Calculation of partial isotope incorporation into peptides measured by mass spectrometry

<p>Abstract</p> <p>Background</p> <p>Stable isotope probing (SIP) technique was developed to link function, structure and activity of microbial cultures metabolizing carbon and nitrogen containing substrates to synthesize their biomass. Currently, available methods are restricted solely to the estimation of fully saturated heavy stable isotope incorporation and convenient methods with sufficient accuracy are still missing. However in order to track carbon fluxes in microbial communities new methods are required that allow the calculation of partial incorporation into biomolecules.</p> <p>Results</p> <p>In this study, we use the characteristics of the so-called 'half decimal place rule' (HDPR) in order to accurately calculate the partial¹³C incorporation in peptides from enzymatic digested proteins. Due to the clade-crossing universality of proteins within bacteria, any available high-resolution mass spectrometry generated dataset consisting of tryptically-digested peptides can be used as reference.</p> <p>We used a freely available peptide mass dataset from <it>Mycobacterium tuberculosis </it>consisting of 315,579 entries. From this the error of estimated versus known heavy stable isotope incorporation from an increasing number of randomly drawn peptide sub-samples (100 times each; no repetition) was calculated. To acquire an estimated incorporation error of less than 5 atom %, about 100 peptide masses were needed. Finally, for testing the general applicability of our method, peptide masses of tryptically digested proteins from <it>Pseudomonas putida </it>ML2 grown on labeled substrate of various known concentrations were used and¹³C isotopic incorporation was successfully predicted. An easy-to-use script <abbrgrp><abbr bid="B1">1</abbr></abbrgrp> was further developed to guide users through the calculation procedure for their own data series.</p> <p>Conclusion</p> <p>Our method is valuable for estimating¹³C incorporation into peptides/proteins accurately and with high sensitivity. Generally, our method holds promise for wider applications in qualitative and especially quantitative proteomics.</p

Finding one's way in proteomics: a protein species nomenclature

Our knowledge of proteins has greatly improved in recent years, driven by new technologies in the fields of molecular biology and proteome research. It has become clear that from a single gene not only one single gene product but many different ones - termed protein species - are generated, all of which may be associated with different functions. Nonetheless, an unambiguous nomenclature for describing individual protein species is still lacking. With the present paper we therefore propose a systematic nomenclature for the comprehensive description of protein species. The protein species nomenclature is flexible and adaptable to every level of knowledge and of experimental data in accordance with the exact chemical composition of individual protein species. As a minimum description the entry name (gene name + species according to the UniProt knowledgebase) can be used, if no analytical data about the target protein species are available

Histoplasma capsulatum proteome response to decreased iron availability

<p>Abstract</p> <p>Background</p> <p>A fundamental pathogenic feature of the fungus <it>Histoplasma capsulatum </it>is its ability to evade innate and adaptive immune defenses. Once ingested by macrophages the organism is faced with several hostile environmental conditions including iron limitation. <it>H. capsulatum </it>can establish a persistent state within the macrophage. A gap in knowledge exists because the identities and number of proteins regulated by the organism under host conditions has yet to be defined. Lack of such knowledge is an important problem because until these proteins are identified it is unlikely that they can be targeted as new and innovative treatment for histoplasmosis.</p> <p>Results</p> <p>To investigate the proteomic response by <it>H. capsulatum </it>to decreasing iron availability we have created <it>H. capsulatum </it>protein/genomic databases compatible with current mass spectrometric (MS) search engines. Databases were assembled from the <it>H. capsulatum </it>G217B strain genome using gene prediction programs and expressed sequence tag (EST) libraries. Searching these databases with MS data generated from two dimensional (2D) in-gel digestions of proteins resulted in over 50% more proteins identified compared to searching the publicly available fungal databases alone. Using 2D gel electrophoresis combined with statistical analysis we discovered 42 <it>H. capsulatum </it>proteins whose abundance was significantly modulated when iron concentrations were lowered. Altered proteins were identified by mass spectrometry and database searching to be involved in glycolysis, the tricarboxylic acid cycle, lysine metabolism, protein synthesis, and one protein sequence whose function was unknown.</p> <p>Conclusion</p> <p>We have created a bioinformatics platform for <it>H. capsulatum </it>and demonstrated the utility of a proteomic approach by identifying a shift in metabolism the organism utilizes to cope with the hostile conditions provided by the host. We have shown that enzyme transcripts regulated by other fungal pathogens in response to lowering iron availability are also regulated in <it>H. capsulatum </it>at the protein level. We also identified <it>H. capsulatum </it>proteins sensitive to iron level reductions which have yet to be connected to iron availability in other pathogens. These data also indicate the complexity of the response by <it>H. capsulatum </it>to nutritional deprivation. Finally, we demonstrate the importance of a strain specific gene/protein database for <it>H. capsulatum </it>proteomic analysis.</p

Identification of T-Cell Antigens Specific for Latent Mycobacterium Tuberculosis Infection

BACKGROUND: T-cell responses against dormancy-, resuscitation-, and reactivation-associated antigens of Mycobacterium tuberculosis are candidate biomarkers of latent infection in humans. METHODOLOGY/PRINCIPAL FINDINGS: We established an assay based on two rounds of in vitro restimulation and intracellular cytokine analysis that detects T-cell responses to antigens expressed during latent M. tuberculosis infection. Comparison between active pulmonary tuberculosis (TB) patients and healthy latently M. tuberculosis-infected donors (LTBI) revealed significantly higher T-cell responses against 7 of 35 tested M. tuberculosis latency-associated antigens in LTBI. Notably, T cells specific for Rv3407 were exclusively detected in LTBI but not in TB patients. The T-cell IFNgamma response against Rv3407 in individual donors was the most influential factor in discrimination analysis that classified TB patients and LTBI with 83% accuracy using cross-validation. Rv3407 peptide pool stimulations revealed distinct candidate epitopes in four LTBI. CONCLUSIONS: Our findings further support the hypothesis that the latency-associated antigens can be exploited as biomarkers for LTBI

A Systematic Proteomic Study of Irradiated DNA Repair Deficient Nbn-Mice

BACKGROUND: The NBN gene codes for the protein nibrin, which is involved in the detection and repair of DNA double strand breaks (DSBs). The NBN gene is essential in mammals. METHODOLOGY/PRINCIPAL FINDINGS: We have used a conditional null mutant mouse model in a proteomics approach to identify proteins with modified expression levels after 4 Gy ionizing irradiation in the absence of nibrin in vivo. Altogether, amongst approximately 8,000 resolved proteins, 209 were differentially expressed in homozygous null mutant mice in comparison to control animals. One group of proteins significantly altered in null mutant mice were those involved in oxidative stress and cellular redox homeostasis (p<0.0001). In substantiation of this finding, analysis of Nbn null mutant fibroblasts indicated an increased production of reactive oxygen species following induction of DSBs. CONCLUSIONS/SIGNIFICANCE: In humans, biallelic hypomorphic mutations in NBN lead to Nijmegen breakage syndrome (NBS), an autosomal recessive genetic disease characterised by extreme radiosensitivity coupled with growth retardation, immunoinsufficiency and a very high risk of malignancy. This particularly high cancer risk in NBS may be attributable to the compound effect of a DSB repair defect and oxidative stress

Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

<p>Abstract</p> <p>Background</p> <p>Tuberculosis is an infectious bacterial disease in humans caused primarily by <it>Mycobacterium tuberculosis</it>, and infects one-third of the world's total population. <it>Mycobacterium bovis </it>bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated <it>M. bovis </it>BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions.</p> <p>Results</p> <p>Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well in our work.</p> <p>Conclusions</p> <p>In this study, we utilized LC-MS/MS in combination with blue native PAGE to characterize modular components of multiprotein complexes in BCG membrane fractions. The results demonstrated that the proteomic strategy was a reliable and reproducible tool for analysis of BCG multiprotein complexes. The identification in our study may provide some evidence for further study of BCG protein interaction.</p

Mycobacterium tuberculosis Rv3802c Encodes a Phospholipase/Thioesterase and Is Inhibited by the Antimycobacterial Agent Tetrahydrolipstatin

The cell wall of M. tuberculosis is central to its success as a pathogen. Mycolic acids are key components of this cell wall. The genes involved in joining the α and mero mycolates are located in a cluster, beginning with Rv3799c and extending at least until Rv3804c. The role of each enzyme encoded by these five genes is fairly well understood, except for Rv3802c. Rv3802 is one of seven putative cutinases encoded by the genome of M. tuberculosis. In phytopathogens, cutinases hydrolyze the waxy layer of plants, cutin. In a strictly mammalian pathogen, such as M. tuberculosis, it is likely that these proteins perform a different function. Of the seven, we chose to focus on Rv3802c because of its location in a mycolic acid synthesis gene cluster, its putative essentiality, its ubiquitous presence in actinomycetes, and its conservation in the minimal genome of Mycobacterium leprae. We expressed Rv3802 in Escherichia coli and purified the enzymatically active form. We probed its activities and inhibitors characterizing those relevant to its possible role in mycolic acid biosynthesis. In addition to its reported phospholipase A activity, Rv3802 has significant thioesterase activity, and it is inhibited by tetrahydrolipstatin (THL). THL is a described anti-tuberculous compound with an unknown mechanism, but it reportedly targets cell wall synthesis. Taken together, these data circumstantially support a role for Rv3802 in mycolic acid synthesis and, as the cell wall is integral to M. tuberculosis pathogenesis, identification of a novel cell wall enzyme and its inhibition has therapeutic and diagnostic implications

PhoP: A Missing Piece in the Intricate Puzzle of Mycobacterium tuberculosis Virulence

Inactivation of the transcriptional regulator PhoP results in Mycobacterium tuberculosis attenuation. Preclinical testing has shown that attenuated M. tuberculosis phoP mutants hold promise as safe and effective live vaccine candidates. We focused this study to decipher the virulence networks regulated by PhoP. A combined transcriptomic and proteomic analysis revealed that PhoP controls a variety of functions including: hypoxia response through DosR crosstalking, respiratory metabolism, secretion of the major T-cell antigen ESAT-6, stress response, synthesis of pathogenic lipids and the M. tuberculosis persistence through transcriptional regulation of the enzyme isocitrate lyase. We also demonstrate that the M. tuberculosis phoP mutant SO2 exhibits an antigenic capacity similar to that of the BCG vaccine. Finally, we provide evidence that the SO2 mutant persists better in mouse organs than BCG. Altogether, these findings indicate that PhoP orchestrates a variety of functions implicated in M. tuberculosis virulence and persistence, making phoP mutants promising vaccine candidates

Computational Comparative Study of Tuberculosis Proteomes Using a Model Learned from Signal Peptide Structures

Secretome analysis is important in pathogen studies. A fundamental and convenient way to identify secreted proteins is to first predict signal peptides, which are essential for protein secretion. However, signal peptides are highly complex functional sequences that are easily confused with transmembrane domains. Such confusion would obviously affect the discovery of secreted proteins. Transmembrane proteins are important drug targets, but very few transmembrane protein structures have been determined experimentally; hence, prediction of the structures is essential. In the field of structure prediction, researchers do not make assumptions about organisms, so there is a need for a general signal peptide predictor

Portrait of a Pathogen: The Mycobacterium tuberculosis Proteome In Vivo

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo.We used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection.Cumulatively, the results of this work may provide the basis for rational drug design - identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection