X
%X Campylobacter jejuni is the major worldwide cause of bacterial gastroenteritis. C. jejuni possesses an extensive repertoire of carbohydrate structures that decorate both protein and non-protein surface-exposed structures. An N-linked glycosylation system encoded by the pgl gene cluster mediates the synthesis of a rigidly conserved heptasaccharide that is attached to protein substrates, or released as free oligosaccharide, in the periplasm. Removal of N-glycosylation results in reduced virulence and impeded host cell attachment. Since the N-glycan is conserved, the N-glycosylation system is also an attractive option for glycoengineering recombinant vaccines in Escherichia coli. To determine whether non-canonical N-glycans are present in C. jejuni, we utilized high-throughput glycoproteomics to characterize C. jejuni JHH1 and identified 93 glycosylation sites, including 34 not previously reported. Interrogation of these data allowed the identification of a phosphoethanolamine (pEtN)-modified variant of the N-glycan that was attached to multiple proteins. The pEtN moiety was attached to the terminal GalNAc of the canonical N-glycan. Deletion of the pEtN transferase eptC removed all evidence of the pEtN-glycan, but did not globally influence protein reactivity to patient sera, while deletion of the pglB oligosaccharyltransferase significantly reduced reactivity. Transfer of eptC and the pgl gene cluster to E. coli confirmed the addition of the pEtN-glycan to a target C. jejuni protein. Significantly reduced, yet above background, levels of pEtN-glycan were also observed in E. coli not expressing eptC, suggesting that endogenous E. coli pEtN transferases can mediate addition of pEtN to N-glycans. Addition of pEtN must be considered in the context of glycoengineering and may alter C. jejuni glycan-mediated structure-function interactions.
%Z FOR Codes: 60501 60109 110106


%0 Journal Article
%~ PubMed
%A Hare, Nathan J
%A Solis, Nestor
%A Harmer, Christopher
%A Marzook, N Bishara
%A Rose, Barbara
%A Harbour, Colin
%A Crossett, Ben
%A Manos, Jim
%A Cordwell, Stuart J
%T Proteomic profiling of Pseudomonas aeruginosa AES-1R, PAO1 and PA14 reveals potential virulence determinants associated with a transmissible cystic fibrosis-associated strain.
%B BMC Microbiology
%D 2012
%C United Kingdom
%I BioMed Central Ltd.
%V 12
%N 1
%P 16
%@ 1471-2180
%X ABSTRACT: BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-person transmissible strains have been identified in CF clinics worldwide. The molecular basis for transmissibility and colonization of the CF lung remains poorly understood. RESULTS: A dual proteomics approach consisting of gel-based and gel-free comparisons were undertaken to analyse protein profiles in a transmissible, early (acute) isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1. Over 1700 P. aeruginosa proteins were confidently identified. AES-1R protein profiles revealed elevated abundance of proteins associated with virulence and siderophore biosynthesis and acquisition, antibiotic resistance and lipopolysaccharide and fatty acid biosynthesis. The most abundant protein in AES-1R was confirmed as a previously hypothetical protein with sequence similarity to carbohydrate-binding proteins and database search revealed this gene is only found in the CF-associated strain PA2192. The link with CF infection may suggest that transmissible strains have acquired an ability to rapidly interact with host mucosal glycoproteins. CONCLUSIONS: Our data suggest that AES-1R expresses higher levels of proteins, such as those involved in antibiotic resistance, iron acquisition and virulence that may provide a competitive advantage during early infection in the CF lung. Identification of novel proteins associated with transmissibility and acute infection may aid in deciphering new strategies for intervention to limit P. aeruginosa infections in CF patients.
%Z FOR Codes: 30406 60501 110106


%0 Journal Article
%~ PubMed
%A Hare, Nathan J
%A Soe, Cho Zin
%A Rose, Barbara
%A Harbour, Colin
%A Codd, Rachel
%A Manos, Jim
%A Cordwell, Stuart J
%T Proteomics of <i>Pseudomonas aeruginosa</i> Australian epidemic strain 1 (AES-1) cultured under conditions mimicking the cystic fibrosis lung reveals increased iron acquisition via the siderophore pyochelin.
%B Journal of proteome research
%D 2012
%C United States
%I American Chemical Society
%V 11
%N 2
%P 776-95
%@ 1535-3893
%X Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-to-person transmissible strains have been identified in CF clinics worldwide, and the molecular basis for transmissibility remains poorly understood. We undertook a complementary proteomics approach to characterize protein profiles from a transmissible, acute isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1 when grown in an artificial medium that mimics the CF lung environment compared to growth in standard laboratory medium. Proteins elevated in abundance in AES-1R included those involved in methionine and S-adenosylmethionine biosynthesis and in the synthesis of phenazines. Proteomic data were validated by measuring culture supernatant levels of the virulence factor pyocyanin, which is the final product of the phenazine pathway. AES-1R and PAO1 released higher extracellular levels of pyocyanin compared to PA14 when grown in conditions that mimic the CF lung. Proteins associated with biosynthesis of the iron-scavenging siderophore pyochelin (PchDEFGH and FptA) were also present at elevated abundance in AES-1R and at much higher levels than in PAO1, whereas they were reduced in PA14. These protein changes resulted phenotypically in increased extracellular iron acquisition potential and, specifically, elevated pyochelin levels in AES-1R culture supernatants as detected by chrome azurol-S assay and fluorometry, respectively. Transcript analysis of pyochelin genes (pchDFG and fptA) showed they were highly expressed during the early stage of growth in artificial sputum medium (18 h) but returned to basal levels following the establishment of microcolony growth (72 h) consistent with that observed in the CF lung. This provides further evidence that iron acquisition by pyochelin may play a role in the early stages of transmissible CF infection associated with AES-1R.
%Z FOR Codes: 30406 110801 60109


%0 Journal Article
%~ PubMed
%A Cordwell, Stuart J
%A Edwards, Alistair V G
%A Liddy, Kiersten A
%A Moshkanbaryans, Lia
%A Solis, Nestor
%A Parker, Benjamin L
%A Yong, Andy S C
%A Wong, Clement
%A Kritharides, Leonard
%A Hambly, Brett D
%A White, Melanie Y
%T Release of tissue-specific proteins into coronary perfusate as a model for biomarker discovery in myocardial ischemia / reperfusion injury.
%B Journal of Proteome Research
%D 2012
%C United States
%I American Chemical Society
%V 11
%N 4
%P 2114-2126
%@ 1535-3893
%X Diagnosis of acute coronary syndromes is based on protein biomarkers, such as the cardiac troponins (cTnI/cTnT) and creatine kinase (CK-MB) that are released into the circulation. Biomarker discovery is focused on identifying very low abundance tissue-derived analytes from within albumin-rich plasma, in which the wide dynamic range of the native protein complement hinders classical proteomic investigations. We employed an <i>ex vivo</i> rabbit model of myocardial ischemia / reperfusion (I/R) injury using Langendorff buffer perfusion. Non-recirculating perfusate was collected over a temporal profile of 60 minutes reperfusion following brief, reversible ischemia (15 minutes; 15I/60R) for comparison with irreversible I/R (60I/60R). Perfusate proteins were separated using two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS), revealing 26 tissue-specific proteins released during reperfusion post-15I. Proteins released during irreversible I/R (60I/60R) were profiled using gel-based (2-DE and one-dimensional gel electrophoresis coupled to liquid chromatography and tandem mass spectrometry; geLC-MS) and gel-free (LC/MS-MS) methods. A total of 192 tissue-specific proteins were identified during reperfusion post-60I. Identified proteins included those previously associated with I/R (myoglobin, CK-MB, cTnI and cTnT), in addition to examples currently under investigation in large cohort studies (heart-type fatty acid binding protein; FABPH). The post-ischemic release profile of a novel cardiac-specific protein, cysteine and glycine-rich protein 3 (Csrp3; cardiac LIM domain protein) was validated by Western blot analysis. We also identified Csrp3 in serum from 6 of 8 patients post-reperfusion following acute myocardial infarction. These studies indicate that animal modeling of biomarker release using ex vivo buffer perfused tissue to limit the presence of obfuscating plasma proteins may identify candidates for further study in humans.
%Z FOR Codes: 1102 1101


%0 Journal Article
%A Edwards, Alistair VG
%A Edwards, Gregory J
%A Larsen, Martin R
%A Cordwell, Stuart
%T ReportSites - a computational method to extract positional and physico- chemical information from large-scale proteomic post-translational modification datasets
%B Journal of Proteomics and Bioinformatics
%D 2012
%C United States
%I Omics Publishing Group
%V 5
%N 4
%P 104-107
%@ 0974-276X
%X 
%Z FOR Codes: 10401


%0 Journal Article
%~ PubMed
%A Solis, Nestor
%A Cordwell, Stuart J
%T Current methodologies for proteomics of bacterial surface-exposed and cell envelope proteins.
%B Proteomics
%D 2011
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 11
%N 15
%P 3169-89
%@ 1615-9853
%X The study of surface-exposed proteins has received increasing attention following the advent of genomic sequencing, which in turn has enabled predictive tools and facilitated the technologies for their analysis by proteomics. The exterior topology of a bacterial pathogen is the interface between the cell and environment and thus is the initial mediator for infection, providing an important reservoir for components that may be used for novel vaccine development as well as the characterization of new drug targets. The study of such biological molecules has however, been under-represented in proteomics studies due to the difficulty involved in their analysis. Cell-envelope proteins in bacteria are typically difficult to characterize due to their low abundance, poor solubility, and the problematic isolation of pure surface fractions with only minimal contamination. Here, we describe different cell envelope preparations for proteomic characterization, focused principally on gel-free technologies. Fractionation techniques popularly used in proteomics are also explained with emphasis on surface and membrane-derived proteins/peptides. Conditional confirmation of localization is also explored with emphasis on different prediction algorithms as well as on analyses of surface peptide fractions by the use of different search programs and their implications for the unambiguous identification of surface-exposed and membrane-embedded proteins. Finally, different quantification techniques are discussed that are important for the validation of identifications and for highlighting novel proteins that may warrant further study by independent techniques.
%Z FOR Codes: 60109


%0 Journal Article
%~ PubMed
%A Edwards, Alistair V G
%A Cordwell, Stuart J
%A White, Melanie Y
%T Phosphoproteomic profiling of the myocyte.
%B Circulation: Cardiovascular Genetics
%D 2011
%C United States
%I Lippincott Williams & Wilkins
%V 4
%N 5
%P 575
%@ 1942-325X
%X 
%Z FOR Codes: 60109


%0 Journal Article
%~ PubMed
%A Hare, Nathan J
%A Scott, Nichollas E
%A Shin, Eun Hye H
%A Connolly, Angela M
%A Larsen, Martin R
%A Palmisano, Giuseppe
%A Cordwell, Stuart J
%T Proteomics of the oxidative stress response induced by hydrogen peroxide and paraquat reveals a novel AhpC-like protein in Pseudomonas aeruginosa.
%B Proteomics
%D 2011
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 11
%N 15
%P 3056-3069
%@ 1615-9853
%X Pseudomonas aeruginosa is a ubiquitous pathogen most typically associated with wound infections, but also the main cause of mortality in patients suffering from cystic fibrosis (CF). The ability to adapt to oxidative stress associated with host immune defense may be one mechanism by which P. aeruginosa establishes infection in the cystic fibrosis lung and eventually out-competes other pathogenic bacteria to persist into chronic infection. We utilized a proteomics approach to identify the proteins associated with the oxidative stress response of P. aeruginosa PAO1 to hydrogen peroxide and superoxide-inducing paraquat. 2-DE and MS allowed for the identification of 59 and 58 protein spots that were statistically significantly altered following H(2) O(2) and paraquat treatment, respectively. We observed a unique mass and pI pattern for alkylhydroperoxide reductase C (AhpC) that was replicated by hypothetical protein PA3529 following treatment with 10???mM H(2) O(2) . AhpC belongs to the 2-Cys peroxiredoxin family and is a redox enzyme responsible for removing peroxides in bacterial cells. MS analysis showed that PA3529 was altered by the formation of a dimer via a disulfide bond in a manner analogous to that known for AhpC, and by cysteine overoxidation to Cys-sulfonic acid (SO(3) H) postoxidative stress. PA3529 is therefore a functional AhpC paralog expressed under H(2) O(2) stress. Following paraquat-induced oxidative stress, we also observed the overabundance and likely oxidative modification of a second hypothetical antioxidant protein (PA3450) that shares sequence similarity with 1-Cys peroxiredoxins. Other induced proteins included known oxidative stress proteins (superoxide dismutase and catalase), as well as those involved in iron acquisition (siderophore biosynthesis and receptor proteins FpvA and FptA) and hypothetical proteins, including others predicted to be antioxidants (PA0848). These data suggest that P. aeruginosa contains a plethora of novel antioxidant proteins that contribute to its increased resistance against oxidative stress.
%Z FOR Codes: 60109


%0 Journal Article
%~ PubMed
%A Naughton, Sharna
%A Parker, Dane
%A Seemann, Torsten
%A Thomas, Torsten
%A Turnbull, Lynne
%A Rose, Barbara
%A Bye, Peter
%A Cordwell, Stuart
%A Whitchurch, Cynthia
%A Manos, Jim
%T Pseudomonas aeruginosa AES-1 Exhibits Increased Virulence Gene Expression during Chronic Infection of Cystic Fibrosis Lung.
%B PLoS One
%D 2011
%C United States
%I Public Library of Science
%V 6
%N 9
%P e24526
%@ 1932-6203
%X Pseudomonas aeruginosa, the leading cause of morbidity and mortality in people with cystic fibrosis (CF), adapts for survival in the CF lung through both mutation and gene expression changes. Frequent clonal strains such as the Australian Epidemic Strain-1 (AES-1), have increased ability to establish infection in the CF lung and to superimpose and replace infrequent clonal strains. Little is known about the factors underpinning these properties. Analysis has been hampered by lack of expression array templates containing CF-strain specific genes. We sequenced the genome of an acute infection AES-1 isolate from a CF infant (AES-1R) and constructed a non-redundant micro-array (PANarray) comprising AES-1R and seven other sequenced P. aeruginosa genomes. The unclosed AES-1R genome comprised 6.254Mbp and contained 6957 putative genes, including 338 not found in the other seven genomes. The PANarray contained 12,543 gene probe spots; comprising 12,147 P. aeruginosa gene probes, 326 quality-control probes and 70 probes for non-P. aeruginosa genes, including phage and plant genes. We grew AES-1R and its isogenic pair AES-1M, taken from the same patient 10.5 years later and not eradicated in the intervening period, in our validated artificial sputum medium (ASMDM) and used the PANarray to compare gene expression of both in duplicate. 675 genes were differentially expressed between the isogenic pairs, including upregulation of alginate, biofilm, persistence genes and virulence-related genes such as dihydroorotase, uridylate kinase and cardiolipin synthase, in AES-1M. Non-PAO1 genes upregulated in AES-1M included pathogenesis-related (PAGI-5) genes present in strains PACS2 and PA7, and numerous phage genes. Elucidation of these genes'' roles could lead to targeted treatment strategies for chronically infected CF patients.
%Z FOR Codes: 60408 60405 110801


%0 Journal Article
%~ PubMed
%A Parker, Benjamin L
%A Gupta, Pankaj
%A Cordwell, Stuart J
%A Larsen, Martin R
%A Palmisano, Giuseppe
%T Purification and Identification of O-GlcNAc-Modified Peptides Using Phosphate-Based Alkyne CLICK Chemistry in Combination with Titanium Dioxide Chromatography and Mass Spectrometry.
%B Journal of Proteome Research
%D 2011
%C United States
%I American Chemical Society
%V 10
%N 4
%P 1449-1458
%@ 1535-3893
%X A selective method for the enrichment of O-GlcNAcylated peptides using a novel CLICK chemistry reagent is described. Peptides modified by O-GlcNAc were enzymatically labeled with N-azidoacetylgalactosamine. The azide was then reacted with a phospho-alkyne using CLICK chemistry and O-GlcNAcGalNAzPO(4)-containing peptides were enriched using titanium dioxide chromatography. Modified peptides were analyzed using a combination of higher energy collision dissociation for identification and electron transfer dissociation to localize the site of O-GlcNAc attachment. The enrichment method was developed and optimized using an alpha-crystallin standard protein and then applied to a soluble protein preparation of mouse brain tissue and a nuclear preparation generated from HeLa cells. A total of 42 unique O-GlcNAcylated peptides were identified, including 7 novel O-GlcNAc sites.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Parker, Benjamin L
%A Palmisano, Giuseppe
%A Edwards, Alistair V G
%A White, Melanie Y
%A Engholm-Keller, Kasper
%A Lee, Albert
%A Scott, Nichollas E
%A Kolarich, Daniel
%A Hambly, Brett D
%A Packer, Nicolle H
%A Larsen, Martin R
%A Cordwell, Stuart J
%T Quantitative N-linked glycoproteomics of myocardial ischemia / reperfusion injury reveals early remodeling in the extracellular environment.
%B Molecular & Cellular Proteomics
%D 2011
%C United States
%I American Society for Biochemistry and Molecular Bi
%V 10
%N 8
%P M110.006833
%@ 1535-9484
%X Extracellular and cell surface proteins are generally modified with N-linked glycans and glycopeptide enrichment is an attractive tool to analyze these proteins. The role of N-linked glycoproteins in cardiovascular disease, particularly ischemia and reperfusion injury, is poorly understood. Observation of glycopeptides by mass spectrometry is challenging due to the presence of abundant, nonglycosylated analytes, and robust methods for purification are essential. We employed digestion with multiple proteases to increase glycoproteome coverage coupled with parallel glycopeptide enrichments using hydrazide capture, titanium dioxide, and hydrophilic interaction liquid chromatography with and without an ion-pairing agent. Glycosylated peptides were treated with PNGase F and analyzed by liquid chromatography-MS/MS. This allowed the identification of 1556 nonredundant N-linked glycosylation sites, representing 972 protein groups from ex vivo rat left ventricular myocardium. False positive "glycosylations" were observed on 44 peptides containing a deamidated Asn-Asp in the N-linked sequon by analysis of samples without PNGase F treatment. We used quantitation via isobaric tags for relative and absolute quantitation (iTRAQ) and validation with dimethyl labeling to analyze changes in glycoproteins from tissue following prolonged ischemia and reperfusion (40 mins ischemia and 20 mins reperfusion) indicative of myocardial infarction. The iTRAQ approach revealed 80 of 437 glycopeptides with altered abundance, while dimethyl labeling confirmed 46 of these and revealed an additional 62 significant changes. These were mainly from predicted extracellular matrix and basement membrane proteins that are implicated in cardiac remodeling. Analysis of N-glycans released from myocardial proteins suggest that the observed changes were not due to significant alterations in N-glycan structures. Altered proteins included the collagen-laminin-integrin complexes and collagen assembly enzymes, cadherins, mast cell proteases, proliferation-associated secreted protein acidic and rich in cysteine, and microfibril-associated proteins. The data suggest that cardiac remodeling is initiated earlier during reperfusion than previously hypothesized.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Bogema, Daniel R
%A Scott, Nichollas E
%A Padula, Matthew P
%A Tacchi, Jessica L
%A Raymond, Benjamin B A
%A Jenkins, Cheryl
%A Cordwell, Stuart J
%A Minion, F Chris
%A Walker, Mark J
%A Djordjevic, Steven P
%T Sequence TTKF&#8595; QE Defines the Site of Proteolytic Cleavage in Mhp683 Protein, a Novel Glycosaminoglycan and Cilium Adhesin of Mycoplasma hyopneumoniae.
%B Journal of Biological Chemistry
%D 2011
%C United States
%I American Society for Biochemistry and Molecular Bi
%V 286
%N 48
%P 41217-41229
%@ 0021-9258
%X Mycoplasma hyopneumoniae colonizes the ciliated respiratory epithelium of swine, disrupting mucociliary function and inducing chronic inflammation. P97 and P102 family members are major surface proteins of M. hyopneumoniae and play key roles in colonizing cilia via interactions with glycosaminoglycans and mucin. The p102 paralog, mhp683, and homologs in strains from different geographic origins encode a 135-kDa pre-protein (P135) that is cleaved into three fragments identified here as P45(683), P48(683), and P50(683). A peptide sequence (TTKF???QE) was identified surrounding both cleavage sites in Mhp683. N-terminal sequences of P48(683) and P50(683), determined by Edman degradation and mass spectrometry, confirmed cleavage after the phenylalanine residue. A similar proteolytic cleavage site was identified by mass spectrometry in another paralog of the P97/P102 family. Trypsin digestion and surface biotinylation studies showed that P45(683), P48(683), and P50(683) reside on the M. hyopneumoniae cell surface. Binding assays of recombinant proteins F1(683)-F5(683), spanning Mhp683, showed saturable and dose-dependent binding to biotinylated heparin that was inhibited by unlabeled heparin, fucoidan, and mucin. F1(683)-F5(683) also bound porcine epithelial cilia, and antisera to F2(683) and F5(683) significantly inhibited cilium binding by M. hyopneumoniae cells. These data suggest that P45(683), P48(683), and P50(683) each display cilium- and proteoglycan-binding sites. Mhp683 is the first characterized glycosaminoglycan-binding member of the P102 family.
%Z FOR Codes: 60109 110801 60501


%0 Journal Article
%~ PubMed
%A Scott, Nichollas E
%A Parker, Benjamin L
%A Connolly, Angela M
%A Paulech, Jana
%A Edwards, Alistair V G
%A Crossett, Ben
%A Falconer, Linda
%A Kolarich, Daniel
%A Djordjevic, Steven P
%A Hojrup, Peter
%A Packer, Nicolle H
%A Larsen, Martin R
%A Cordwell, Stuart J
%T Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, HCD and ETD-MS applied to the N-linked glycoproteome of Campylobacter jejuni.
%B Molecular & Cellular Proteomics
%D 2011
%C United States
%I American Society for Biochemistry and Molecular B
%V 10
%N 2
%P M000031-MCP201
%@ 1535-9484
%X Campylobacter jejuni is a gastrointestinal pathogen that is able to modify membrane and periplasmic proteins by the N-linked addition of a 7-residue glycan at the strict attachment motif (D/E)XNX(S/T). Strategies for a comprehensive analysis of the targets of glycosylation, however, are hampered by the resistance of the glycan-peptide bond to enzymatic digestion or ??-elimination and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction chromatography and examined novel fragmentation, including collision-induced dissociation (CID) and higher energy collisional (C-trap) dissociation (HCD) as well as CID/electron transfer dissociation (ETD) mass spectrometry. CID/HCD enabled the identification of glycan structure and peptide backbone, allowing glycopeptide identification, whereas CID/ETD enabled the elucidation of glycosylation sites by maintaining the glycan-peptide linkage. A total of 130 glycopeptides, representing 75 glycosylation sites, were identified from LC-MS/MS using zwitterionic hydrophilic interaction chromatography coupled to CID/HCD and CID/ETD. CID/HCD provided the majority of the identifications (73 sites) compared with ETD (26 sites). We also examined soluble glycoproteins by soybean agglutinin affinity and two-dimensional electrophoresis and identified a further six glycosylation sites. This study more than doubles the number of confirmed N-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled to novel mass spectrometric fragmentation techniques may be suitable for application to eukaryotic glycoproteins for simultaneous elucidation of glycan structures and peptide sequence.
%Z FOR Codes: 60101


%0 Journal Article
%~ PubMed
%A Witchell, Timothy D
%A Hoke, David E
%A Bulach, Dieter M
%A Coutts, Scott A J
%A Driesen, Steve J
%A Cordwell, Stuart J
%A Adler, Ben
%T The major surface Vsp proteins of Brachyspira hyodysenteriae form antigenic protein complexes.
%B Veterinary Microbiology
%D 2011
%C Netherlands
%I Elsevier BV
%V 149
%N 1-2
%P 157-162
%@ 1873-2542
%X The Vsp proteins are the major outer membrane proteins of Brachyspira hyodysenteriae, the causative agent of swine dysentery. Eight vsp genes have been identified in B. hyodysenteriae strain B204, arranged into two four-gene loci, and at least two of the corresponding proteins are produced in vitro. The aims of this study were to characterise the vsp genes of the virulent Australian B. hyodysenteriae strain X576 and their corresponding proteins, Genomic sequence comparison with strains B204 and WA1 demonstrated that the number of vsp genes varies between B. hyodysenteriae strains, although the chromosomal locations of the vsp gene loci are consistent. We identified two additional vsp-like genes, designated vspI and vspJ, in each of the three strains. Double SDS-PAGE was used to demonstrate that Vsp proteins of B. hyodysenteriae strain X576 form multimeric protein complexes in the outer membrane that are stable in 6M urea but dissociate after boiling. The Vsp complexes primarily consisted of VspF but also contain VspE and VspI. VspD was also found in a series of complexes slightly larger than the more abundant VspF complexes. Vsp proteins are purported to be antigenic; however little direct data are available to support this claim. In this study convalescent pig sera did not bind denatured Vsp proteins by Western blotting, but did bind the Vsp complexes on Western blots, showing that conformational epitopes may be important in immune recognition of these major outer membrane proteins. This is the first definitive demonstration of the antigenicity of these proteins in swine dysentery.
%Z FOR Codes: 60501


%0 Journal Article
%~ PubMed
%A Solis, Nestor
%A Larsen, Martin R
%A Cordwell, Stuart J
%T Improved accuracy of cell surface shaving proteomics in Staphylococcus aureus using a false positive control.
%B Proteomics
%D 2010
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 10
%N 10
%P 2037-49
%@ 1615-9853
%X Proteolytic treatment of intact bacterial cells is an ideal means for identifying surface-exposed peptide epitopes and has potential for the discovery of novel vaccine targets. Cell stability during such treatment, however, may become compromised and result in the release of intracellular proteins that complicate the final analysis. Staphylococcus aureus is a major human pathogen, causing community and hospital-acquired infections, and is a serious healthcare concern due to the increasing prevalence of multiple antibiotic resistances amongst clinical isolates. We employed a cell surface "shaving" technique with either trypsin or proteinase-K combined with LC-MS/MS. Trypsin-derived data were controlled using a "false-positive" strategy where cells were incubated without protease, removed by centrifugation and the resulting supernatants digested. Peptides identified in this fraction most likely result from cell lysis and were removed from the trypsin-shaved data set. We identified 42 predicted S. aureus COL surface proteins from 260 surface-exposed peptides. Trypsin and proteinase-K digests were highly complementary with ten proteins identified by both, 16 specific to proteinase-K treatment, 13 specific to trypsin and three identified in the control. The use of a subtracted false-positive strategy improved enrichment of surface-exposed peptides in the trypsin data set to approximately 80% (124/155 peptides). Predominant surface proteins were those associated with methicillin resistance-surface protein SACOL0050 (pls) and penicillin-binding protein 2'' (mecA), as well as bifunctional autolysin and the extracellular matrix-binding protein Ebh. The cell shaving strategy is a rapid method for identifying surface-exposed peptide epitopes that may be useful in the design of novel vaccines against S. aureus.
%Z FOR Codes: 60109 60501 110106


%0 Journal Article
%~ PubMed
%A Scott, Nichollas E
%A Marzook, N Bishara
%A Deutscher, Ania
%A Falconer, Linda
%A Crossett, Ben
%A Djordjevic, Steven P
%A Cordwell, Stuart J
%T Mass spectrometric characterization of the Campylobacter jejuni adherence factor CadF reveals post-translational processing that removes immunogenicity while retaining fibronectin binding.
%B Proteomics
%D 2010
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 10
%N 2
%P 277-288
%@ 1615-9853
%X Campylobacter jejuni is a major gastrointestinal pathogen that colonizes host mucosa via interactions with extracellular matrix proteins, such as fibronectin (Fn). Fn-binding is mediated by a 37 kDa outer membrane protein termed Campylobacter adherence Factor (CadF). The outer membrane protein profile of a recent gastrointestinal C. jejuni clinical isolate (JHH1) was analysed using 2-DE and MS. Several spots were identified as products of the cadF gene. These included mass and pI variants of 34 and 30 kDa, as well as 24 kDa (CadF(24)) and 22 kDa (CadF(22)) mass variants. CadF variants were fully characterized by MALDI-TOF MS and MALDI-MS/MS. These data confirmed that CadF forms re-folding variants resulting in spots with lower mass and varying pI that are identical at the amino acid sequence level and are not modified post-translationally. CadF(22) and CadF(24), however, were characterized as N-terminal, membrane-associated polypeptides resulting from cleavage between serine(195) and leucine(196), and glycine(201) and phenylalanine(202), respectively. These variants were more abundant in the virulent (O) isolate of C. jejuni NCTC11168 when compared with the avirulent (genome sequenced) isolate. Hexahistidine fusion constructs of full-length CadF (34 kDa), CadF(24), and the deleted C-terminal OmpA domain (14 kDa; CadF(14)) were created in Escherichia coli. Recombinant CadF variants were probed against patient sera and revealed that only full-length CadF retained reactivity. Binding assays showed that CadF(24) retained Fn-binding capability, while CadF(14) did not bind Fn. These data suggest that the immunogenic epitope of CadF is cleaved to generate smaller Fn-binding polypeptides, which are not recognized by the host humoral response. CadF cleavage therefore may be associated with virulence in C. jejuni.
%Z FOR Codes: 60501


%0 Journal Article
%A Hare, Nathan J.
%A Cordwell, Stuart
%T Proteomics of bacterial pathogens: Pseudomonas aeruginosa infections in cystic fibrosis – A case study
%B Proteomics - Clinical Applications
%D 2010
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 4
%N 2
%P 228-248
%@ 1862-8346
%X 
%Z FOR Codes: 60109


%0 Journal Article
%~ PubMed
%A Cordwell, Stuart J
%A Thingholm, Tine E
%T Technologies for plasma membrane proteomics.
%B Proteomics
%D 2010
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 10
%N 0
%P 611-27
%@ 1615-9853
%X Cell-cell and intracellular signaling are critical mechanisms by which an organism can respond quickly and appropriately to internal or environmental stimuli. Transmission of the stimulus to effector proteins must be coordinated, rapid and transient such that the response is not exaggerated and the overall balance of the cell or tissue is retained. Proteomics technology has traditionally been adept at analyzing effector proteins (such as cytoskeletal and heat shock proteins, and those involved in metabolic processes) in studies examining the effects of altered environmental or nutritional conditions, drugs, or genetic manipulation, since these proteins are often highly abundant, soluble and therefore amenable to analysis. Conversely, the proteins mediating the transmission of the signal have been generally under-represented, typically because of their low abundance. One mechanism that has overcome this to some extent is the advent of very high-resolution phosphoproteomics techniques, which have enabled temporal profiling of intracellular signal pathways via quantitative assessment of peptide phosphorylation sites. One group of proteins, however, that still remains under-represented in proteomics studies are those found in the plasma membrane (PM). Such proteins are crucial in sensing changes in the external environment and in stimulating the transmission of the signal intracellularly. This review examines PM proteins and appraises the proteomics approaches currently available for providing a comprehensive analysis of these crucial mediators of signal pathways. We discuss different strategies for enrichment and solubilization of these proteins and include discussion on cross-linking of PM complexes and glycoproteomics as the basis for purification prior to proteomic analyses.
%Z FOR Codes: 1004


%0 Journal Article
%~ PubMed
%A Faulkner, Christine R
%A Blackman, Leila M
%A Collings, David A
%A Cordwell, Stuart J
%A Overall, Robyn L
%T Anti-tropomyosin antibodies co-localise with actin microfilaments and label plasmodesmata.
%B European journal of cell biology
%D 2009
%C Germany
%I Urban und Fischer Verlag
%V 88
%N 6
%P 357-69
%@ 0171-9335
%X The actin cytoskeleton and associated actin-binding proteins form a complex network involved in a number of fundamental cellular processes including intracellular trafficking. In plants, both actin and myosin have been localised to plasmodesmata, and thus it is likely that other actin-binding proteins are also associated with plasmodesmata structure or function. A 75-kDa protein, enriched in plasmodesmata-rich cell wall extracts from the green alga Chara corallina, was sequenced and found to contain three peptides with similarity to the animal actin-binding protein tropomyosin. Western blot analysis with anti-tropomyosin antibodies confirmed the identity of this 75-kDa protein as a tropomyosin-like protein and further identified an additional 55-kDa protein, while immunofluorescence microscopy localised the antibodies to plasmodesmata and to the subcortical actin bundles and associated structures. The anti-tropomyosin antibodies detected a single protein at 42.5 kDa in Arabidopsis thaliana extracts and two proteins at 58.5 and 54 kDa in leek extracts, and these localised to plasmodesmata and the cell plate in A. thaliana and to plasmodesmata in leek tissue. Tropomyosin is an actin-binding protein thought to be involved in a range of functions associated with the actin cytoskeleton, including the regulation of myosin binding to actin filaments, but to date no tropomyosin-like proteins have been conclusively identified in plant genomes. Our data suggests that a tropomyosin-like protein is associated with plasmodesmata.
%Z FOR Codes: 601


%0 Journal Article
%~ PubMed
%A Scott, Nichollas E
%A Cordwell, Stuart J
%T Campylobacter proteomics: guidelines, challenges and future perspectives.
%B Expert Review of Proteomics
%D 2009
%C United Kingdom
%I Expert Reviews Ltd.
%V 6
%N 1
%P 61-74
%@ 1744-8387
%X Campylobacter species are a major cause of disease in mammalian systems. The most common human etiological agent within this genus is Campylobacter jejuni - the leading cause of bacterial gastroenteritis in the developed world. While this organism has been extensively studied at the cellular level, and the genome sequences of several strains have now been elucidated, little is known regarding the role of individual proteins in virulence processes, such as adhesion, colonization and toxicity towards host cells. Proteomics encompasses the global analysis of proteins at the organism level. The technologies included under this term have now started to be utilized for understanding how Campylobacter species respond to changes in the environment, with an emphasis on the human host, as well as to map subcellular locations of proteins, in particular those that are surface-associated. C. jejuni is also of great significance as, unlike most other bacteria, it is able to post-translationally modify its proteins. The analysis of such proteins represents a major challenge in understanding this organism at the proteomic and cellular levels. This review will examine the state-of-the-art in Campylobacter proteomics, as well as provide insights into strategies that need to be undertaken to provide a comprehensive understanding of this organism at the molecular and functional level.
%Z FOR Codes: 601


%0 Journal Article
%~ PubMed
%A Lo, Miranda
%A Cordwell, Stuart J
%A Bulach, Dieter M
%A Adler, Ben
%T Comparative transcriptional and translational analysis of leptospiral outer membrane protein expression in response to temperature.
%B PLoS neglected tropical diseases
%D 2009
%C United States
%I Public Library of Science
%V 3
%N 12
%P e560
%@ 1935-2735
%X BACKGROUND: Leptospirosis is a global zoonosis affecting millions of people annually. Transcriptional changes in response to temperature were previously investigated using microarrays to identify genes potentially expressed upon host entry. Past studies found that various leptospiral outer membrane proteins are differentially expressed at different temperatures. However, our microarray studies highlighted a divergence between protein abundance and transcript levels for some proteins. Given the abundance of post-transcriptional expression control mechanisms, this finding highlighted the importance of global protein analysis systems. METHODOLOGY/PRINCIPAL FINDINGS: To complement our previous transcription study, we evaluated differences in the proteins of the leptospiral outer membrane fraction in response to temperature upshift. Outer membrane protein-enriched fractions from Leptospira interrogans grown at 30 degrees C or overnight upshift to 37 degrees C were isolated and the relative abundance of each protein was determined by iTRAQ analysis coupled with two-dimensional liquid chromatography and tandem mass spectrometry (2-DLC/MS-MS). We identified 1026 proteins with 99% confidence; 27 and 66 were present at elevated and reduced abundance respectively. Protein abundance changes were compared with transcriptional differences determined from the microarray studies. While there was some correlation between the microarray and iTRAQ data, a subset of genes that showed no differential expression by microarray was found to encode temperature-regulated proteins. This set of genes is of particular interest as it is likely that regulation of their expression occurs post-transcriptionally, providing an opportunity to develop hypotheses about the molecular dynamics of the outer membrane of Leptospira in response to changing environments. CONCLUSIONS/SIGNIFICANCE: This is the first study to compare transcriptional and translational responses to temperature shift in L. interrogans. The results thus provide an insight into the mechanisms used by L. interrogans to adapt to conditions encountered in the host and to cause disease. Our results suggest down-regulation of protein expression in response to temperature, and decreased expression of outer membrane proteins may facilitate minimal interaction with host immune mechanisms.
%Z FOR Codes: 601


%0 Journal Article
%~ PubMed
%A Scott, Nichollas E
%A Bogema, Daniel R
%A Connolly, Angela M
%A Falconer, Linda
%A Djordjevic, Steven P
%A Cordwell, Stuart J
%T Mass spectrometric characterization of the surface-associated 42 kDa lipoprotein JlpA as a glycosylated antigen in strains of Campylobacter jejuni.
%B Journal of proteome research
%D 2009
%C United States
%I American Chemical Society
%V 8
%N 10
%P 4654-64
%@ 1535-3893
%X Campylobacter jejuni is the most common cause of bacterial gastroenteritis in the developed world. Immunoproteomics highlighted a 42-45 kDa antigen that comigrated on two-dimensional (2-DE) gels with the C. jejuni major outer membrane protein (MOMP). Predictive analysis revealed two candidates for the identity of the antigen, the most likely of which was the surface-associated lipoprotein, JlpA. Recombinant JlpA (rJlpA) reacted with patient sera, confirming that JlpA is antigenic. Polyclonal antibodies raised against rJlpA reacted against 3 JlpA mass variants from multiple C. jejuni. These variants differed by approximately 1.5 kDa, suggesting the presence of the N-linked C. jejuni glycan on two sites. Soybean agglutinin affinity and 2-DE purified 2 JlpA glycoforms (43.5 and 45 kDa). Their identities were confirmed using mass spectrometry following trypsin digest. Glycopeptides within JlpA variants were identified by proteinase-K digestion, graphite micropurification and MS-MS. Sites of glycosylation were confirmed as asparagines 107 and 146, both of which are flanked by the N-linked sequon. Sequence analysis confirmed that the N146 sequon is conserved in all C. jejuni genomes examined to date, while the N107 sequon is absent in the reference strain NCTC 11168. Western blotting confirmed the presence of only a single JlpA glycoform in both virulent (O) and avirulent (GS) isolates of NCTC 11168. MS analysis showed that JlpA exists as 3 discrete forms, unmodified, glycosylated at N146, and glycosylated at both N(146/107), suggesting glycan addition at N146 is necessary for N107 glycosylation. Glycine extracts and Western blotting revealed that doubly glycosylated JlpA was the predominant form on the C. jejuni JHH1 surface; however, glycosylation is not required for antigenicity. This is the first study to identify N-linked glycosylation of a surface-exposed C. jejuni virulence factor and to show strain variation in glycosylation sites.
%Z FOR Codes: 1004


%0 Journal Article
%~ PubMed
%A Wilton, Jody
%A Jenkins, Cheryl
%A Cordwell, Stuart J
%A Falconer, Linda
%A Minion, F Chris
%A Oneal, David C
%A Djordjevic, Michael A
%A Connolly, Angela
%A Barchia, Idris
%A Walker, Mark J
%A Djordjevic, Steven P
%T Mhp493 (P216) is a proteolytically processed, cilium and heparin binding protein of Mycoplasma hyopneumoniae.
%B Molecular microbiology
%D 2009
%C United Kingdom
%I Wiley-Blackwell Publishing Ltd.
%V 71
%N 3
%P 566-82
%@ 0950-382X
%X Mycoplasma hyopneumoniae induces respiratory disease in swine by colonizing cilia causing ciliostasis, cilial loss and epithelial cell death. Heparin binds to M. hyopneumoniae cells in a dose-dependent manner and blocks its ability to adhere to porcine cilia. We show here that Mhp493 (P216), a paralogue of the cilium adhesin P97 (Mhp183), is cleaved between amino acids 1040 and 1089 generating surface-accessible, heparin-binding proteins P120 and P85. Antiphosphoserine antibodies recognized P85 in 2-D immunoblotting studies and TiO(2) chromatography of trypsin digests of P85 isolated a single peptide with an m/z of 917.3. A phosphoserine residue in the tryptic peptide (90)VSELpSFR(96) (position 94 in P85) was identified by MALDI-MS/MS. Polyhistidine fusion proteins (F1(P216), F2(P216), F3(P216)) spanning Mhp493 bound heparin with biologically significant Kd values, and heparin, fucoidan and mucin inhibited this interaction. Latex beads coated with F1(P216), F2(P216) and F3(P216) adhered to and entered porcine kidney epithelial-like (PK15) cell monolayers. Microtitre plate-based assays showed that sequences within P120 and P85 bind to porcine cilia and are recognized by serum antibodies elicited during infection by M. hyopneumoniae. Mhp493 contributes significantly to the surface architecture of M. hyopneumoniae and is the first cilium adhesin to be described that lacks an R1 cilium-binding domain.
%Z FOR Codes: 601


%0 Book Section
%A Cordwell, Stuart
%A Crossett, Ben
%A White, Melanie
%T Gel-Based Approaches
%B Proteomics
%D 2008
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 
%N 
%P 33-57
%@ 1615-9853
%E O'Connor, C.D.
%X 
%Z FOR Codes: 60109


%0 Journal Article
%~ PubMed
%A Cordwell, Stuart J
%A Len, Alice C L
%A Touma, Rachel G
%A Scott, Nichollas E
%A Falconer, Linda
%A Jones, David
%A Connolly, Angela
%A Crossett, Ben
%A Djordjevic, Steven P
%T Identification of membrane-associated proteins from Campylobacter jejuni strains using complementary proteomics technologies.
%B Proteomics
%D 2008
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 8
%N 1
%P 122-139
%@ 1615-9853
%X Campylobacter jejuni is the leading cause of food- and water-borne illness world-wide. The membrane-associated proteome of a recent C. jejuni gastrointestinal isolate (JHH1) was generated by sodium carbonate precipitation and ultracentrifugation followed by 2-DE and MALDI-TOF MS as well as 2-DLC (strong cation exchange followed by RP chromatography) of trypsin digests coupled to MS/MS (2-DLC/MS/MS). 2-DE/MS identified 77 proteins, 44 of which were predicted membrane proteins, while 2-DLC/MS/MS identified 432 proteins, of which 206 were predicted to be membrane associated. A total of 453 unique proteins (27.4% of the C. jejuni theoretical proteome), including 187 bona fide membrane proteins were identified in this study. Membrane proteins were also compared between C. jejuni JHH1 and ATCC 700297 to identify factors potentially associated with increased gastrointestinal virulence. We identified 28 proteins that were significantly (>two-fold) more abundant in, or unique to, JHH1, including eight proteins involved in chemotaxis signal transduction and flagellar motility, the amino acid-binding surface antigens CjaA and CjaC, and four outer membrane proteins (OMPs) of unknown function (Cj0129c, Cj1031, Cj1279c, and Cj1721c). Immunoblotting using convalescent patient sera generated post-gastrointestinal infection revealed 13 (JHH1) and 12 (ATCC 700297) immunoreactive proteins. These included flagellin (FlaA) and CadF as well as Omp18, Omp50, Cj1721c, PEB1A, PEB2, and PEB4A. This study provides a comprehensive analysis of membrane-associated proteins from C. jejuni.
%Z FOR Codes: 110801


%0 Journal Article
%~ PubMed
%A Upritchard, Hamish G
%A Cordwell, Stuart J
%A Lamont, Iain L
%T Immunoproteomics to examine cystic fibrosis host interactions with extracellular Pseudomonas aeruginosa proteins.
%B Infection and immunity
%D 2008
%C Customer Relations Manager,Bay 15, Shannon Industrialestate Coclare, Ireland
%I Elsevier Sci Ireland Ltd
%V 76
%N 10
%P 4624-32
%@ 1098-5522
%X The lungs of patients with cystic fibrosis (CF) are typically chronically infected with Pseudomonas aeruginosa. We used an immunoproteomics approach to analyze the responses of patients to secreted P. aeruginosa proteins. Extracellular proteins from P. aeruginosa strain PAO1 that had been grown to stationary phase were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by Western blotting using sera from four chronically infected patients. Sera from all four patients detected multiple extracellular proteins. The identities of selected proteins recognized by antisera were determined. Production of at least four of these proteins (azurin and three proteases: elastase, PrpL, and PasP) is governed by quorum sensing, consistent with active bacterial quorum sensing in the lungs of CF patients. The CF lung is generally thought to be an iron-deficient environment for infecting bacteria, and growing the bacteria in the presence of an iron-chelating agent, ethylene-diamine-di(o-hydroxyphenylacetic acid), enabled detection of additional proteins that were recognized by patient sera. The sera also detected multiple proteins from cells in the logarithmic growth phase, and protein identification suggested that most of these were the result of cell lysis or secretion in membrane vesicles. Comparison with extracellular proteins from a second P. aeruginosa strain, strain Pa4, showed that many proteins recognized by patient sera are common to both strains, although there are also some strain-specific extracellular proteins. Our data show that while there are some differences in the responses of different patients to P. aeruginosa, there are also many similarities, and that an immunoproteomics approach enables the identification of proteins that are made by P. aeruginosa during infection.
%Z FOR Codes: 110106 1108


%0 Journal Article
%A White, Melanie
%A Edwards, Alistair
%A Cordwell, Stuart
%A Van Eyk, Jennifer
%T Mitochondria: A mirror into cellular dysfunction in heart disease
%B Proteomics - Clinical Applications
%D 2008
%C Germany
%I WILEY-VCH Verlag GmbH & Co. KGaA
%V 2
%N 6
%P 845-861
%@ 1862-8346
%X 
%Z FOR Codes: 110106 111601


%0 Journal Article
%~ PubMed
%A Cordwell, Stuart J
%T Sequential extraction of proteins by chemical reagents.
%B Methods in molecular biology (Clifton, N.J.)
%D 2008
%C United States
%I Humana Press, Inc.
%V 424
%N 
%P 139-46
%@ 1064-3745
%X Reproducible techniques for the prefractionation of proteins prior to two-dimensional gel electrophoresis (2-DE) are essential for increasing the number of unique proteins that can be identified and assayed following biological experimentation. A simple and robust technique for separating highly soluble (hydrophilic) cytoplasmic proteins from poorly soluble (hydrophobic) membrane-associated proteins uses differential solubility in a progressive series of extraction buffers, each containing more potent solubilizing chaotropes and detergents. This "sequential extraction" procedure is based on protein solubility in Tris buffer for the initial removal of highly soluble proteins, whereas proteins from the insoluble pellet are then extracted in 2-DE sample buffers containing urea and CHAPS. The final step of the procedure uses thiourea and amidosulfobetaine-14 (ASB-14) to solubilize CHAPS-insoluble proteins. This procedure has been optimized for the analysis of outer membrane porins from Gram negative bacteria, as well as the separation of plasma membrane proteins from mammalian cells grown in culture, and finally for the removal of insoluble cytoskeletal structures from mammalian heart tissue.
%Z FOR Codes: 110106


%0 Book Section
%A Crossett, Ben
%A Edwards, Alistair
%A White, Melanie
%A Cordwell, Stuart
%T Statistical Analysis of Image Data Provided by Two-Dimensional Gel Electrophoresis for Discovery Proteomics
%B Methods in Molecular Medicine: Clinical Bioinformatics
%D 2008
%C New Jersey, USA
%I Humana Press
%V 
%N 
%P 271-285
%@ 9781588297914
%E Trent, Ronald A
%X 
%Z FOR Codes: 1108


%0 Journal Article
%~ PubMed
%A Edwards, Alistair V G
%A White, Melanie Y
%A Cordwell, Stuart J
%T The role of proteomics in clinical cardiovascular biomarker discovery.
%B Molecular & cellular proteomics : MCP
%D 2008
%C United States
%I American Society for Biochemistry and Molecular B
%V 7
%N 10
%P 1824-37
%@ 1535-9484
%X Cardiovascular disease remains the most common cause of death in the developed world and is predicted by the World Health Organization to kill approximately 20 million people worldwide each year until at least 2015. In light of these figures, work on producing superior tools for clinical use in the cardiovascular field is intensive. As proteins are the primary effectors of cellular function, a significant majority of this work focuses on the role of proteins in the cardiovascular system in physiological and pathological states in order to outline both mechanisms and markers of disease. One of the most effective ways to investigate these on a global basis is through proteomic analysis, which allows for broad spectrum screening of cellular protein or peptide complements during cardiovascular pathogenesis. Furthermore, specific technologies are now available to screen animal model or human blood samples for novel, improved markers of chronic disease states, such as atherosclerosis or for earlier indicators of acute myocardial stress, including ischemia/reperfusion injury and heart failure. This review summarizes current literature on the key aspects of proteomics and peptidomics related to clinical cardiovascular science.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Alexander-Kaufman, Kimberley
%A Cordwell, Stuart
%A Harper, Clive
%A Matsumoto, Izuru
%T A proteome analysis of the dorsolateral prefrontal cortex in human alcoholic patients.
%B Proteomics. Clinical applications
%D 2007
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 1
%N 1
%P 62-72
%@ 1862-8346
%X Alcoholic patients commonly experience cognitive decline. It is postulated that cognitive dysfunction is caused by an alcohol-induced region-selective brain damage, particularly to the prefrontal region, and grey and white matter may be affected differently. We used a proteomics-based approach to compare protein expression profiles of the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)) from human alcoholic and healthy control brains. Changes in the relative expression of 110 protein ''spots'' were identified in the BA9 grey matter, of which 54 were identified as 44 different proteins. In our recent article, 60 protein spots were differentially expressed in the BA9 white matter and 18 of these were identified (Alexander-Kaufman, K., James, G., Sheedy, D., Harper, C., Matsumoto, I., Mol. Psychiatry 2006, 11, 56-65). Additional BA9 white matter proteins are identified here and discussed in conjunction to our grey matter results. Thiamine-dependent enzymes transketolase and pyruvate dehydrogenase (E1? ubunit) were among the proteins identified. To our knowledge, this is the first time a disruption in thiamine-dependent enzymes has been demonstrated in the brains of ''neurologically uncomplicated'' alcoholics. By identifying protein expression changes in prefrontal grey and white matter separately, hypotheses may draw upon more mechanistic explanations as to how alcoholism causes the structural alterations associated with alcohol-related brain damage and cognitive dysfunction.
%Z FOR Codes: 60109


%0 Journal Article
%~ Isi
%A Sivagnanasundaram, S
%A Crossett, B
%A Dedova, I
%A Cordwell, S
%A Matsumoto, I
%T Abnormal pathways in the genu of the corpus callosum in schizophrenia pathogenesis: a proteome study
%B PROTEOMICS CLINICAL APPLICATIONS
%D 2007
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 1
%N 10
%P 1291-1305
%@ 1862-8346
%X 
%Z FOR Codes: 110903


%0 Journal Article
%~ Isi
%A Clark, D
%A Dedova, I
%A Cordwell, S
%A Matsumoto, I
%T Altered proteins of the anterior cingulate cortex white matter proteome in schizophrenia
%B PROTEOMICS CLINICAL APPLICATIONS
%D 2007
%C Germany
%I WILEY-V C H VERLAG GMBH
%V 1
%N 2
%P 157-166
%@ 1862-8346
%X 
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Cole, Jason N
%A Aquilina, John A
%A Hains, Peter G
%A Henningham, Anna
%A Sriprakash, Kadaba S
%A Caparon, Michael G
%A Nizet, Victor
%A Kotb, Malak
%A Cordwell, Stuart J
%A Djordjevic, Steven P
%A Walker, Mark J
%T Role of group A Streptococcus HtrA in the maturation of SpeB protease.
%B Proteomics
%D 2007
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 7
%N 24
%P 4488-98
%@ 1615-9853
%X The serine protease high-temperature requirement A (HtrA) (DegP) of the human pathogen Streptococcus pyogenes (group A Streptococcus; GAS) is localized to the ExPortal secretory microdomain and is reportedly essential for the maturation of cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Here, we utilize HSC5 (M5 serotype) and the in-frame isogenic mutant HSC5DeltahtrA to determine whether HtrA contributes to the maturation of other GAS virulence determinants. Mutanolysin cell wall extracts and secreted proteins were arrayed by 2-DE and identified by MALDI-TOF PMF analysis. HSC5DeltahtrA had elevated levels of cell wall-associated M protein, whilst the supernatant had higher concentrations of M protein fragments and a reduced amount of mature SpeB protease, compared to wild-type (WT). Western blot analysis and protease assays revealed a delay in the maturation of SpeB in the HSC5DeltahtrA supernatant. HtrA was unable to directly process SpeB zymogen (proSpeB) to the active form in vitro. We therefore conclude that HtrA plays an indirect role in the maturation of cysteine protease SpeB.
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Henrich, Silke
%A Cordwell, Stuart J
%A Crossett, Ben
%A Baker, Mark S
%A Christopherson, Richard I
%T The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells.
%B Biochimica et biophysica acta
%D 2007
%C Netherlands
%I Elsevier BV
%V 1774
%N 4
%P 413-432
%@ 0006-3002
%X Purification of organelles and analysis of their proteins is an important initial step for biological proteomics, simplifying the proteome prior to analysis by established techniques such as two-dimensional liquid chromatography (2-DLC) or two-dimensional gel electrophoresis (2-DE). Nuclear proteins play a central role in regulating gene expression, but are often under-represented in proteomic studies due to their lower abundance in comparison to cellular ''housekeeping'' metabolic enzymes and structural proteins. A reliable procedure for separation and proteomic analysis of nuclear proteins would be useful for investigations of cell proliferation and differentiation during disease processes (e.g., human cancer). In this study, we have purified nuclei from the human Burkitt''s lymphoma B-cell line, Raji, using sucrose density gradient centrifugation. The integrity and purity of the nuclei were assessed by light microscopy and proteins from the nuclear fractions were separated by 2-DE and identified using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A total of 124 unique proteins were identified, of which 91% (n=110) were predicted to be nuclear using PSORT. Proteins from the nuclear fraction were subjected to affinity chromatography on DNA-agarose to isolate DNA-binding proteins. From this purified fraction, 131 unique proteins were identified, of which 69% (n=90) were known or predicted DNA-binding proteins. Purification of nuclei and subsequent enrichment of DNA-binding proteins allowed identification of a total of 209 unique proteins, many involved in transcription and/or correlated with lymphoma, leukemia or cancer in general. The data obtained should be valuable for identification of biomarkers and targets for cancer therapy, and for furthering our understanding of the molecular mechanisms underlying lymphoma development and progression.
%Z FOR Codes: 110106


%0 Journal Article
%~ PubMed
%A Clark, D
%A Dedova, I
%A Cordwell, S
%A Matsumoto, I
%T A proteome analysis of the anterior cingulate cortex gray matter in schizophrenia.
%B Molecular psychiatry
%D 2006
%C United Kingdom
%I Nature Publishing Group
%V 11
%N 5
%P 459-70, 423
%@ 1359-4184
%X The Anterior Cingulate Cortex (ACC, Brodmans Area 24) is implicated in the pathogenesis of schizophrenia due to its normal functions and connectivity together with reports of structural, morphological and neurotransmitter aberrations within this brain area in the disease state. Two-dimensional gel electrophoresis (2DE) was employed to scan and compare the ACC gray matter proteomes between schizophrenia (n = 10) and control (n = 10) post-mortem human tissue. This proteomic approach has detected 42 protein spots with altered levels in the schizophrenia cohort, which to our knowledge is the first proteomic analysis of the ACC in schizophrenia. Thirty nine of these proteins were subsequently identified using mass spectrometry and functionally classified into metabolism and oxidative stress, cytoskeletal, synaptic, signalling, trafficking and glial-specific groups. Some of the identified proteins have previously been implicated in the disease pathogenesis and some offer new insights into schizophrenia. Investigating these proteins, the genes encoding these proteins, their functions and interactions may shed light on the molecular mechanisms underlying the heterogeneous symptoms characteristic of schizophrenia.
%Z FOR Codes: 110903 110106


%0 Journal Article
%~ Isi
%A Caixeiro, N.
%A Dedova, I.
%A Cordwell, S.
%A Matsumoto, I.
%A Sivagnanasundaram, S.
%T A proteomic analysis of Brodmann's area 46 (BA46) in schizophrenia.
%B Australian and New Zealand Journal of Psychiatry
%D 2006
%C UK
%I Taylor & Francis Ltd.
%V 40
%N 
%P A136-A136
%@ 0004-8674
%X 
%Z FOR Codes: 110903


%0 Journal Article
%~ PubMed
%A O'brien, Elizabeth
%A Dedova, Irina
%A Duffy, Liesl
%A Cordwell, Stuart
%A Karl, Tim
%A Matsumoto, Izuru
%T Effects of chronic risperidone treatment on the striatal protein profiles in rats.
%B Brain research
%D 2006
%C Netherlands
%I Elsevier Science Bv
%V 1113
%N 1
%P 24-32
%@ 0006-8993
%X Extrapyramidal symptoms (EPS) commonly occur as side effects of antipsychotic drugs (APDs) and are most likely to arise when the occupancy of dopamine D(2) receptors in the striatum by these drugs exceeds 80%. We aimed to characterize changes in the protein expression profile in the striatum of rats after chronic (4 week) supra-therapeutic (EPS-inducing) treatment with risperidone (RIS), an atypical antipsychotic drug. Administration of RIS (2.1 mg/kg/day, via subcutaneous osmotic minipumps) induced significant vacuous chewing movements and catalepsy in male Sprague-Dawley rats over a 28-day treatment period compared with a vehicle (VEH) control group (n=12) (Karl et al., unpublished observation). Using two-dimensional gel electrophoresis (2DE), total protein extracts from the rat brain striatum were separated and protein expression was analyzed by Phoretix 2D Expression and Image Beta V4.02 software followed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). 2DE gels resolved up to 450 protein spots, presumably different proteins and/or their isoforms. There were 30 protein spots showing statistically significant different densities between the RIS- and VEH-treated groups. All 30 proteins were successfully identified by MALDI-TOF MS, 28 of these were divided into groups based on their known functions. These included metabolic, signaling, transport, protein metabolism, chaperone, DNA binding and cell cycle categories. We conclude that chronic risperidone treatment accompanied by an EPS-like behavioral phenotype results in alterations in the striatal protein profile possibly subsequent to blockade of dopaminergic systems. These results suggest that possible mechanisms involved in APD-induced EPS include metabolic dysfunction and oxidative stress.
%Z FOR Codes: 110903


%0 Journal Article
%~ Isi
%A Nesvaderani, M.
%A Dedova, I.
%A Cordwell, S.
%A Matsumoto, I.
%A Sivagnanasundaram, S.
%T Identification of proteins differentially expressed in the schizophrenic hippocampus.
%B Australian and New Zealand Journal of Psychiatry
%D 2006
%C UK
%I Taylor & Francis Ltd.
%V 40
%N 
%P A137-A137
%@ 0004-8674
%X 
%Z FOR Codes: 110903


%0 Journal Article
%~ PubMed
%A White, Melanie Y
%A Hambly, Brett D
%A Jeremy, Richmond W
%A Cordwell, Stuart J
%T Ischemia-specific phosphorylation and myofilament translocation of heat shock protein 27 precedes alpha B-crystallin and occurs independently of reactive oxygen species in rabbit myocardium.
%B Journal of molecular and cellular cardiology
%D 2006
%C United Kingdom
%I Academic Press
%V 40
%N 6
%P 761-74
%@ 0022-2828
%X Heat shock protein 27 (Hsp27) and alpha B-crystallin (alphaBC) are small heat shock proteins that stabilize the myofilament during stress. We utilized two-dimensional gel electrophoresis (2-DE), phospho-fluorescence staining, titanium dioxide (TiO(2)) phosphopeptide purification and mass spectrometry (MS) to fully characterize isoelectric point (pI) variants of Hsp27 and alphaBC in rabbit myocardium subjected to brief ischemia/reperfusion (I/R) injury. Four variants of Hsp27 were detected, two of which were phosphorylated: HSP1 (at three sites, Ser15, Ser78 and Ser82) and HSP2 (at Ser15 and Ser82, but not Ser78). Three variants of alphaBC were detected: alphaBC1 was phosphorylated (at Ser59 alone) and alphaBC2 was deamidated (at Asn146). No modifications were found in the remaining variants. Both phospho-Hsp27 variants increased in abundance in tissue subjected to brief I/R injury (15 min I/60 min R) and ischemia without subsequent reflow (15I/0R), and these increases were not affected by addition of the potent antioxidant, N-(2-mercaptopropionyl) glycine (MPG; 15I/60R + MPG and 15I/0R + MPG). Abundance of native and phosphorylated (but not deamidated) alphaBC was elevated following 15I/60R; however, these increases were ameliorated by the presence of MPG, and did not occur in tissue subjected to 15I/0R. Both phospho-Hsp27 variants and phospho-alphaBC translocated to the myofilament following 15I/60R. Increased myofilament association of phospho-Hsp27 was not influenced by MPG, and there was a greater proportion of HSP2 than HSP1 in this fraction. MPG inhibited phospho-alphaBC translocation and increased alphaBC association with the myofilament did not occur during 15I/0R. Increased phosphorylation of Hsp27 is ischemia-specific and not influenced by reactive oxygen species (ROS), while increased expression and phosphorylation of alphaBC are ROS-dependant.
%Z FOR Codes: 110106


%0 Journal Article
%~ Isi
%A Burnett, T. A.
%A Dinkla, K.
%A Rohde, M.
%A Chhatwal, G. S.
%A Uphoff, C.
%A Srivastava, M.
%A Cordwell, S. J.
%A Geary, S.
%A Liao, X.
%A Minion, F. C.
%A Walker, M. J.
%A Djordjevic, S. P.
%T P159 is a proteolytically processed, surface adhesin of Mycoplasma hyopneumoniae: defined domains of P159 bind heparin and promote adherence to eukaryote cells.
%B Molecular Microbiology
%D 2006
%C UK
%I Blackwell Publishing Ltd
%V 60
%N 3
%P 669-686
%@ 1365-2958
%X 
%Z FOR Codes: 110801


%0 Journal Article
%~ PubMed
%A Clark, D
%A Dedova, I
%A Cordwell, S
%A Matsumoto, I
%T Protein expression profile in the anterior cingulate cortex.
%B Molecular psychiatry
%D 2006
%C United Kingdom
%I Nature Publishing Group
%V 11
%N 5
%P 423
%@ 1359-4184
%X 
%Z FOR Codes: 110903 110106 110903 110106


%0 Journal Article
%~ Isi
%A Sivagnanasundaram, S.
%A Dedova, I.
%A Cordwell, S.
%A Matsumoto, I.
%T Proteome analysis of the corpus callosum of schizophrenia.
%B Australian and New Zealand Journal of Psychiatry
%D 2006
%C United Kingdom
%I Blackwell Publishing Ltd.
%V 40
%N 
%P A124-A124
%@ 0004-8674
%X 
%Z FOR Codes: 110903


%0 Journal Article
%~ Isi
%A Sivagnanasundaram, S.
%A Caixeiro, N.
%A Nesvaderani, M.
%A Dedova, I.
%A Cordwell, S.
%A Matsumoto, I.
%T Proteome profiling of the prefrontal cortex (PFC) and hippocampus of schizophrenia brains.
%B American Journal of Medical Genetics Part B-Neuropsychiatric Genetics
%D 2006
%C USA
%I John Wiley & Sons, Inc.
%V 141B
%N 7
%P 698-698
%@ 1552-4841
%X 
%Z FOR Codes: 110903


%0 Journal Article
%~ PubMed
%A White, Melanie Y
%A Tchen, Adrian S
%A McCarron, Hugh C K
%A Hambly, Brett D
%A Jeremy, Richmond W
%A Cordwell, Stuart J
%T Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger.
%B Proteomics
%D 2006
%C Germany
%I Wiley - VCH Verlag GmbH & Co. KGaA
%V 6
%N 23
%P 6221-33
%@ 1615-9853
%X A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.
%Z FOR Codes:


%0 Journal Article
%~ PubMed
%A Cordwell, Stuart J
%T Technologies for bacterial surface proteomics.
%B Current opinion in microbiology
%D 2006
%C London, UK
%I Elsevier Ltd.
%V 9
%N 3
%P 320-329
%@ 1369-5274
%X Proteins from bacterial membranes are notoriously difficult to analyze using the traditional technologies encompassed under the term ''proteomics''. This is because of several factors, including the comparatively low abundance of most membrane proteins within a complex mixture containing cytoplasmic metabolic enzymes, the poor solubility of membrane components such as phospholipids, lipopolysaccharides and peptidoglycans, and the inherent hydrophobicity of many integral membrane proteins that contain up to 15 transmembrane-spanning regions. Recent advances in gel-based and chromatographic separations, coupled with protein and peptide labelling and the exquisite sensitivity of mass spectrometry, are finally beginning to overcome these problems. New technologies in membrane proteomics enable comparative analysis of these recalcitrant proteins from bacteria under a variety of biological conditions.
%Z FOR Codes: 110106


%0 Journal Article
%~ Isi
%A White, M. Y.
%A Gundry, R. L.
%A Cordwell, S. J.
%T When does a fingerprint constitute a diagnostic?
%B Lancet
%D 2006
%C 84 THEOBALDS RD, LON
%I Lancet Ltd
%V 368
%N 9540
%P 971-973
%@ 0140-6736
%X 
%Z FOR Codes: 110106