Electron-Activated Dissociation and Collision-Induced Dissociation Glycopeptide Fragmentation for Improved Glycoproteomics.

Tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) has proven a versatile tool for the identification and quantification of proteins and their post-translational modifications (PTMs). Protein glycosylation is a critical PTM for the stability and biological function of many proteins, but full characterization of site-specific glycosylation of proteins remains analytically challenging. Collision-induced dissociation (CID) is the most common fragmentation method used in LC-MS/MS workflows, but the loss of labile modifications renders CID inappropriate for detailed characterization of site-specific glycosylation. Electron-based dissociation methods provide alternatives that retain intact glycopeptide fragments for unambiguous site localization, but these methods often underperform CID due to increased reaction times and reduced efficiency. Electron-activated dissociation (EAD) is another strategy for glycopeptide fragmentation. Here, we use a ZenoTOF 7600 SCIEX instrument to compare the performance of various fragmentation techniques for the analysis of a complex mixture of mammalian O- and N-glycopeptides. We found CID fragmentation identified the most glycopeptides and generally produced higher quality spectra, but EAD provided improved confidence in glycosylation site localization. Supplementing EAD with CID fragmentation (EAciD) further increased the number and quality of glycopeptide identifications, while retaining localization confidence. These methods will be useful for glycoproteomics workflows for either optimal glycopeptide identification or characterization.

N-glycosylation on Oryza sativa root germin-like protein 1 is conserved but not required for stability or activity.

Germin and germin-like proteins (GLPs) are a broad family of extracellular glycoproteins ubiquitously distributed in plants. Overexpression of Oryza sativa root germin like protein 1 (OsRGLP1) enhances superoxide dismutase (SOD) activity in transgenic plants. Here, we report bioinformatic analysis and heterologous expression of OsRGLP1 to study the role of glycosylation on OsRGLP1 protein stability and activity. Sequence analysis of OsRGLP1 homologs identified diverse N-glycosylation sequons, one of which was highly conserved. We therefore expressed OsRGLP1 in glycosylation-competent Saccharomyces cerevisiae as a Maltose Binding Protein (MBP) fusion. Mass spectrometry analysis of purified OsRGLP1 showed it was expressed by S. cerevisiae in both N-glycosylated and unmodified forms. Glycoprotein thermal profiling showed little difference in the thermal stability of the glycosylated and unmodified protein forms. Circular Dichroism spectroscopy of MBP-OsRGLP1 and a N-Q glycosylation-deficient variant showed that both glycosylated and unmodified MBP-OsRGLP1 had similar secondary structure, and both forms had equivalent SOD activity. Together, we concluded that glycosylation was not critical for OsRGLP1 protein stability or activity, and it could therefore likely be produced in Escherichia coli without glycosylation. Indeed, we found that OsRGLP1 could be efficiently expressed and purified from K12 shuffle E. coli with a specific activity of 1251 ± 70 Units/mg. In conclusion, we find that some highly conserved N-glycosylation sites are not necessarily required for protein stability or activity, and describe a suitable method for production of OsRGLP1 which paves the way for further characterization and use of this protein.

A Unique Relative of Rotifer Birnavirus Isolated from Australian Mosquitoes.

The family Birnaviridae are a group of non-enveloped double-stranded RNA viruses which infect poultry, aquatic animals and insects. This family includes agriculturally important pathogens of poultry and fish. Recently, next-generation sequencing technologies have identified closely related birnaviruses in Culex, Aedes and Anopheles mosquitoes. Using a broad-spectrum system based on detection of long double-stranded RNA, we have discovered and isolated a birnavirus from Aedes notoscriptus mosquitoes collected in northern New South Wales, Australia. Phylogenetic analysis of Aedes birnavirus (ABV) showed that it is related to Rotifer birnavirus, a pathogen of microscopic aquatic animals. In vitro cell infection assays revealed that while ABV can replicate in Aedes-derived cell lines, the virus does not replicate in vertebrate cells and displays only limited replication in Culex- and Anopheles-derived cells. A combination of SDS-PAGE and mass spectrometry analysis suggested that the ABV capsid precursor protein (pVP2) is larger than that of other birnaviruses and is partially resistant to trypsin digestion. Reactivity patterns of ABV-specific polyclonal and monoclonal antibodies indicate that the neutralizing epitopes of ABV are SDS sensitive. Our characterization shows that ABV displays a number of properties making it a unique member of the Birnaviridae and represents the first birnavirus to be isolated from Australian mosquitoes.

Evolution for improved secretion and fitness may be the selective pressures leading to the emergence of two NDM alleles.

The New Delhi metallo-ß-lactamase (NDM-1) mediates resistance to ß-lactam antibiotics. NDM-1 was likely formed as the result of a gene fusion between sequences encoding the first six amino acids of cytoplasm-localised aminoglycosidase, AphA6, and a periplasmic metallo-ß -lactamase. We show that NDM-1 has an atypical signal peptide and is inefficiently secreted. Two new blaNDM-1 alleles that have polymorphisms in the signal peptide; NDM-1(P9R), a proline to arginine substitution, and NDM-2, a proline to alanine substitution (P28A) were studied. Here, we show that both the P9R and P28A substitutions improve secretion compared to NDM-1 and display higher resistance to some ß-lactam antibiotics. Mass spectrometry analysis of these purified NDM proteins showed that the P28A mutation in NDM-2 creates new signal peptide cleavage sites at positions 27 and 28. For NDM-1, we detected a signal peptide cleavage site between L21/M22 of the precursor protein. We find no evidence that NDM-1 is a lipoprotein, as has been reported elsewhere. In addition, expression of NDM-2 improves the fitness of E. coli, compared to NDM-1, in the absence of antibiotic selection. This study shows how optimization of the secretion efficiency of NDM-1 leads to increased resistance and increased fitness.

Process Proteomics of Beer Reveals a Dynamic Proteome with Extensive Modifications.

Modern beer production is a complex industrial process. However, some of its biochemical details remain unclear. Using mass spectrometry proteomics, we have performed a global untargeted analysis of the proteins present across time during nanoscale beer production. Samples included sweet wort produced by a high temperature infusion mash, hopped wort, and bright beer. This analysis identified over 200 unique proteins from barley and yeast, emphasizing the complexity of the process and product. We then used data independent SWATH-MS to quantitatively compare the relative abundance of these proteins throughout the process. This identified large and significant changes in the proteome at each process step. These changes described enrichment of proteins by their biophysical properties, and identified the appearance of dominant yeast proteins during fermentation. Altered levels of malt modification also quantitatively changed the proteomes throughout the process. Detailed inspection of the proteomic data revealed that many proteins were modified by protease digestion, glycation, or oxidation during the processing steps. This work demonstrates the opportunities offered by modern mass spectrometry proteomics in understanding the ancient process of beer production.

High-performance targeted mass spectrometry with precision data-independent acquisition reveals site-specific glycosylation macroheterogeneity.

Protein glycosylation is a critical post-translational modification that regulates the structure, stability, and function of many proteins. Mass spectrometry is currently the preferred method for qualitative and quantitative characterization of glycosylation. However, the inherent heterogeneity of glycosylation makes its analysis difficult. Quantification of glycosylation occupancy, or macroheterogeneity, has proven to be especially challenging. Here, we used a variation of high-resolution multiple reaction monitoring (MRM(HR)) or pseudo-MRM for targeted data-independent acquisition that we term SWAT (sequential window acquisition of targeted fragment ions). We compared the analytical performance of SWATH (sequential window acquisition of all theoretical fragment ions), SWAT, and SRM (selected reaction monitoring) using a suite of synthetic peptides spiked at various concentrations into a complex yeast tryptic digest sample. SWAT provided superior analytical performance to SWATH in a targeted approach. We then used SWAT to measure site-specific N-glycosylation occupancy in cell wall glycoproteins from yeast with defects in the glycosylation biosynthetic machinery. SWAT provided robust measurement of occupancy at more N-glycosylation sites and with higher precision than SWATH, allowing identification of novel glycosylation sites dependent on the Ost3p and Ost6p regulatory subunits of oligosaccharyltransferase.

SWATH analysis of the synaptic proteome in Alzheimer's disease.

Brain tissue from Alzheimer's disease patients exhibits synaptic degeneration in selected regions. Synaptic dysfunction occurs early in the disease and is a primary pathological target for treatment. The molecular mechanisms underlying this degeneration remain unknown. Quantifying the synaptic proteome in autopsy brain and comparing tissue from Alzheimer's disease cases and subjects with normal aging are critical to understanding the molecular mechanisms associated with Alzheimer pathology. We isolated synaptosomes from hippocampus and motor cortex so as to reduce sample complexity relative to whole-tissue homogenates. Synaptosomal extracts were subjected to strong cation exchange (SCX) fractionation to further partition sample complexity; each fraction received SWATH-based information-dependent acquisition to generate a comprehensive peptide-ion library. The expression of synaptic proteins from AD hippocampus and motor cortex was then compared between groups. A total of 2077 unique proteins were identified at a critical local false discovery rate <5%. Thirty of these, including 17 novel proteins, exhibited significant expression differences between cases and controls; these proteins are involved in cellular functions including structural maintenance, signal transduction, autophagy, oxidative stress, and proteasome activity, or they have synaptic-vesicle related or energy-related functions. Differentially expressed proteins were subjected to pathway analysis to identify protein-protein interactions. This revealed that the most perturbed molecular and cellular functions were cellular assembly and organization. Core analysis revealed RhoA signaling to be the top canonical pathway. Network analysis showed that differentially expressed proteins were related to cellular assembly and organization, and cellular function and maintenance. This is the first study to combine SCX fractionation with SWATH analysis. SWATH is a promising new technique that can greatly enhance protein identification in any proteome, and has many other benefits; however, there are limitations yet to be resolved.

High-antibody-producing Chinese hamster ovary cells up-regulate intracellular protein transport and glutathione synthesis.

Chinese hamster ovary (CHO) cells are the preferred production host for therapeutic monoclonal antibodies (mAb) due to their ability to perform post-translational modifications and their successful approval history. The completion of the genome sequence for CHO cells has reignited interest in using quantitative proteomics to identify markers of good production lines. Here we applied two different proteomic techniques, iTRAQ and SWATH, for the identification of expression differences between a high- and low-antibody-producing CHO cell lines derived from the same transfection. More than 2000 proteins were quantified with 70 of them classified as differentially expressed in both techniques. Two biological processes were identified as differentially regulated by both methods: up-regulation of glutathione biosynthesis and down-regulation of DNA replication. Metabolomic analysis confirmed that the high producing cell line displayed higher intracellular levels of glutathione. SWATH further identified up-regulation of actin filament processes and intracellular transport and down regulation of several growth-related processes. These processes may be important for conferring high mAb production and as such are promising candidates for targeted engineering of high-expression cell lines.

Targeted quantitative analysis of synaptic proteins in Alzheimer's disease brain.

Brain tissue from Alzheimer's disease (AD) patients shows significant loss of synapses in selected regions. Synaptic degeneration is the best predictor for loss of cognitive functions ante mortem. The molecular mechanisms underlying this degeneration remain unknown. Our previous two-dimensional gel-electrophoresis proteomics study found that 26 synaptic proteins are differentially expressed in Alzheimer's brain. It is difficult to quantify global protein expression using this technique because (a) several proteins can migrate together and (b) isoforms of the same protein can migrate to different places. The present study estimated global synaptic protein levels by label-free multiple reaction monitoring. Multiple reaction monitoring is a powerful and sensitive mass spectrometry technique that specifically targets multiple protein of interests. The severely AD-affected hippocampus was compared with motor cortex, a relatively spared region. We targeted ten proteins in autopsy brain based on the earlier study. Analytes separated by high performance liquid-chromatography were monitored on a hybrid triple quadrupole linear ion trap mass spectrometer in multiple reaction monitoring mode. With the use of an internal standard protein, linear and highly reproducible (CV<9%) label-free assays were achieved. Data were contrasted with the gel-based study to highlight differences and similarities. Significantly higher expression levels of peroxiredoxin-1 (may provide antioxidant protection) and dihydropyrimidinase-related protein-1 (associated with cytoskeletal remodeling) were found in AD hippocampus. Significantly lower levels of peroxiredoxin-1 and the energy-related enzymes creatine kinase B and fructose-bisphosphate aldolase C were found in non-AD hippocampus. Our previously reported difference in synaptotagmin expression is probably isoform-specific. These findings suggest potential roles of key proteins in synaptic loss in AD, and/or a protective mechanism in non-AD brain tissue.

Metabolic adaptation and trophic strategies of soil bacteria-C1- metabolism and sulfur chemolithotrophy in Starkeya novella.

The highly diverse and metabolically versatile microbial communities found in soil environments are major contributors to the global carbon, nitrogen, and sulfur cycles. We have used a combination of genome -based pathway analysis with proteomics and gene expression studies to investigate metabolic adaptation in a representative of these bacteria, Starkeya novella, which was originally isolated from agricultural soil. This bacterium was the first facultative sulfur chemolithoautotroph that was isolated and it is also able to grow with methanol and on over 39 substrates as a heterotroph. However, using glucose, fructose, methanol, thiosulfate as well as combinations of the carbon compounds with thiosulfate as growth substrates we have demonstrated here that contrary to the previous classification, S. novella is not a facultative sulfur chemolitho- and methylotroph, as the enzyme systems required for these two growth modes are always expressed at high levels. This is typical for key metabolic pathways. In addition enzymes for various pathways of carbon dioxide fixation were always expressed at high levels, even during heterotrophic growth on glucose or fructose, which suggests a role for these pathways beyond the generation of reduced carbon units for cell growth, possibly in redox balancing of metabolism. Our results then indicate that S. novella, a representative of the Xanthobacteraceae family of methylotrophic soil and freshwater dwelling bacteria, employs a mixotrophic growth strategy under all conditions tested here. As a result the contribution of this bacterium to either carbon sequestration or the release of climate active substances could vary very quickly, which has direct implications for the modeling of such processes if mixotrophy proves to be the main growth strategy for large populations of soil bacteria.

Arginine methylation of hnRNP A2 does not directly govern its subcellular localization.

The hnRNP A/B paralogs A1, A2/B1 and A3 are key components of the nuclear 40S hnRNP core particles. Despite a high degree of sequence similarity, increasing evidence suggests they perform additional, functionally distinct roles in RNA metabolism. Here we identify and study the functional consequences of differential post-translational modification of hnRNPs A1, A2 and A3. We show that while arginine residues in the RGG box domain of hnRNP A1 and A3 are almost exhaustively, asymmetrically dimethylated, hnRNP A2 is dimethylated at only a single residue (Arg-254) and this modification is conserved across cell types. It has been suggested that arginine methylation regulates the nucleocytoplasmic distribution of hnRNP A/B proteins. However, we show that transfected cells expressing an A2(R254A) point mutant exhibit no difference in subcellular localization. Similarly, immunostaining and mass spectrometry of endogenous hnRNP A2 in transformed cells reveals a naturally-occurring pool of unmethylated protein but an exclusively nuclear pattern of localization. Our results suggest an alternative role for post-translational arginine methylation of hnRNPs and offer further evidence that the hnRNP A/B paralogs are not functionally redundant.

The molybdoproteome of Starkeya novella--insights into the diversity and functions of molybdenum containing proteins in response to changing growth conditions.

Molybdenum enzymes are known to underpin key reactions in the biological carbon, nitrogen and sulfur cycles, however, the diversity of these enzymes and the reactions they catalyze especially in bacteria is much greater than currently known. We have analysed the molybdoproteome of the soil bacterium Starkeya novella as a function of growth mode and identified a complete pathway for Mo-PPT synthesis, a Mo transporter and 18 gene loci encoding mononuclear Mo-enzymes of the Xanthine Oxidase, Sulfite Oxidase and DMSO Reductase enzyme families. This relatively high number of Mo enzymes may be a specific property of the taxonomic group (Xanthobacteraceae) to which S. novella belongs. About 70% of S. novella Mo enzymes have no characterized close relatives, and two thirds of them are expressed under the conditions analysed, which included heterotrophy, methylotrophy, chemolithotrophy and mixotrophy. Many enzymes were clearly regulated in response to either the type of carbon source present in the growth medium or the presence of thiosulfate, and two, in particular, including an uncharacterized enzyme of the XO family (Snov_3370) were highly abundant under all growth conditions tested. We also uncovered novel enzymes with links to growth in the presence of thiosulfate, such as a PaoABC-type aldehyde oxidoreductase and an uncharacterized group 1 sulfite oxidase family enzyme, although the function of these enzymes during sulfur oxidation is unclear at present. Clearly, further work is needed to uncover the significance of these enzymes for cell metabolism.

The synaptic proteome in Alzheimer's disease.

BACKGROUND: Synaptic dysfunction occurs early in Alzheimer's disease (AD) and is recognized to be a primary pathological target for treatment. Synapse degeneration or dysfunction contributes to clinical signs of dementia through altered neuronal communication; the degree of synaptic loss correlates strongly with cognitive impairment. The molecular mechanisms underlying synaptic degeneration are still unclear, and identifying abnormally expressed synaptic proteins in AD brain will help to elucidate such mechanisms and to identify therapeutic targets that might slow AD progression. METHODS: Synaptosomal fractions from human autopsy brain tissue from subjects with AD (n = 6) and without AD (n = 6) were compared using two-dimensional differential in-gel electrophoresis. AD pathology is region specific; human subjects can be highly variable in age, medication, and other factors. To counter these factors, two vulnerable areas (the hippocampus and the temporal cortex) were compared with two relatively spared areas (the motor and occipital cortices) within each group. Proteins exhibiting significant changes in expression were identified (≥20% change, Newman-Keuls P value < .05) using either matrix-assisted laser desorption ionization time-of-flight or electrospray ionisation quadrupole-time of flight mass spectrometry. RESULTS: Twenty-six different synaptic proteins exhibited more than twofold differences in expression between AD and normal subjects. These proteins are involved in regulating different cellular functions, including energy metabolism, signal transduction, vesicle transport, structure, and antioxidant activity. CONCLUSION: Comparative proteome analysis uncovered markers of pathogenic mechanisms involved in synaptic dysfunction.

The transcriptome and proteome are altered in marine polychaetes (Annelida) exposed to elevated metal levels.

Polychaetes are often used in toxicological studies to understand mechanisms of resistance and for biomarker detection, however, we know of only a few genetic pathways involved in resistance. We found the marine polychaete Ophelina sp.1 (Opheliidae) in sediment containing high copper levels and investigated this phenomenon by measuring metal accumulation in the worms and changes in gene and protein expression. We sequenced the transcriptome of Ophelina sp.1 from both the impacted and reference sediments using 454-sequencing and analysed their proteomes using differential in gel electrophoresis (DIGE). We used the sequenced transcriptome to guide protein identification. Transcripts coding for the copper chaperone, Atox1, were up-regulated in the worms inhabiting the high copper sediment. In addition, genes coding for respiratory proteins, detoxification proteins and cytoskeletal proteins were significantly altered in metal-exposed worms; many of these changes were also detected in the proteome. This dual approach has provided a better understanding of heavy metal resistance in polychaetes and we now have a wider range of suitable indicator genes and proteins for future biomarker development.

Direct involvement of the TEN domain at the active site of human telomerase.

Telomerase is a ribonucleoprotein that adds DNA to the ends of chromosomes. The catalytic protein subunit of telomerase (TERT) contains an N-terminal domain (TEN) that is important for activity and processivity. Here we describe a mutation in the TEN domain of human TERT that results in a greatly increased primer K(d), supporting a role for the TEN domain in DNA affinity. Measurement of enzyme kinetic parameters has revealed that this mutant enzyme is also defective in dNTP polymerization, particularly while copying position 51 of the RNA template. The catalytic defect is independent of the presence of binding interactions at the 5'-region of the DNA primer, and is not a defect in translocation rate. These data suggest that the TEN domain is involved in conformational changes required to position the 3'-end of the primer in the active site during nucleotide addition, a function which is distinct from the role of the TEN domain in providing DNA binding affinity.

NS1' of flaviviruses in the Japanese encephalitis virus serogroup is a product of ribosomal frameshifting and plays a role in viral neuroinvasiveness.

Flavivirus NS1 is a nonstructural protein involved in virus replication and regulation of the innate immune response. Interestingly, a larger NS1-related protein, NS1', is often detected during infection with the members of the Japanese encephalitis virus serogroup of flaviviruses. However, how NS1' is made and what role it performs in the viral life cycle have not been determined. Here we provide experimental evidence that NS1' is the product of a -1 ribosomal frameshift event that occurs at a conserved slippery heptanucleotide motif located near the beginning of the NS2A gene and is stimulated by a downstream RNA pseudoknot structure. Using site-directed mutagenesis of these sequence elements in an infectious clone of the Kunjin subtype of West Nile virus, we demonstrate that NS1' plays a role in viral neuroinvasiveness.

Amplification of telomerase reverse transcriptase gene in human mammary epithelial cells with limiting telomerase RNA expression levels.

Activation of telomerase is a crucial step during cellular immortalization, and in some tumors this results from amplification of the human telomerase reverse transcriptase (hTERT) gene. Immortalization of normal human cells has been achieved by transduction with hTERT cDNA under the control of a strong heterologous enhancer/promoter, but this is sometimes an inefficient process, with periods of poor growth or even crisis occurring before immortalization. Here, we showed that normal human mammary epithelial cells expressing exogenous hTERT amplified the transgene extensively and expressed high levels of hTERT mRNA and protein. Paradoxically, the cells had low levels of telomerase activity and very short telomeres, indicating that telomerase activity did not correlate with hTERT expression. These cells contained only approximately 20 human telomerase RNA (hTR) molecules/cell (compared with approximately 120 hTR molecules per 293 cell). Expression of exogenous hTR caused increased telomerase activity and telomere lengthening. These data indicate that some hTERT-transduced normal cells may express high levels of the transgene but fail to up-regulate endogenous hTR expression sufficiently to enable expression of robust levels of telomerase activity.

Application of proteomics to Pseudomonas aeruginosa.

The recent completion of the Pseudomonas Genome Project, in conjunction with the Pseudomonas Community Annotation Project (PseudoCAP) has fast-tracked our ability to apply the tools encompassed under the term 'proteomics' to this pathogen. Such global approaches will allow the research community to answer long-standing questions regarding the ability of Pseudomonas aeruginosa to survive diverse habitats, its high intrinsic resistance to antibiotics and its pathogenic nature towards humans. Proteomics provides an array of tools capable of confirming the expression of Open Reading Frames (ORF), the relative levels of their expression, the environmental conditions required for this expression and the sub-cellular location of the encoded gene-products. Since proteins are important cellular effectors, the biological questions we pose can be defined in terms of changes in protein expression detectable by separation to purity using two-dimensional gel electrophoresis (2-DGE) and relation to gene sequences via mass spectrometry. As such, we can compare strains with well-characterized phenotypic differences, growth under a variety of stresses, protein interactions and complexes and aid in defining proteins of unknown function. While the complete genome has only recently been finished, a number of studies have already utilized this information and examined various protein gene-products using proteomics. This review summarizes the application of proteomics to P. aeruginosa and highlights potential areas of future research, including overcoming the traditional technical limitations associated with 2-DGE. More focused approaches that target sub-cellular fractions ('sub-proteomes') prior to 2-DGE can provide further functional information. A review of current and previous proteomic projects on P. aeruginosa is presented, as well as theoretical considerations of the importance of sub-proteomic approaches to enhance these investigations.

Proteome analysis of extracellular proteins regulated by the las and rhl quorum sensing systems in Pseudomonas aeruginosa PAO1.

The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhlRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown QS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Deltalas mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-I) could not be detected when any of the lasRI or rhlRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of QS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a QS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.

Proteomic comparison of membrane and extracellular proteins from invasive (PAO1) and cytotoxic (6206) strains of Pseudomonas aeruginosa.

Strains of Pseudomonas aeruginosa can be phenotypically classified by their mode of pathogenicity as either invasive, where the bacterium is internalised by host cells, or cytotoxic, where the host cell is killed without internalisation through the expression of cytotoxicity factors. These phenotypes are thought to depend primarily on the interactions of pseudomonal membrane and secreted proteins with host cells. We report here comparisons of outer membrane and extracellular protein-enriched fractions from invasive (PAO1) and cytotoxic (6206) strains of P. aeruginosa separated by two-dimensional (2-D) gel electrophoresis. Gel image comparisons revealed the two strains express essentially identical membrane protein profiles under the conditions investigated. Membrane protein strain differences were typically the result of minor amino acid sequence variations resulting in small mass and isoelectric point shifts visible on 2-D gels. Analysis of extracellular proteins from stationary phase growth, however, revealed significantly different protein profiles. Extracellular fractions from the invasive PAO1 strain were dominated by extracellular proteases including elastase (LasB), LasA protease and chitin-binding protein, as well as several previously designated 'hypothetical' proteins. LasB appeared to be highly processed with 28 discrete mass and isoelectric point forms detected in this study. The significant number of active extracellular proteases (including LasB itself) may account for this processing. Conversely, extracellular fractions from strain 6206 consisted mainly of cellular and membrane exposed proteins including GroEL, DnaK and flagellar subunits. These are thought to result from cellular turnover during growth and the reliance on the secretory mechanisms of this strain to produce high levels of cytotoxicity factors, such as ExoU, which may be produced only upon specific interactions with host cells. These studies will aid in elucidating the differences between invasive and cytotoxic P. aeruginosa at the proteome level.