A pilot study to show that asymptomatic sexually transmitted infections alter the foreskin epithelial proteome.

There is limited data on the role of asymptomatic STIs (aSTIs) on the risk of human immunodeficiency virus (HIV) acquisition in the male genital tract (MGT). The impact of foreskin removal on lowering HIV acquisition is well described, but molecular events leading to HIV acquisition are unclear. Here, in this pilot study, we show that asymptomatic urethral infection with Chlamydia trachomatis (CT) significantly impacts the foreskin proteome composition. We developed and optimized a shotgun liquid chromatography coupled tandem mass spectrometry (MS)-based proteomics approach and utilized this on foreskins collected at medical male circumcision (MMC) from 16 aSTI+ men and 10 age-matched STI- controls. We used a novel bioinformatic metaproteomic pipeline to detect differentially expressed (DE) proteins. Gene enrichment ontology analysis revealed proteins associated with inflammatory and immune activation function in both inner and outer foreskin from men with an aSTI. Neutrophil activation/degranulation and viral-evasion proteins were significantly enriched in foreskins from men with aSTI, whereas homotypic cell-cell adhesion proteins were enriched in foreskin tissue from men without an aSTI. Collectively, our data show that asymptomatic urethral sexually transmitted infections result in profound alterations in epithelial tissue that are associated with depletion of barrier integrity and immune activation.

Strongylopus grayii tadpole blastema extract exerts cytotoxic effects on embryonal rhabdomyosarcoma cells.

Amphibians have regenerative capacity and are resistant to developing cancer. This suggests that the developing blastema, located at the tissue regeneration site, may secrete anti-cancer factors. Here, we investigate the anti-cancer potential of tadpole tail blastema extracts (TAD) from the stream frog, Strongylopus grayii, in embryonal rhabdomyosarcoma (ERMS) cells. ERMS originates in skeletal muscle tissue and is a common pediatric soft tissue sarcoma. We show using MTT assays that TAD inhibited ERMS cell viability in a concentration-dependent manner, and phase contrast/fluorescent microscopy revealed that it induced morphological markers of senescence and apoptosis. Western blotting showed that this was associated with DNA damage (γH2AX) and activation of the p38/MAPK stress signaling pathway as well as molecular markers of senescence (p16INK4a), apoptosis (cleaved PARP), and inhibition of cell cycle promoters (cyclin A, CDK2, and cyclin B1). Furthermore, proteomics followed by gene ontology analyses showed that TAD treatment inhibited known tumor promoters and proteins required for cancer cell survival. Lastly, using the LINCS drug perturbation library, we show that there is an overlap between the proteomics signature induced by TAD and common anti-cancer drugs. Taken together, this study provides novel evidence that TAD exhibits cytotoxicity in ERMS cells.

Identification of novel salivary candidate protein biomarkers for tuberculosis diagnosis: A preliminary biomarker discovery study.

BACKGROUND: There is an urgent need for new, accurate, rapid, and affordable tuberculosis (TB) diagnostic tests. The aim of the present study was to use mass spectrometry to identify new preliminary candidate TB diagnostic protein biomarkers in saliva obtained from individuals with TB, and patients with other respiratory diseases (ORD). METHODS: Saliva samples were collected from 22 individuals who self-presented with symptoms suggestive of TB as part of a larger TB biomarker project. Purified salivary proteins were subjected to tryptic digestion peptides were analyzed using a QExactive Orbitrap Mass Spectrometer. Data are available via ProteomeXchange with identifier PXD027294. Identified proteins were subjected to gene ontology and ingenuity pathway analysis for functional enrichment analysis. RESULTS: 26 of the 652 identified proteins significantly discriminated individuals with TB from those with ORD after Benjamini Hochberg correction (5% FDR), with five of these proteins diagnosing TB with an AUC ≥ 0.80. A 5-protein biosignature comprising of P01011, Q8NCW5, P28072, A0A2Q2TTZ9, and Q99574 diagnosed TB with an AUC of 1.00 (95% CI, 1.00-1.00), sensitivity of 100% (95% CI, 76.2-100%) and specificity of 90.9% (95% CI, 58.7-99.8%) after leave-one-out cross validation. CONCLUSIONS: We identified novel candidate salivary protein biomarkers and biosignatures with strong potential as TB diagnostic candidates. Our results are preliminary and require validation in larger studies.

Protocols for Preparation and Mass Spectrometry Analysis of Clinical Urine Samples to Identify Candidate Biomarkers of Schistosoma-Associated Bladder Cancer.

Advances in mass spectrometry instrumentation have revolutionized analytical capability in clinical proteomics. In parallel, various sample preparation methods have been developed to try to address the inherent complexity and dynamic range of clinical samples, typically involving a combination of depletion of abundant proteins followed by extensive prefractionation. However, the depth of coverage routinely achieved in discovery proteomics experiments on peripheral fluids such as serum, still leaves something to be desired, especially if no depletion or prefractionation is done in order to increase the throughput of clinical samples. Remarkably, despite being an easily accessible, typically sterile and diagnostically rich clinical sample, urine is often overlooked and as such has received less development effort. As an ultrafiltrate of blood, urine contains proteins and protein fragments originating from all parts of the body which may have diagnostic or prognostic potential if accurately and reproducibly quantified. Here, we describe an efficient and simple method for the concentration of urine samples by methanol-chloroform precipitation and subsequent in-solution tryptic digestion prior to discovery or targeted mass spectrometry analysis. We exemplify this method by reference to the discovery of novel candidate urinary biomarkers of schistosomiasis. Importantly, the methods described here have been used to identify >1900 protein groups in human urine by label-free discovery proteomics, without requiring any prior depletion or prefractionation, making this approach amenable to high throughput clinical biomarker studies in many diseases.

Analysis of the regenerative capacity of human serum exosomes after a simple multistep separation from lipoproteins.

Due to the abundance of lipoproteins in blood, it is challenging to characterize the biological functions and components of blood-derived extracellular vesicles. The aim of this study was to develop a multiple-step purification protocol to separate serum exosomes from serum proteins and lipoproteins and assess their regenerative potential. Exosomes were isolated by concentrating them in human serum using ultracentrifugation (UC), followed sequentially by density gradient (DG) UC and size exclusion chromatography (SEC). Purity and characterization were assessed by western blots, Lipoprint®, enzyme-linked immunosorbent assay, electron microscopy, mass spectrometry, and nanoparticle tracking analysis. Functionality was assessed by cell proliferation analysis and with an in vivo subcutaneous angiogenesis model. SEC alone isolated nano-sized vesicles possessing vesicle markers TSG101 and CD9, but there was a substantial presence of apolipoprotein B, predominantly derived from very-low- and intermediate-density lipoprotein particles. This was reduced to an undetectable level using the combined UC DG SEC approach. Mass spectrometry identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with considerable increases in exosome-related proteins and reductions in lipoproteins. A consistent but limited increase in human dermal fibroblast proliferation and evidence of neovascularization enhancement were observed after exposure to UC DG SEC exosomes. An UC DG SEC purification protocol considerably improved the removal of lipoproteins during isolation of serum exosomes. The purified exosomes stimulated cell proliferation and potentially increased an in vivo angiogenic response. This multistep purification allows for more accurate identification of serum exosome functional activity and composition.

Microbial function and genital inflammation in young South African women at high risk of HIV infection.

BACKGROUND: Female genital tract (FGT) inflammation is an important risk factor for HIV acquisition. The FGT microbiome is closely associated with inflammatory profile; however, the relative importance of microbial activities has not been established. Since proteins are key elements representing actual microbial functions, this study utilized metaproteomics to evaluate the relationship between FGT microbial function and inflammation in 113 young and adolescent South African women at high risk of HIV infection. Women were grouped as having low, medium, or high FGT inflammation by K-means clustering according to pro-inflammatory cytokine concentrations. RESULTS: A total of 3186 microbial and human proteins were identified in lateral vaginal wall swabs using liquid chromatography-tandem mass spectrometry, while 94 microbial taxa were included in the taxonomic analysis. Both metaproteomics and 16S rRNA gene sequencing analyses showed increased non-optimal bacteria and decreased lactobacilli in women with FGT inflammatory profiles. However, differences in the predicted relative abundance of most bacteria were observed between 16S rRNA gene sequencing and metaproteomics analyses. Bacterial protein functional annotations (gene ontology) predicted inflammatory cytokine profiles more accurately than bacterial relative abundance determined by 16S rRNA gene sequence analysis, as well as functional predictions based on 16S rRNA gene sequence data (p < 0.0001). The majority of microbial biological processes were underrepresented in women with high inflammation compared to those with low inflammation, including a Lactobacillus-associated signature of reduced cell wall organization and peptidoglycan biosynthesis. This signature remained associated with high FGT inflammation in a subset of 74 women 9 weeks later, was upheld after adjusting for Lactobacillus relative abundance, and was associated with in vitro inflammatory cytokine responses to Lactobacillus isolates from the same women. Reduced cell wall organization and peptidoglycan biosynthesis were also associated with high FGT inflammation in an independent sample of ten women. CONCLUSIONS: Both the presence of specific microbial taxa in the FGT and their properties and activities are critical determinants of FGT inflammation. Our findings support those of previous studies suggesting that peptidoglycan is directly immunosuppressive, and identify a possible avenue for biotherapeutic development to reduce inflammation in the FGT. To facilitate further investigations of microbial activities, we have developed the FGT-DB application that is available at http://fgtdb.org/ . Video Abstract.

A Human Lung Challenge Model to Evaluate the Safety and Immunogenicity of PPD and Live Bacillus Calmette-Guérin.

Rationale: A human model to better understand tuberculosis immunopathogenesis and facilitate vaccine development is urgently needed.Objectives: We evaluated the feasibility, safety, and immunogenicity of live bacillus Calmette-Guérin (BCG) in a lung-oriented controlled human infection model.Methods: We recruited 106 healthy South African participants with varying degrees of tuberculosis susceptibility. Live BCG, sterile PPD, and saline were bronchoscopically instilled into separate lung segments (n = 65). A control group (n = 34) underwent a single bronchoscopy without challenge. The primary outcome was safety. Cellular and antibody immune signatures were identified in BAL before and 3 days after challenge using flow cytometry, ELISA, RNA sequencing, and mass spectrometry.Measurements and Main Results: The frequency of adverse events was low (9.4%; n = 10), similar in the challenge versus control groups (P = 0.8), and all adverse events were mild and managed conservatively in an outpatient setting. The optimal PPD and BCG dose was 0.5 TU and 104 cfu, respectively, based on changes in BAL cellular profiles (P = 0.02) and antibody responses (P = 0.01) at incremental doses before versus after challenge. At 104 versus 103 cfu BCG, there was a significant increase in number of differentially expressed genes (367 vs. 3; P < 0.001) and dysregulated proteins (64 vs. 0; P < 0.001). Immune responses were highly setting specific (in vitro vs. in vivo) and compartment specific (BAL vs. blood) and localized to the challenged lung segments.Conclusions: A lung-oriented mycobacterial controlled human infection model using live BCG and PPD is feasible and safe. These data inform the study of tuberculosis immunopathogenesis and strategies for evaluation and development of tuberculosis vaccine candidates.

Associating H2O2-and NO-related changes in the proteome of Mycobacterium smegmatis with enhanced survival in macrophage.

Mycobacterium manages to evade the host cell immune system, partially owing to its ability to survive redox stress after macrophage engulfment. Exposure to redox stress has been linked to later replication, persistence, and latent infection. In this work, mass spectrometry was used to elucidate the cell-wide changes that occur in response to sublethal doses of hydrogen peroxide and nitric oxide over time, with Mycobacterium smegmatis being used as a model organism. A total of 3135 proteins were confidently assigned, of which 1713, 1674, and 1713 were identified under NO, H2O2, and control conditions, respectively. Both treatment conditions resulted in changes of protein expression from the DosR regulon as well as those related to lipid metabolism. Complementary to the changes in the proteome, sublethal exposure to NO and H2O2 improved the survival of the bacteria after macrophage infection. Our data indicate that pre-exposure to sublethal doses of these redox stressors causes an alteration in the expression of proteins related to lipid metabolism, suggesting a link between altered lipid metabolism and enhanced survival in macrophages.

The Role of Tumor Microenvironment in Chemoresistance: 3D Extracellular Matrices as Accomplices.

BACKGROUND: The functional interplay between tumor cells and their adjacent stroma has been suggested to play crucial roles in the initiation and progression of tumors and the effectiveness of chemotherapy. The extracellular matrix (ECM), a complex network of extracellular proteins, provides both physical and chemicals cues necessary for cell proliferation, survival, and migration. Understanding how ECM composition and biomechanical properties affect cancer progression and response to chemotherapeutic drugs is vital to the development of targeted treatments. METHODS: 3D cell-derived-ECMs and esophageal cancer cell lines were used as a model to investigate the effect of ECM proteins on esophageal cancer cell lines response to chemotherapeutics. Immunohistochemical and qRT-PCR evaluation of ECM proteins and integrin gene expression was done on clinical esophageal squamous cell carcinoma biopsies. Esophageal cancer cell lines (WHCO1, WHCO5, WHCO6, KYSE180, KYSE 450 and KYSE 520) were cultured on decellularised ECMs (fibroblasts-derived ECM; cancer cell-derived ECM; combinatorial-ECM) and treated with 0.1% Dimethyl sulfoxide (DMSO), 4.2 µM cisplatin, 3.5 µM 5-fluorouracil and 2.5 µM epirubicin for 24 h. Cell proliferation, cell cycle progression, colony formation, apoptosis, migration and activation of signaling pathways were used as our study endpoints. RESULTS: The expression of collagens, fibronectin and laminins was significantly increased in esophageal squamous cell carcinomas (ESCC) tumor samples compared to the corresponding normal tissue. Decellularised ECMs abrogated the effect of drugs on cancer cell cycling, proliferation and reduced drug induced apoptosis by 20⁻60% that of those plated on plastic. The mitogen-activated protein kinase-extracellular signal-regulated kinase (MEK-ERK) and phosphoinositide 3-kinase-protein kinase B (PI3K/Akt) signaling pathways were upregulated in the presence of the ECMs. Furthermore, our data show that concomitant addition of chemotherapeutic drugs and the use of collagen- and fibronectin-deficient ECMs through siRNA inhibition synergistically increased cancer cell sensitivity to drugs by 30⁻50%, and reduced colony formation and cancer cell migration. CONCLUSION: Our study shows that ECM proteins play a key role in the response of cancer cells to chemotherapy and suggest that targeting ECM proteins can be an effective therapeutic strategy against chemoresistant tumors.

Challenges and Opportunities for Biological Mass Spectrometry Core Facilities in the Developing World.

The developing world is seeing rapid growth in the availability of biological mass spectrometry (MS), particularly through core facilities. As proteomics and metabolomics becomes locally feasible for investigators in these nations, application areas associated with high burden in these nations, such as infectious disease, will see greatly increased research output. This article evaluates the rapid growth of MS in South Africa (currently approaching 20 laboratories) as a model for establishing MS core facilities in other nations of the developing world. Facilities should emphasize new services rather than new instruments. The reduction of the delays associated with reagent and other supply acquisition would benefit both facilities and the users who make use of their services. Instrument maintenance and repair, often mediated by an in-country business for an international vendor, is also likely to operate on a slower schedule than in the wealthiest nations. A key challenge to facilities in the developing world is educating potential facility users in how best to design experiments for proteomics and metabolomics, what reagents are most likely to introduce problematic artifacts, and how to interpret results from the facility. Here, we summarize the experience of 6 different institutions to raise the level of biological MS available to researchers in South Africa.

Identification of Novel Physiological Substrates of Mycobacterium bovis BCG Protein Kinase G (PknG) by Label-free Quantitative Phosphoproteomics.

Mycobacterial Ser/Thr kinases play a critical role in bacterial physiology and pathogenesis. Linking kinases to the substrates they phosphorylate in vivo, thereby elucidating their exact functions, is still a challenge. The aim of this work was to associate protein phosphorylation in mycobacteria with important subsequent macro cellular events by identifying the physiological substrates of PknG in Mycobacterium bovis BCG. The study compared the phosphoproteome dynamics during the batch growth of M. bovis BCG versus the respective PknG knock-out mutant (ΔPknG-BCG) strains. We employed TiO2 phosphopeptide enrichment techniques combined with label-free quantitative phosphoproteomics workflow on LC-MS/MS. The comprehensive analysis of label-free data identified 603 phosphopeptides on 307 phosphoproteins with high confidence. Fifty-five phosphopeptides were differentially phosphorylated, of these, 23 phosphopeptides were phosphorylated in M. bovis BCG wild-type only and not in the mutant. These were further validated through targeted mass spectrometry assays (PRMs). Kinase-peptide docking studies based on a published crystal structure of PknG in complex with GarA revealed that the majority of identified phosphosites presented docking scores close to that seen in previously described PknG substrates, GarA, and ribosomal protein L13. Six out of the 22 phosphoproteins had higher docking scores than GarA, consistent with the proteins identified here being true PknG substrates. Based on protein functional analysis of the PknG substrates identified, this study confirms that PknG plays an important regulatory role in mycobacterial metabolism, through phosphorylation of ATP binding proteins and enzymes in the TCA cycle. This work also reinforces PknG's regulation of protein translation and folding machinery.

Proteomic comparison of three clinical diarrhoeagenic drug-resistant Escherichia coli isolates grown on CHROMagar™STEC media.

Shiga-toxin-producing Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) are key diarrhoea-causing foodborne pathogens. We used proteomics to characterize the virulence and antimicrobial resistance protein profiles of three clinical pathogenic E. coli isolates (two EPEC [one resistant to ciprofloxacin] and one STEC) cultured on CHROMagar™STEC solid media after minimal laboratory passage. We identified 4767 unique peptides from 1630 protein group across all three clinical E. coli strains. Label-free proteomic analysis allowed the identification of virulence and drug resistance proteins that were unique to each of the clinical isolates compared in this study. The B subunit of Shiga toxin, ToxB, was uniquely detected in the STEC strain while several other virulence factors including SheA, OmpF, OmpC and OmpX were significantly more abundant in the STEC strain. The ciprofloxacin resistant EPEC isolate possessed reduced levels of key virulence proteins compared to the ciprofloxacin susceptible EPEC and STEC strains. Parallel reaction monitoring assays validated the presence of biologically relevant proteins across biologically-replicated cultures. Propagation of clinical isolates on a relevant solid medium followed by mass spectrometry analysis represents a convenient means to quantify virulence factors and drug resistance determinants that might otherwise be lost through extensive in vitro passage in enteropathogenic bacteria. SIGNIFICANCE: Through the use of quantitative proteomics, we have characterized the virulence and antimicrobial resistance attributes of three clinically isolated, pathogenic E. coli strains cultured on solid media. Our results provide new, quantitative data on the expressed proteomes of these tellurite-resistant, diarrhoeagenic E. coli strains and reveal a subset of antimicrobial resistance and virulence proteins that are differentially abundant between these clinical strains. Our quantitative proteomics-based approach should thus have applicability in microbiological diagnostic labs for the identification of pathogenic/drug resistant E. coli in the future.

Global proteome and phosphoproteome dynamics indicate novel mechanisms of vitamin C induced dormancy in Mycobacterium smegmatis.

Vitamin C has been found to affect mycobacteria in multiple ways, including increasing susceptibility to antimicrobial drugs, inducing dormancy, and having a bactericidal effect. However, the regulatory events mediating vitamin C related adaptations remain largely elusive. Ser/Thr/Tyr protein phosphorylation plays an important regulatory role in mycobacteria, contributing to environmental adaptation, including dormancy and drug resistance. This study utilised the model organism, Mycobacterium smegmatis, and TiO2 phosphopeptide enrichment combined with mass spectrometry-based proteomics methods to elucidate the mycobacterial signalling and regulatory response to sub-lethal concentrations of vitamin C. After initial validation of peptide spectra, 224 non-redundant phosphosites in 154 proteins were retained with high confidence. Data analysis revealed that 30 peptides were differentially phosphorylated with Vitamin C treatment, including novel phosphosites found on both PknG and GarA. Of these significant proteins, we validated 11 by parallel reaction monitoring of high-confidence phosphopeptides. Interestingly, 17/30 phosphopeptides were annotated as part of transmembrane proteins, suggesting that it is likely vitamin C triggers typical signal transduction events in which the protein periplasmic domain perceives environmental signals and the cytoplasmic domain is then phosphorylated. Finally, the diverse nature of phosphorylated proteins involved in signalling, transport, and carbohydrate biosynthesis indicates the extent of such regulatory phosphorylation events. BIOLOGICAL SIGNIFICANCE: Our findings provide new mechanistic insight into a coordinated network of signalling and regulatory responses to sub-lethal vitamin C in Mycobacterium smegmatis and provide evidence that vitamin C is able to act as a novel extracellular signalling molecule. Vitamin C treatment caused changes in both the proteome and phosphoproteome associated with response to oxidative stress, a shift in metabolic regulation and progression toward dormancy, as well as phospho-dependent activation of specific secretory pathways and activation of specific two component and Ser/Thr/Tyr protein kinase activities. This study confirms the potential of vitamin C as convenient means to study aspects of mycobacterial dormancy, including those regulated at post-translational level.

Quantitative label-free proteomic analysis of human urine to identify novel candidate protein biomarkers for schistosomiasis.

BACKGROUND: Schistosomiasis is a chronic neglected tropical disease that is characterized by continued inflammatory challenges to the exposed population and it has been established as a possible risk factor in the aetiology of bladder cancer. Improved diagnosis of schistosomiasis and its associated pathology is possible through mass spectrometry to identify biomarkers among the infected population, which will influence early detection of the disease and its subtle morbidity. METHODOLOGY: A high-throughput proteomic approach was used to analyse human urine samples for 49 volunteers from Eggua, a schistosomiasis endemic community in South-West, Nigeria. The individuals were previously screened for Schistosoma haematobium and structural bladder pathologies via microscopy and ultrasonography respectively. Samples were categorised into schistosomiasis, schistosomiasis with bladder pathology, bladder pathology, and a normal healthy control group. These samples were analysed to identify potential protein biomarkers. RESULTS: A total of 1306 proteins and 9701 unique peptides were observed in this study (FDR = 0.01). Fifty-four human proteins were found to be potential biomarkers for schistosomiasis and bladder pathologies due to schistosomiasis by label-free quantitative comparison between groups. Thirty-six (36) parasite-derived potential biomarkers were also identified, which include some existing putative schistosomiasis biomarkers that have been previously reported. Some of these proteins include Elongation factor 1 alpha, phosphopyruvate hydratase, histone H4 and heat shock proteins (HSP 60, HSP 70). CONCLUSION: These findings provide an in-depth analysis of potential schistosoma and human host protein biomarkers for diagnosis of chronic schistosomiasis caused by Schistosoma haematobium and its pathogenesis.

Identification of Quantitative Proteomic Differences between Mycobacterium tuberculosis Lineages with Altered Virulence.

Evidence currently suggests that as a species Mycobacterium tuberculosis exhibits very little genomic sequence diversity. Despite limited genetic variability, members of the M. tuberculosis complex (MTBC) have been shown to exhibit vast discrepancies in phenotypic presentation in terms of virulence, elicited immune response and transmissibility. Here, we used qualitative and quantitative mass spectrometry tools to investigate the proteomes of seven clinically-relevant mycobacterial strains-four M. tuberculosis strains, M. bovis, M. bovis BCG, and M. avium-that show varying degrees of pathogenicity and virulence, in an effort to rationalize the observed phenotypic differences. Following protein preparation, liquid chromatography mass spectrometry (LC MS/MS) and data capture were carried out using an LTQ Orbitrap Velos. Data analysis was carried out using a novel bioinformatics strategy, which yielded high protein coverage and was based on high confidence peptides. Through this approach, we directly identified a total of 3788 unique M. tuberculosis proteins out of a theoretical proteome of 4023 proteins and identified an average of 3290 unique proteins for each of the MTBC organisms (representing 82% of the theoretical proteomes), as well as 4250 unique M. avium proteins (80% of the theoretical proteome). Data analysis showed that all major classes of proteins are represented in every strain, but that there are significant quantitative differences between strains. Targeted selected reaction monitoring (SRM) assays were used to quantify the observed differential expression of a subset of 23 proteins identified by comparison to gene expression data as being of particular relevance to virulence. This analysis revealed differences in relative protein abundance between strains for proteins which may promote bacterial fitness in the more virulent W. Beijing strain. These differences may contribute to this strain's capacity for surviving within the host and resisting treatment, which has contributed to its rapid spread. Through this approach, we have begun to describe the proteomic portrait of a successful mycobacterial pathogen. Data are available via ProteomeXchange with identifier PXD004165.

Mass Spectrometry Offers Insight into the Role of Ser/Thr/Tyr Phosphorylation in the Mycobacteria.

Phosphorylation is a post translational modification which can rapidly regulate biochemical pathways by altering protein function, and has been associated with pathogenicity in bacteria. Once engulfed by host macrophages, pathogenic bacteria are exposed to harsh conditions and must respond rapidly in order to survive. The causative agent of TB, Mycobacterium tuberculosis, is unusual amongst the bacteria because it can survive within the host macrophage for decades in a latent state, demonstrating a remarkable capacity to successfully evade the host immune response. This ability may be mediated in part by regulatory mechanisms such as ser/thr/tyr phosphorylation. Mass spectrometry-based proteomics has afforded us the capacity to identify hundreds of phosphorylation sites in the bacterial proteome, allowing for comparative phosphoproteomic studies in the mycobacteria. There remains an urgent need to validate the reported phosphosites, and to elucidate their biological function in the context of pathogenicity. However, given the sheer number of putative phosphorylation events in the mycobacterial proteome, and the technical difficulty of assigning biological function to a phosphorylation event, it will not be trivial to do so. There are currently six published phosphoproteomic investigations of a member of mycobacteria. Here, we combine the datasets from these studies in order to identify commonly detected phosphopeptides and phosphosites in order to present high confidence candidates for further validation. By applying modern mass spectrometry-based techniques to improve our understanding of phosphorylation and other PTMs in pathogenic bacteria, we may identify candidates for therapeutic intervention.

In silico verification and parallel reaction monitoring prevalidation of potential prostate cancer biomarkers.

PURPOSE: Targeted proteomics of potential biomarkers is often challenging. Hence, we developed an intermediate workflow to streamline potential urinary biomarkers of prostate cancer (PCa). MATERIALS & METHODS: Using previously discovered potential PCa biomarkers; we selected proteotypic peptides for targeted validation. Preliminary in silico immunohistochemical and single reaction monitoring (SRM) verification was performed. Successful PTPs were then prevalidated using parallel reaction monitoring (PRM) and reconfirmed in 15 publicly available databases. RESULTS: Stringency-based targetable potential biomarkers were shortlisted following in silico screening. PRM reveals top 12 potential biomarkers including the top ranking seven in silico verification-based biomarkers. Database reconfirmation showed differential expression between PCa and benign/normal prostatic urine samples. CONCLUSION: The pragmatic penultimate screening step, described herein, would immensely improve targeted proteomics validation of potential disease biomarkers.

Mycobacterial proteomics: analysis of expressed proteomes and post-translational modifications to identify candidate virulence factors.

The Mycobacterium tuberculosis bacillus has a number of unique features that make it a particularly effective human pathogen. Although genomic analysis has added to our current understanding of the molecular basis by which M. tuberculosis damages its host, proteomics may be better suited to describe the dynamic interactions between mycobacterial and host systems that underpin this disease. The M. tuberculosis proteome has been investigated using proteomics for over a decade, with increasingly sophisticated mass spectrometry technology and sensitive methods for comparative proteomic profiling. Deeper coverage of the M. tuberculosis proteome has led to the identification of hundreds of putative virulence determinants, as well as an unsurpassed coverage of post-translational modifications. Proteomics is therefore uniquely poised to contribute to our understanding of this pathogen, which may ultimately lead to better management of the disease.