Histone H3 clipping is a novel signature of human neutrophil extracellular traps.

Neutrophils are critical to host defence, executing diverse strategies to perform their antimicrobial and regulatory functions. One tactic is the production of neutrophil extracellular traps (NETs). In response to certain stimuli, neutrophils decondense their lobulated nucleus and release chromatin into the extracellular space through a process called NETosis. However, NETosis, and the subsequent degradation of NETs, can become dysregulated. NETs are proposed to play a role in infectious as well as many non-infection related diseases including cancer, thrombosis, autoimmunity and neurological disease. Consequently, there is a need to develop specific tools for the study of these structures in disease contexts. In this study, we identified a NET-specific histone H3 cleavage event and harnessed this to develop a cleavage site-specific antibody for the detection of human NETs. By microscopy, this antibody distinguishes NETs from chromatin in purified and mixed cell samples. It also detects NETs in tissue sections. We propose this antibody as a new tool to detect and quantify NETs.

High resolution quantitative proteomics of HeLa cells protein species using stable isotope labeling with amino acids in cell culture(SILAC), two-dimensional gel electrophoresis(2DE) and nano-liquid chromatograpohy coupled to an LTQ-OrbitrapMass spectrometer.

The proteomics field has shifted over recent years from two-dimensional gel electrophoresis (2-DE)-based approaches to SDS-PAGE or gel-free workflows because of the tremendous developments in isotopic labeling techniques, nano-liquid chromatography, and high-resolution mass spectrometry. However, 2-DE still offers the highest resolution in protein separation. Therefore, we combined stable isotope labeling with amino acids in cell culture of controls and apoptotic HeLa cells with 2-DE and the subsequent analysis of tryptic peptides via nano-liquid chromatography coupled to an LTQ-Orbitrap mass spectrometer to obtain quantitative data using the methods with the highest resolving power on all levels of the proteomics workflow. More than 1,200 proteins with more than 2,700 protein species were identified and quantified from 816 Coomassie Brilliant Blue G-250 stained 2-DE spots. About half of the proteins were identified and quantified only in single 2-DE spots. The majority of spots revealed one to five proteins; however, in one 2-DE spot, up to 23 proteins were identified. Only half of the 2-DE spots represented a dominant protein with more than 90% of the whole protein amount. Consequently, quantification based on staining intensities in 2-DE gels would in approximately half of the spots be imprecise, and minor components could not be quantified. These problems are circumvented by quantification using stable isotope labeling with amino acids in cell culture. Despite challenges, as shown in detail for lamin A/C and vimentin, the quantitative changes of protein species can be detected. The combination of 2-DE with high-resolution nano-liquid chromatography-mass spectrometry allowed us to identify proteomic changes in apoptotic cells that would be unobservable using any of the other previously employed proteomic workflows.

Quantitative phosphoproteomics reveals link between Helicobacter pylori infection and RNA splicing modulation in host cells.

The Gram-negative, spiral-shaped bacterium Helicobacter pylori is a common human pathogen that causes chronic inflammation of the human gastric mucosa, leading to peptic ulceration and/or gastric cancer. Here, we analyzed changes in the phosphoproteome of gastric epithelial cells (AGS) upon infection with H. pylori using a combination of SILAC, phosphoprotein enrichment, 2-DE, and MALDI TOF/TOF-MS. From a total of 526 spots we identified 391 protein species (143 proteins) and quantified 332 (127 proteins). Nearly, one-third of the identified proteins (40/143) were associated with the spliceosome or RNA splicing. The abundance of 20 proteins was altered by H. pylori infection, in particular, a number of serine arginine-rich (SR) proteins involved in the regulation and control of alternative splicing. Importantly, the combined methodologies enabled the detection of infection-dependent protein species-specific regulation, suggesting functional modulation of individual protein species. These findings reveal unexpected new insights into the mechanisms of host cell manipulation by H. pylori, which are likely associated with gastric pathologies, including gastric cancer.

Quantitative proteome analysis of the 20S proteasome of apoptotic Jurkat T cells.

Regulated proteolysis plays important roles in cell biology and pathological conditions. A crosstalk exists between apoptosis and the ubiquitin-proteasome system, two pathways responsible for regulated proteolysis executed by different proteases. To investigate whether the apoptotic process also affects the 20S proteasome, we performed three independent SILAC-based quantitative proteome approaches: 1-DE/MALDI-MS, small 2-DE/MALDI-MS and large 2-DE/nano-LC-ESI-MS. Taking the results of all experiments together, no quantitative changes were observed for the α- and ß-subunits of the 20S proteasome except for subunit α7. This protein was identified in two protein spots with a down-regulation of the more acidic protein species (α7a) and up-regulation of the more basic protein species (α7b) during apoptosis. The difference in these two α7 protein species could be attributed to oxidation of cysteine-41 to cysteine sulfonic acid and phosphorylation at serine-250 near the C terminus in α7a, whereas these modifications were missing in α7b. These results pointed to the biological significance of posttranslational modifications of proteasome subunit α7 after induction of apoptosis.

Proteomic identification of secreted proteins of Propionibacterium acnes.

BACKGROUND: The anaerobic Gram-positive bacterium Propionibacterium acnes is a human skin commensal that resides preferentially within sebaceous follicles; however, it also exhibits many traits of an opportunistic pathogen, playing roles in a variety of inflammatory diseases such as acne vulgaris. To date, the underlying disease-causing mechanisms remain ill-defined and knowledge of P. acnes virulence factors remains scarce. Here, we identified proteins secreted during anaerobic cultivation of a range of skin and clinical P. acnes isolates, spanning the four known phylogenetic groups. RESULTS: Culture supernatant proteins of P. acnes were separated by two-dimensional electrophoresis (2-DE) and all Coomassie-stained spots were subsequently identified by MALDI mass spectrometry (MALDI-MS). A set of 20 proteins was secreted in the mid-exponential growth phase by the majority of strains tested. Functional annotation revealed that many of these common proteins possess degrading activities, including glycoside hydrolases with similarities to endoglycoceramidase, ß-N-acetylglucosaminidase and muramidase; esterases such as lysophospholipase and triacylglycerol lipase; and several proteases. Other secreted factors included Christie-Atkins-Munch-Petersen (CAMP) factors, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and several hypothetical proteins, a few of which are unique to P. acnes. Strain-specific differences were apparent, mostly in the secretion of putative adhesins, whose genes exhibit variable phase variation-like sequence signatures. CONCLUSIONS: Our proteomic investigations have revealed that the P. acnes secretome harbors several proteins likely to play a role in host-tissue degradation and inflammation. Despite a large overlap between the secretomes of all four P. acnes phylotypes, distinct differences between predicted host-tissue interacting proteins were identified, providing potential insight into the differential virulence properties of P. acnes isolates. Thus, our data presents a rich resource for guiding much-needed investigations on P. acnes virulence factors and host interacting properties.

Helicobacter pylori proteomics by 2-DE/MS, 1-DE-LC/MS and functional data mining.

With its predicted proteome of 1550 proteins (data set Etalon) Helicobacter pylori 26695 represents a perfect model system of medium complexity for investigating basic questions in proteomics. We analyzed urea-solubilized proteins by 2-DE/MS (data set 2-DE) and by 1-DE-LC/MS (Supprot); proteins insoluble in 9 M urea but solubilized by SDS (Pellet); proteins precipitating in the Sephadex layer at the application side of IEF (Sephadex) by 1-DE-LC/MS; and proteins precipitating close to the application side within the IEF gel by LC/MS (Startline). The experimental proteomics data of H. pylori comprising 567 proteins (protein coverage: 36.6%) were stored in the Proteome Database System for Microbial Research (http://www.mpiib-berlin.mpg.de/2D-PAGE/), which gives access to raw mass spectra (MALDI-TOF/TOF) in T2D format, as well as to text files of peak lists. For data mining the protein mapping and comparison tool PROMPT (http://webclu.bio.wzw.tum.de/prompt/) was used. The percentage of proteins with transmembrane regions, relative to all proteins detected, was 0, 0.2, 0, 0.5, 3.8 and 6.3% for 2-DE, Supprot, Startline, Sephadex, Pellet, and Etalon, respectively. 2-DE does not separate membrane proteins because they are insoluble in 9 M urea/70 mM DTT and 2% CHAPS. SDS solubilizes a considerable portion of the urea-insoluble proteins and makes them accessible for separation by SDS-PAGE and LC. The 2-DE/MS analysis with urea-solubilized proteins and the 1-DE-LC/MS analysis with the urea-insoluble protein fraction (Pellet) are complementary procedures in the pursuit of a complete proteome analysis. Access to the PROMPT-generated diagrams in the Proteome Database allows the mining of experimental data with respect to other functional aspects.

Classical proteomics: two-dimensional electrophoresis/MALDI mass spectrometry.

The rapid development in proteomics over the last 10 years has led to a series of new technologies and combinations of them designed to unravel as much as possible of the proteins of an organism or otherwise specified biological material. Despite being a little tricky at certain steps, 2-DE has a very high resolution power with more than 10,000 spots per gel and is able to separate one protein into its different protein species caused by posttranslational modifications, alternative splicing or genetic variability. This high-resolution separation is combined with a highly sensitive identification method using peptide mass fingerprinting combined with sequence information by MS/MS, which results in high sequence coverage: the key to elucidate protein species structures. The off-line measurement by MALDI-TOFTOF-MS allows the repeated measurement of each sample and therefore provides more complete structure information for each protein species. The presented protocols represent the basic technology consisting of 2-DE, two staining methods, tryptic digestion and MALDI-TOFTOF-MS.

Identification of proteins that modify cataract of mouse eye lens.

The occurrence of a nuclear cataract in the eye lens due to disruption of the alpha3Cx46 connexin gene, Gja3, is dependent on strain background in a mouse model, implicating factors that modify the pathology. The differences upon cataractogenesis in the urea soluble proteins of the lens of two mouse strains, C57BL/6J and 129/SvJ, were analyzed by a comparative proteomics approach. Determination of the complete proteome of an organ offers the opportunity to characterize at a molecular level, differences in gene expression and PTMs occurring during pathology and between individuals. The abundance of 63 protein species was altered between the strains. A unique aspect of this study is the identification of chaperonin subunit 6A, mortalin, ERp29, and syntaxin-binding protein 6 in the eye lens. DNA polymorphisms resulting in nonconservative amino acid changes that led to altered physicochemical properties of the proteins were detected for mortalin, chaperonin subunit 6A, annexin A1, and possibly gamma-N crystallin. The results show HSP27/25 and/or ERp29 are the likely major modifying factors for cataractogenesis. Extension of the results suggests that small heat-shock proteins have a major role for influencing cataract formation in humans.

Transgenic, fluorescent Leishmania mexicana allow direct analysis of the proteome of intracellular amastigotes.

Investigating the proteome of intracellular pathogens is often hampered by inadequate methodologies to purify the pathogen free of host cell material. This has also precluded direct proteome analysis of the intracellular, amastigote form of Leishmania spp., protozoan parasites that cause a spectrum of diseases that affect some 12 million patients worldwide. Here a method is presented that combines classic, isopycnic density centrifugation with fluorescent particle sorting for purification by exploiting transgenic, fluorescent parasites to allow direct proteome analysis of the purified organisms. By this approach the proteome of intracellular Leishmania mexicana amastigotes was compared with that of extracellular promastigotes that are transmitted by insect vectors. In total, 509 different proteins were identified by mass spectrometry and database search. This number corresponds to approximately 6% of gene products predicted from the reference genome of Leishmania major. Intracellular amastigotes synthesized significantly more proteins with basic pI and showed a greater abundance of enzymes of fatty acid catabolism, which may reflect their living in acidic habitats and metabolic adaptation to nutrient availability, respectively. Bioinformatics analyses of the genes corresponding to the protein data sets produced clear evidence for skewed codon usage and translational bias in these organisms. Moreover analysis of the subset of genes whose products were more abundant in amastigotes revealed characteristic sequence motifs in 3'-untranslated regions that have been linked to translational control elements. This suggests that proteome data sets may be used to identify regulatory elements in mRNAs. Last but not least, at 6% coverage the proteome identified all vaccine antigens tested to date. Thus, the present data set provides a valuable resource for selection of candidate vaccine antigens.

Gene expression and protein profiling of AGS gastric epithelial cells upon infection with Helicobacter pylori.

Helicobacter pylori, one of the most common bacterial pathogens, colonizes the human stomach and causes a variety of gastric diseases. This pathogen elicits a range of phenotypic responses in infected cultured AGS gastric epithelial cells, including expression of proinflammatory genes and changes in the actin cytoskeleton. Some of these responses are mediated by the type IV secretion system (T4SS) encoded by the cag pathogenicity island. We have used two global approaches, namely 2-DE combined with PMF and cDNA expression array analyses, to study in both a comprehensive and quantitative manner the protein profile and the temporal patterns of mRNA accumulation in AGS cells upon infection with H. pylori and isogenic T4SS mutants. We identified 140 transcripts and detected 190 protein species that were differentially regulated upon infection. Infection with wild-type H. pylori induced expression of a variety of host genes and changes in protein pattern involved in transcriptional responses, cell shape regulation and signal transduction. Among them, some were differentially regulated in a cag PAI-dependent manner, as shown by both the proteomic and cDNA expression array approaches. While 2-DE and PMF allowed us to examine the protein profiles in the infected host, array analysis enabled us to demonstrate dynamic temporal changes in host gene expression profile. In conclusion, our combined application of the two global approaches provides further molecular details on how the host cell responds to infection by H. pylori and its isogenic T4SS mutants on both transcriptional and protein levels. The findings pinpoint host proteins such as serine/threonine and tyrosine kinases, transcription factors, cell cycle related components and actin cytoskeletal signaling molecules as potential targets of individual H. pylori virulence determinants. This study serves as a basis for future work on transcription and proteome analyses of the H. pylori infection model.

Identification of Helicobacter pylori surface proteins by selective proteinase K digestion and antibody phage display.

Five surface proteins of Helicobacter pylori were identified by proteinase K treatment of live H. pylori followed by proteome analysis. One of the identified proteins, HopQ, is also recognized by an antibody selected by phage display screening of intact H. pylori.

Eye lens proteomics: from global approach to detailed information about phakinin and gamma E and F crystallin genes.

Exploration of the lenticular proteome poses a challenging and worthwhile undertaking as cataracts, the products of a disease phenotype elicited by this proteome, remains the leading cause of vision impairment worldwide. The complete ten day old lens proteome of Mus musculus C57BL/6J was resolved into 900 distinct spots by large gel carrier ampholyte based 2-DE. The predicted amino acid sequences of all 16 crystallins ubiquitous in mammals were corroborated by mass spectrometry (MS). In detailed individual spot analyses, the primary structure of the full murine C57BL/6J beaded filament component phakinin CP49 was sequenced by liquid chromatography/electrospray ionization-tandem MS and amended at two positions. This definitive polypeptide sequence was aligned to the mouse genome, thus identifying the entire C57BL/6J genomic coding region. Also, two murine C57/6J polypeptides, both previously classified as gamma F crystallin, were clearly distinguished by MS and electrophoretic mobility. Both were assigned to their respective genes, one of the polypeptides was reclassified as C57BL/6J gamma E crystallin. Building on these data and previous investigations an updated crystallin reference map was put forth and several non crystallin lenticular components were examined. These results represent the first part of a comprehensive investigation of the mouse lens proteome (http://www.mpiib-berlin.mpg.de/2D-PAGE) with emphasis on understanding genetic effects on proteins and disease development.

Proteomic analysis of the sarcosine-insoluble outer membrane fraction of the bacterial pathogen Bartonella henselae.

Bartonella henselae is an emerging zoonotic pathogen causing a wide range of disease manifestations in humans. In this study, we report on the analysis of the sarcosine-insoluble outer membrane fraction of B. henselae ATCC 49882 Houston-1 by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-D SDS-PAGE) and two-dimensional nonequilibrium pH gradient polyacrylamide gel electrophoresis (2-D NEPHGE). Protein species were identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and subsequent database query against the B. henselae genome sequence. Subcellular fractionation, application of the ionic detergent lauryl sarcosine, assessment of trypsin sensitivity, and heat modifiability of surface-exposed proteins represented valuable tools for the analysis of the outer membrane subproteome of B. henselae. 2-D NEPHGE was applied to display and catalogue a substantial number of proteins associated with the B. henselae sarcosine-insoluble outer membrane fraction, resulting in the establishment of a first 2-D reference map of this compartment. Thus, 53 distinct protein species associated with the outer membrane subproteome fraction were identified. This study provides novel insights into the membrane biology and the associated putative virulence factors of this pathogen of increasing medical importance.

Identification of candidate antigens for serologic detection of Helicobacter pylori-infected patients with gastric carcinoma.

Helicobacter pylori colonizes the stomach of almost half the world population and is a causative agent of gastric carcinomas and duodenal ulcers. Only a small fraction of infected people will develop these severe illnesses and a predictive test to identify people at high risk would greatly benefit disease management. Our study aimed to identify conserved bacterial antigens that may be useful for the development of such a diagnostic test. High-resolution immunoproteomics by 2-dimensional electrophoresis of H. pylori 26695 proteins was carried out with sera from infected patients with either duodenal ulcer (n=30) or gastric carcinoma (n=30), 2 clinically divergent conditions. According to their antigen recognition patterns clear groups of patients were identified. Although this classification did not correspond to the clinical status, it may be correlated to other bacterial or host factors that influence the outcome of infection. In general antigen recognition patterns were found to be highly variable, however by utilizing powerful image analysis and statistical tests the recognition of 14 antigenic protein species was found to differ significantly (p<0.01) between both diseases. Particular protein species of GroEL, HyuA, GroES and AtpA appear to be useful surrogate markers for gastric carcinoma detection and consequently should be considered for further prospective studies to assess their predictive value. For one protein species of AtpA, evidence was found that different post-translational modifications may confer different immunogenicities.

A comparison of murine and human immunoproteomes of Helicobacter pylori validates the preclinical murine infection model for antigen screening.

Preclinical mouse infection models are widely used for Helicobacter vaccine development, but how well such models mimic important aspects of human infections is unknown. A comparison of Helicobacter pylori immunoproteomes of infected mice with previously reported patient data reveals a high agreement in the antigens recognized, suggesting that H. pylori in vivo protein composition and recognition by the host immune system are comparable in mice and humans. Murine Helicobacter models may thus be valid to screen antigens for human vaccination.

Identification of surface proteins of Helicobacter pylori by selective biotinylation, affinity purification, and two-dimensional gel electrophoresis.

Helicobacter pylori is a widespread human pathogen that can cause gastric ulcers and cancer. To identify surface proteins that may play a role in pathogen-host interactions and represent potential targets for the control of this infection, we selectively biotinylated intact H. pylori with the hydrophilic reagent sulfosuccinimidyl-6-(biotinamido)-hexanoate and purified the labeled proteins by membrane isolation, solubilization, and affinity chromatography. After separation of 82 biotinylated proteins on two-dimensional gels, 18 were identified with comparison to proteome data and peptide mass fingerprinting. Among the identified proteins, 9 have previously been shown to be surface-exposed, 7 are associated with virulence, and 11 are highly immunogenic in infected patients. In conclusion, this generally applicable combined proteome approach facilitates the rapid identification of promising targets for the control of H. pylori and might be applicable to numerous other human pathogens although larger biotinylation reagents might be required in some cases to prevent permeation of porin channels in the outer membrane.

Link between organ-specific antigen processing by 20S proteasomes and CD8(+) T cell-mediated autoimmunity.

Adoptive transfer of cross-reactive HSP60-specific CD8(+) T cells into immunodeficient mice causes autoimmune intestinal pathology restricted to the small intestine. We wondered whether local immunopathology induced by CD8(+) T cells can be explained by tissue-specific differences in proteasome-mediated processing of major histocompatibility complex class I T cell epitopes. Our experiments demonstrate that 20S proteasomes of different organs display a characteristic composition of alpha and beta chain subunits and produce distinct peptide fragments with respect to both quality and quantity. Digests of HSP60 polypeptides by 20S proteasomes show most efficient generation of the pathology related CD8(+) T cell epitope in the small intestine. Further, we demonstrate that the organ-specific potential to produce defined T cell epitopes reflects quantities that are relevant for cytotoxic T lymphocyte recognition. We propose tissue-specific antigen processing by 20S proteasomes as a potential mechanism to control organ-specific immune responses.

Immunoproteomics of Helicobacter pylori infection and relation to gastric disease.

The Gram negative bacterium Helicobacter pylori is a human pathogen which infects the gastric mucosa and causes an inflammatory process leading to gastritis, ulceration and cancer. A systematic, proteome based approach was chosen to detect candidate antigens of H. pylori for diagnosis, therapy and vaccine development and to investigate potential associations between specific immune responses and manifestations of disease. Sera from patients with active H. pylori infection (n = 24), a control group with unrelated gastric disorders (n = 12) and from patients with gastric cancer (n = 6) were collected and analyzed for the reactivity against proteins of the strain HP 26695 separated by two-dimensional electrophoresis. Overall, 310 antigenic protein species were recognized by H. pylori positive sera representing about 17% of all spots separated. Out of the 32 antigens most frequently recognized by H. pylori positive sera, nine were newly identified and 23 were confirmed from other studies. Three newly identified antigens which belong to the 150 most abundant protein species of H. pylori, were specifically recognized by H. pylori positive sera: the predicted coding region HP0231, serine protease HtrA (HP1019) and Cag3 (HP0522). Other antigens were recognized differently by sera from gastritis and ulcer patients, which may identify them as candidate indicators for clinical manifestations. The data from these immunoproteomic analyses are added to our public database (http://www.mpiib-berlin.mpg.de/2D-PAGE). This platform enables one to compile many protein profiles and to integrate data from other studies, an approach which will greatly assist the search for more immunogenic proteins for diagnostic assays and vaccine design.