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1.
Proc Natl Acad Sci U S A ; 121(22): e2310677121, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38753503

ABSTRACT

Seasonal and pandemic-associated influenza strains cause highly contagious viral respiratory infections that can lead to severe illness and excess mortality. Here, we report on the optimization of our small-molecule inhibitor F0045(S) targeting the influenza hemagglutinin (HA) stem with our Sulfur-Fluoride Exchange (SuFEx) click chemistry-based high-throughput medicinal chemistry (HTMC) strategy. A combination of SuFEx- and amide-based lead molecule diversification and structure-guided design led to identification and validation of ultrapotent influenza fusion inhibitors with subnanomolar EC50 cellular antiviral activity against several influenza A group 1 strains. X-ray structures of six of these compounds with HA indicate that the appended moieties occupy additional pockets on the HA surface and increase the binding interaction, where the accumulation of several polar interactions also contributes to the improved affinity. The compounds here represent the most potent HA small-molecule inhibitors to date. Our divergent HTMC platform is therefore a powerful, rapid, and cost-effective approach to develop bioactive chemical probes and drug-like candidates against viral targets.


Subject(s)
Antiviral Agents , Hemagglutinin Glycoproteins, Influenza Virus , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Hemagglutinin Glycoproteins, Influenza Virus/chemistry , Humans , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Chemistry, Pharmaceutical/methods , High-Throughput Screening Assays/methods , Influenza, Human/drug therapy , Influenza, Human/virology , Crystallography, X-Ray/methods , Click Chemistry/methods , Animals , Influenza A virus/drug effects , Madin Darby Canine Kidney Cells , Viral Fusion Protein Inhibitors/pharmacology , Viral Fusion Protein Inhibitors/chemistry , Dogs
2.
Proc Natl Acad Sci U S A ; 119(37): e2208540119, 2022 09 13.
Article in English | MEDLINE | ID: mdl-36070343

ABSTRACT

Diversity Oriented Clicking (DOC) is a discovery method geared toward the rapid synthesis of functional libraries. It combines the best attributes of both classical and modern click chemistries. DOC strategies center upon the chemical diversification of core "SuFExable" hubs-exemplified by 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs)-enabling the modular assembly of compounds through multiple reaction pathways. We report here a range of stereoselective Michael-type addition pathways from SASF hubs including reactions with secondary amines, carboxylates, 1H-1,2,3-triazole, and halides. These high yielding conjugate addition pathways deliver unprecedented ß-substituted alkenyl sulfonyl fluorides as single isomers with minimal purification, greatly enriching the repertoire of DOC and holding true to the fundamentals of modular click chemistry. Further, we demonstrate the potential for biological function - a key objective of click chemistry - of this family of SASF-derived molecules as covalent inhibitors of human neutrophil elastase.


Subject(s)
Click Chemistry , Fluorides , Leukocyte Elastase , Proteinase Inhibitory Proteins, Secretory , Sulfinic Acids , Click Chemistry/methods , Fluorides/chemical synthesis , Fluorides/chemistry , Fluorides/pharmacology , Humans , Leukocyte Elastase/antagonists & inhibitors , Proteinase Inhibitory Proteins, Secretory/chemical synthesis , Proteinase Inhibitory Proteins, Secretory/chemistry , Proteinase Inhibitory Proteins, Secretory/pharmacology , Sulfinic Acids/chemical synthesis , Sulfinic Acids/chemistry , Sulfinic Acids/pharmacology
3.
Mol Cell Proteomics ; 21(3): 100197, 2022 03.
Article in English | MEDLINE | ID: mdl-35033677

ABSTRACT

The gut microbiota plays an important yet incompletely understood role in the induction and propagation of ulcerative colitis (UC). Organism-level efforts to identify UC-associated microbes have revealed the importance of community structure, but less is known about the molecular effectors of disease. We performed 16S rRNA gene sequencing in parallel with label-free data-dependent LC-MS/MS proteomics to characterize the stool microbiomes of healthy (n = 8) and UC (n = 10) patients. Comparisons of taxonomic composition between techniques revealed major differences in community structure partially attributable to the additional detection of host, fungal, viral, and food peptides by metaproteomics. Differential expression analysis of metaproteomic data identified 176 significantly enriched protein groups between healthy and UC patients. Gene ontology analysis revealed several enriched functions with serine-type endopeptidase activity overrepresented in UC patients. Using a biotinylated fluorophosphonate probe and streptavidin-based enrichment, we show that serine endopeptidases are active in patient fecal samples and that additional putative serine hydrolases are detectable by this approach compared with unenriched profiling. Finally, as metaproteomic databases expand, they are expected to asymptotically approach completeness. Using ComPIL and de novo peptide sequencing, we estimate the size of the probable peptide space unidentified ("dark peptidome") by our large database approach to establish a rough benchmark for database sufficiency. Despite high variability inherent in patient samples, our analysis yielded a catalog of differentially enriched proteins between healthy and UC fecal proteomes. This catalog provides a clinically relevant jumping-off point for further molecular-level studies aimed at identifying the microbial underpinnings of UC.


Subject(s)
Colitis, Ulcerative , Microbiota , Chromatography, Liquid , Colitis, Ulcerative/diagnosis , Colitis, Ulcerative/microbiology , Endopeptidases , Feces/microbiology , Humans , RNA, Ribosomal, 16S/genetics , Serine , Tandem Mass Spectrometry
4.
Proc Natl Acad Sci U S A ; 117(31): 18431-18438, 2020 08 04.
Article in English | MEDLINE | ID: mdl-32690700

ABSTRACT

Influenza hemagglutinin (HA) glycoprotein is the primary surface antigen targeted by the host immune response and a focus for development of novel vaccines, broadly neutralizing antibodies (bnAbs), and therapeutics. HA enables viral entry into host cells via receptor binding and membrane fusion and is a validated target for drug discovery. However, to date, only a very few bona fide small molecules have been reported against the HA. To identity new antiviral lead candidates against the highly conserved fusion machinery in the HA stem, we synthesized a fluorescence-polarization probe based on a recently described neutralizing cyclic peptide P7 derived from the complementarity-determining region loops of human bnAbs FI6v3 and CR9114 against the HA stem. We then designed a robust binding assay compatible with high-throughput screening to identify molecules with low micromolar to nanomolar affinity to influenza A group 1 HAs. Our simple, low-cost, and efficient in vitro assay was used to screen H1/Puerto Rico/8/1934 (H1/PR8) HA trimer against ∼72,000 compounds. The crystal structure of H1/PR8 HA in complex with our best hit compound F0045(S) confirmed that it binds to pockets in the HA stem similar to bnAbs FI6v3 and CR9114, cyclic peptide P7, and small-molecule inhibitor JNJ4796. F0045 is enantioselective against a panel of group 1 HAs and F0045(S) exhibits in vitro neutralization activity against multiple H1N1 and H5N1 strains. Our assay, compound characterization, and small-molecule candidate should further stimulate the discovery and development of new compounds with unique chemical scaffolds and enhanced influenza antiviral capabilities.


Subject(s)
Antiviral Agents/pharmacology , Drug Evaluation, Preclinical/methods , Fluorescence Polarization/methods , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H5N1 Subtype/drug effects , Influenza, Human/virology , Small Molecule Libraries/pharmacology , Antiviral Agents/chemistry , Hemagglutinin Glycoproteins, Influenza Virus/genetics , Hemagglutinin Glycoproteins, Influenza Virus/metabolism , Humans , Influenza A Virus, H1N1 Subtype/genetics , Influenza A Virus, H1N1 Subtype/metabolism , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/metabolism , Small Molecule Libraries/chemistry
5.
Nat Chem Biol ; 16(9): 997-1005, 2020 09.
Article in English | MEDLINE | ID: mdl-32514184

ABSTRACT

Activity-based protein profiling (ABPP) has been used extensively to discover and optimize selective inhibitors of enzymes. Here, we show that ABPP can also be implemented to identify the converse-small-molecule enzyme activators. Using a kinetically controlled, fluorescence polarization-ABPP assay, we identify compounds that stimulate the activity of LYPLAL1-a poorly characterized serine hydrolase with complex genetic links to human metabolic traits. We apply ABPP-guided medicinal chemistry to advance a lead into a selective LYPLAL1 activator suitable for use in vivo. Structural simulations coupled to mutational, biochemical and biophysical analyses indicate that this compound increases LYPLAL1's catalytic activity likely by enhancing the efficiency of the catalytic triad charge-relay system. Treatment with this LYPLAL1 activator confers beneficial effects in a mouse model of diet-induced obesity. These findings reveal a new mode of pharmacological regulation for this large enzyme family and suggest that ABPP may aid discovery of activators for additional enzyme classes.


Subject(s)
Enzyme Activators/chemistry , Enzyme Activators/pharmacology , Lysophospholipase/metabolism , Small Molecule Libraries/pharmacology , Animals , Drug Discovery , Enzyme Activators/pharmacokinetics , Fluorescence Polarization , HEK293 Cells , High-Throughput Screening Assays/methods , Humans , Insulin Resistance , Lysophospholipase/chemistry , Lysophospholipase/genetics , Male , Metabolic Syndrome/drug therapy , Metabolic Syndrome/metabolism , Mice, Inbred C57BL , Mice, Obese , Molecular Dynamics Simulation , Molecular Structure , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacokinetics , Structure-Activity Relationship
6.
Nature ; 534(7608): 570-4, 2016 06 23.
Article in English | MEDLINE | ID: mdl-27309814

ABSTRACT

Small molecules are powerful tools for investigating protein function and can serve as leads for new therapeutics. Most human proteins, however, lack small-molecule ligands, and entire protein classes are considered 'undruggable'. Fragment-based ligand discovery can identify small-molecule probes for proteins that have proven difficult to target using high-throughput screening of complex compound libraries. Although reversibly binding ligands are commonly pursued, covalent fragments provide an alternative route to small-molecule probes, including those that can access regions of proteins that are difficult to target through binding affinity alone. Here we report a quantitative analysis of cysteine-reactive small-molecule fragments screened against thousands of proteins in human proteomes and cells. Covalent ligands were identified for >700 cysteines found in both druggable proteins and proteins deficient in chemical probes, including transcription factors, adaptor/scaffolding proteins, and uncharacterized proteins. Among the atypical ligand-protein interactions discovered were compounds that react preferentially with pro- (inactive) caspases. We used these ligands to distinguish extrinsic apoptosis pathways in human cell lines versus primary human T cells, showing that the former is largely mediated by caspase-8 while the latter depends on both caspase-8 and -10. Fragment-based covalent ligand discovery provides a greatly expanded portrait of the ligandable proteome and furnishes compounds that can illuminate protein functions in native biological systems.


Subject(s)
Cysteine/metabolism , Drug Evaluation, Preclinical/methods , Proteome/chemistry , Proteome/metabolism , Small Molecule Libraries/metabolism , Small Molecule Libraries/pharmacology , T-Lymphocytes/metabolism , Apoptosis , Caspase 10/chemistry , Caspase 10/metabolism , Caspase 8/chemistry , Caspase 8/metabolism , Cells, Cultured , Enzyme Precursors/chemistry , Enzyme Precursors/metabolism , Humans , Ligands , Peptide Fragments/chemistry , Peptide Fragments/metabolism , T-Lymphocytes/chemistry , Transcription Factors/chemistry , Transcription Factors/metabolism
7.
Mar Drugs ; 20(6)2022 May 31.
Article in English | MEDLINE | ID: mdl-35736176

ABSTRACT

The bengamides comprise an interesting family of natural products isolated from sponges belonging to the prolific Jaspidae family. Their outstanding antitumor properties, coupled with their unique mechanism of action and unprecedented molecular structures, have prompted an intense research activity directed towards their total syntheses, analogue design, and biological evaluations for their development as new anticancer agents. Together with these biological studies in cancer research, in recent years, the bengamides have been identified as potential antibiotics by their impressive biological activities against various drug-resistant bacteria such as Mycobacterium tuberculosis and Staphylococcus aureus. This review reports on the new advances in the chemistry and biology of the bengamides during the last years, paying special attention to their development as promising new antibiotics. Thus, the evolution of the bengamides from their initial exploration as antitumor agents up to their current status as antibiotics is described in detail, highlighting the manifold value of these marine natural products as valid hits in medicinal chemistry.


Subject(s)
Antineoplastic Agents , Biological Products , Mycobacterium tuberculosis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Biological Products/chemistry , Biological Products/pharmacology , Molecular Structure
8.
Proc Natl Acad Sci U S A ; 116(38): 18808-18814, 2019 09 17.
Article in English | MEDLINE | ID: mdl-31484779

ABSTRACT

Sulfur fluoride exchange (SuFEx) has emerged as the new generation of click chemistry. We report here a SuFEx-enabled, agnostic approach for the discovery and optimization of covalent inhibitors of human neutrophil elastase (hNE). Evaluation of our ever-growing collection of SuFExable compounds toward various biological assays unexpectedly revealed a selective and covalent hNE inhibitor: benzene-1,2-disulfonyl fluoride. Synthetic derivatization of the initial hit led to a more potent agent, 2-(fluorosulfonyl)phenyl fluorosulfate with IC50 0.24 µM and greater than 833-fold selectivity over the homologous neutrophil serine protease, cathepsin G. The optimized, yet simple benzenoid probe only modified active hNE and not its denatured form.


Subject(s)
Fluorides/chemistry , Leukocyte Elastase/antagonists & inhibitors , Serine Proteinase Inhibitors/chemistry , Sulfur Compounds/chemistry , Click Chemistry , Enzyme Activation/drug effects , Humans , Inhibitory Concentration 50 , Leukocyte Elastase/chemistry , Leukocyte Elastase/metabolism , Molecular Structure , Protein Binding , Protein Folding , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/pharmacology , Sulfinic Acids/chemical synthesis , Sulfinic Acids/chemistry , Sulfinic Acids/pharmacology
9.
J Proteome Res ; 20(2): 1451-1454, 2021 02 05.
Article in English | MEDLINE | ID: mdl-33393790

ABSTRACT

In this Letter, we reanalyze published mass spectrometry data sets of clinical samples with a focus on determining the coinfection status of individuals infected with SARS-CoV-2 coronavirus. We demonstrate the use of ComPIL 2.0 software along with a metaproteomics workflow within the Galaxy platform to detect cohabitating potential pathogens in COVID-19 patients using mass spectrometry-based analysis. From a sample collected from gargling solutions, we detected Streptococcus pneumoniae (opportunistic and multidrug-resistant pathogen) and Lactobacillus rhamnosus (a probiotic component) along with SARS-Cov-2. We could also detect Pseudomonas sps. Bc-h from COVID-19 positive samples and Acinetobacter ursingii and Pseudomonas monteilii from COVID-19 negative samples collected from oro- and nasopharyngeal samples. We believe that the early detection and characterization of coinfections by using metaproteomics from COVID-19 patients will potentially impact the diagnosis and treatment of patients affected by SARS-CoV-2 infection.


Subject(s)
Bacterial Infections/diagnosis , COVID-19/diagnosis , Proteomics/methods , SARS-CoV-2/metabolism , Acinetobacter/isolation & purification , Bacterial Infections/complications , Bacterial Infections/microbiology , COVID-19/complications , COVID-19/virology , Coinfection/microbiology , Coinfection/virology , Humans , Mass Spectrometry/methods , Nasopharynx/microbiology , Nasopharynx/virology , Pseudomonas/isolation & purification , SARS-CoV-2/physiology , Streptococcus pneumoniae/isolation & purification
10.
Bioorg Med Chem Lett ; 40: 127903, 2021 05 15.
Article in English | MEDLINE | ID: mdl-33713779

ABSTRACT

Folate and related derivatives are essential small molecules required for survival. Of significant interest is the biological role and necessity of folate in the crosstalk between commensal organisms and their respective hosts, including the tremendously complex human distal gut microbiome. Here, we designed a folate-based probe consisting of a photo-crosslinker to detect and quantitate folate-binding proteins from proteomic samples. We demonstrate the selectivity of our probe for the well-established human folate-binding protein dihydrofolate reductase and show no promiscuous labeling occurs with human caspase-3 or bovine serum albumin, which served as negative controls. Affinity-based enrichment of folate-binding proteins from an E. coli lysate in combination with mass spectrometry proteomics verified the ability of our probe to isolate low-abundance folate-dependent proteins. We envision that our probe will serve as a tool to elucidate the roles of commensal microbial folate-binding proteins in health and microbiome-related diseases.


Subject(s)
Cross-Linking Reagents/chemistry , Folic Acid Transporters/analysis , Folic Acid/chemistry , Molecular Probes/chemistry , Caspase 3/chemistry , Chromatography, High Pressure Liquid , Escherichia coli/chemistry , Humans , Microbiota/physiology , Photochemical Processes , Proteomics , Serum Albumin, Bovine/metabolism , Tandem Mass Spectrometry , Tetrahydrofolate Dehydrogenase/chemistry
11.
J Am Chem Soc ; 142(25): 10899-10904, 2020 06 24.
Article in English | MEDLINE | ID: mdl-32479075

ABSTRACT

Optimization of small-molecule probes or drugs is a synthetically lengthy, challenging, and resource-intensive process. Lack of automation and reliance on skilled medicinal chemists is cumbersome in both academic and industrial settings. Here, we demonstrate a high-throughput hit-to-lead process based on the biocompatible sulfur(VI) fluoride exchange (SuFEx) click chemistry. A high-throughput screening hit benzyl (cyanomethyl)carbamate (Ki = 8 µM) against a bacterial cysteine protease SpeB was modified with a SuFExable iminosulfur oxydifluoride [RN═S(O)F2] motif, rapidly diversified into 460 analogs in overnight reactions, and the products were directly screened to yield drug-like inhibitors with 480-fold higher potency (Ki = 18 nM). We showed that the improved molecule is active in a bacteria-host coculture. Since this SuFEx linkage reaction succeeds on picomole scale for direct screening, we anticipate our methodology can accelerate the development of robust biological probes and drug candidates.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Exotoxins/antagonists & inhibitors , Sulfur Compounds/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalytic Domain , Click Chemistry , Crystallography, X-Ray , Cysteine Proteinase Inhibitors/metabolism , Cysteine Proteinase Inhibitors/toxicity , Drug Discovery , Exotoxins/chemistry , Exotoxins/metabolism , High-Throughput Screening Assays , Humans , Jurkat Cells , Microsomes, Liver/metabolism , Proof of Concept Study , Protein Binding
12.
Angew Chem Int Ed Engl ; 59(30): 12460-12469, 2020 07 20.
Article in English | MEDLINE | ID: mdl-32301265

ABSTRACT

Diversity Oriented Clicking (DOC) is a unified click-approach for the modular synthesis of lead-like structures through application of the wide family of click transformations. DOC evolved from the concept of achieving "diversity with ease", by combining classic C-C π-bond click chemistry with recent developments in connective SuFEx-technologies. We showcase 2-Substituted-Alkynyl-1-Sulfonyl Fluorides (SASFs) as a new class of connective hub in concert with a diverse selection of click-cycloaddition processes. Through the selective DOC of SASFs with a range of dipoles and cyclic dienes, we report a diverse click-library of 173 unique functional molecules in minimal synthetic steps. The SuFExable library comprises 10 discrete heterocyclic core structures derived from 1,3- and 1,5-dipoles; while reaction with cyclic dienes yields several three-dimensional bicyclic Diels-Alder adducts. Growing the library to 278 discrete compounds through late-stage modification was made possible through SuFEx click derivatization of the pendant sulfonyl fluoride group in 96 well-plates-demonstrating the versatility of the DOC approach for the rapid synthesis of diverse functional structures. Screening for function against MRSA (USA300) revealed several lead hits with improved activity over methicillin.


Subject(s)
Click Chemistry , Sulfinic Acids/chemistry , Cycloaddition Reaction , Molecular Structure
13.
Biochemistry ; 58(13): 1728-1737, 2019 04 02.
Article in English | MEDLINE | ID: mdl-30835452

ABSTRACT

Commensal bacteria secrete proteins and metabolites to influence host intestinal homeostasis, and proteases represent a significant constituent of the components at the host:microbiome interface. Here, we determined the structures of the two secreted C11 cysteine proteases encoded by the established gut commensal Bacteroides thetaiotaomicron. We employed mutational analysis to demonstrate the two proteases, termed "thetapain" and "iotapain", undergo in trans autoactivation after lysine and/or arginine residues, as observed for other C11 proteases. We determined the structures of the active forms of thetapain and iotapain in complex with irreversible peptide inhibitors, Ac-VLTK-AOMK and biotin-VLTK-AOMK, respectively. Structural comparisons revealed key active-site interactions important for peptide recognition are more extensive for thetapain; however, both proteases employ a glutamate residue to preferentially bind small polar residues at the P2 position. Our results will aid in the design of protease-specific probes to ultimately understand the biological role of C11 proteases in bacterial fitness, elucidate their host and/or microbial substrates, and interrogate their involvement in microbiome-related diseases.


Subject(s)
Bacteroides thetaiotaomicron/enzymology , Cysteine Proteases/chemistry , Cysteine Proteinase Inhibitors/pharmacology , Peptides/pharmacology , Bacteroides Infections/microbiology , Bacteroides thetaiotaomicron/chemistry , Bacteroides thetaiotaomicron/drug effects , Bacteroides thetaiotaomicron/metabolism , Catalytic Domain/drug effects , Crystallography, X-Ray , Cysteine Proteases/metabolism , Humans , Molecular Docking Simulation , Protein Conformation/drug effects
14.
J Proteome Res ; 18(2): 616-622, 2019 02 01.
Article in English | MEDLINE | ID: mdl-30525664

ABSTRACT

We designed a metaproteomic analysis method (ComPIL) to accommodate the ever-increasing number of sequences against which experimental shotgun proteomics spectra could be accurately and rapidly queried. Our objective was to create these large databases for the analysis of complex metasamples with unknown composition, including those derived from human, animal, and environmental microbiomes. The amount of high-throughput sequencing data has substantially increased since our original database was assembled in 2014. Here, we present a rebuild of the ComPIL libraries comprised of updated publicly disseminated sequence data as well as a modified version of the search engine ProLuCID-ComPIL optimized for querying experimental spectra. ComPIL 2.0 consists of 113 million protein records and roughly 4.8 billion unique tryptic peptide sequences and is 2.3 times the size of our original version. We searched a data set collected on a healthy human gut microbiome proteomic sample and compared the results to demonstrate that ComPIL 2.0 showed a substantial increase in the number of unique identified peptides and proteins compared to the first ComPIL version. The high confidence of protein identification and accuracy demonstrated by the use of ComPIL 2.0 may encourage the method's application for large-scale proteomic annotation of complex protein systems.


Subject(s)
Complex Mixtures/analysis , Databases, Protein , Proteomics/methods , Amino Acid Sequence , Animals , Bacterial Proteins/analysis , Gastrointestinal Microbiome , Humans , Peptides/analysis , Search Engine
15.
Bioorg Med Chem Lett ; 29(18): 2609-2612, 2019 09 15.
Article in English | MEDLINE | ID: mdl-31387789

ABSTRACT

To identify sialic acid binding proteins from complex proteomes, three photocrosslinking affinity-based probes were constructed using Neu5Ac (5 and 6) and Neu5Ac2en (7) scaffolds. Kinetic inhibition assays and Western blotting revealed the Neu5Ac2en-based 7 to be an effective probe for the labeling of a purified gut microbial sialidase (BDI_2946) and a purified human sialic acid binding protein (hCD33). Additionally, LC-MS/MS affinity-based protein profiling verified the ability of 7 to enrich a low-abundance sialic acid binding protein (complement factor H) from human serum thus validating the utility of this probe in a complex context.


Subject(s)
Enzyme Inhibitors/pharmacology , Molecular Probes/pharmacology , N-Acetylneuraminic Acid/pharmacology , Sialic Acid Binding Ig-like Lectin 3/antagonists & inhibitors , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Humans , Molecular Probes/chemical synthesis , Molecular Probes/chemistry , Molecular Structure , N-Acetylneuraminic Acid/chemical synthesis , N-Acetylneuraminic Acid/chemistry , Neuraminidase/antagonists & inhibitors , Neuraminidase/metabolism , Sialic Acid Binding Ig-like Lectin 3/isolation & purification , Sialic Acid Binding Ig-like Lectin 3/metabolism , Structure-Activity Relationship
16.
Proteomics ; 18(3-4)2018 02.
Article in English | MEDLINE | ID: mdl-29319931

ABSTRACT

Metaproteomics can greatly assist established high-throughput sequencing methodologies to provide systems biological insights into the alterations of microbial protein functionalities correlated with disease-associated dysbiosis of the intestinal microbiota. Here, the authors utilize the well-characterized murine T cell transfer model of colitis to find specific changes within the intestinal luminal proteome associated with inflammation. MS proteomic analysis of colonic samples permitted the identification of ≈10 000-12 000 unique peptides that corresponded to 5610 protein clusters identified across three groups, including the colitic Rag1-/- T cell recipients, isogenic Rag1-/- controls, and wild-type mice. The authors demonstrate that the colitic mice exhibited a significant increase in Proteobacteria and Verrucomicrobia and show that such alterations in the microbial communities contributed to the enrichment of specific proteins with transcription and translation gene ontology terms. In combination with 16S sequencing, the authors' metaproteomics-based microbiome studies provide a foundation for assessing alterations in intestinal luminal protein functionalities in a robust and well-characterized mouse model of colitis, and set the stage for future studies to further explore the functional mechanisms of altered protein functionalities associated with dysbiosis and inflammation.


Subject(s)
Bacterial Proteins/metabolism , Colitis/metabolism , Colon/metabolism , Inflammation/metabolism , Microbiota , Proteome/analysis , Animals , Colitis/microbiology , Colon/microbiology , Disease Models, Animal , Inflammation/microbiology , Mice , Mice, Inbred C57BL
17.
J Proteome Res ; 17(9): 2978-2986, 2018 09 07.
Article in English | MEDLINE | ID: mdl-30019906

ABSTRACT

The lysis and extraction of soluble bacterial proteins from cells is a common practice for proteomics analyses, but insoluble bacterial biomasses are often left behind. Here, we show that with triflic acid treatment, the insoluble bacterial biomass of Gram- and Gram+ bacteria can be rendered soluble. We use LC-MS/MS shotgun proteomics to show that bacterial proteins in the soluble and insoluble postlysis fractions differ significantly. Additionally, in the case of Gram- Pseudomonas aeruginosa, triflic acid treatment enables the enrichment of cell-envelope-associated proteins. Finally, we apply triflic acid to a human microbiome sample to show that this treatment is robust and enables the identification of a new, complementary subset of proteins from a complex microbial mixture.


Subject(s)
Bacillus subtilis/chemistry , Bacterial Proteins/isolation & purification , Membrane Proteins/isolation & purification , Mesylates/chemistry , Proteomics/methods , Pseudomonas aeruginosa/chemistry , Bacillus subtilis/genetics , Bacillus subtilis/metabolism , Chromatography, Liquid , Complex Mixtures/chemistry , Gastrointestinal Microbiome/genetics , Humans , Jurkat Cells , Metagenome , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Sonication/methods , Tandem Mass Spectrometry
18.
J Proteome Res ; 17(12): 4051-4060, 2018 12 07.
Article in English | MEDLINE | ID: mdl-30270626

ABSTRACT

The 2017 Dagstuhl Seminar on Computational Proteomics provided an opportunity for a broad discussion on the current state and future directions of the generation and use of peptide tandem mass spectrometry spectral libraries. Their use in proteomics is growing slowly, but there are multiple challenges in the field that must be addressed to further increase the adoption of spectral libraries and related techniques. The primary bottlenecks are the paucity of high quality and comprehensive libraries and the general difficulty of adopting spectral library searching into existing workflows. There are several existing spectral library formats, but none captures a satisfactory level of metadata; therefore, a logical next improvement is to design a more advanced, Proteomics Standards Initiative-approved spectral library format that can encode all of the desired metadata. The group discussed a series of metadata requirements organized into three designations of completeness or quality, tentatively dubbed bronze, silver, and gold. The metadata can be organized at four different levels of granularity: at the collection (library) level, at the individual entry (peptide ion) level, at the peak (fragment ion) level, and at the peak annotation level. Strategies for encoding mass modifications in a consistent manner and the requirement for encoding high-quality and commonly seen but as-yet-unidentified spectra were discussed. The group also discussed related topics, including strategies for comparing two spectra, techniques for generating representative spectra for a library, approaches for selection of optimal signature ions for targeted workflows, and issues surrounding the merging of two or more libraries into one. We present here a review of this field and the challenges that the community must address in order to accelerate the adoption of spectral libraries in routine analysis of proteomics datasets.


Subject(s)
Databases, Protein/standards , Peptide Library , Proteomics/methods , Animals , Humans , Tandem Mass Spectrometry/methods , Workflow
19.
Anal Chem ; 90(5): 3156-3164, 2018 03 06.
Article in English | MEDLINE | ID: mdl-29381867

ABSTRACT

METLIN originated as a database to characterize known metabolites and has since expanded into a technology platform for the identification of known and unknown metabolites and other chemical entities. Through this effort it has become a comprehensive resource containing over 1 million molecules including lipids, amino acids, carbohydrates, toxins, small peptides, and natural products, among other classes. METLIN's high-resolution tandem mass spectrometry (MS/MS) database, which plays a key role in the identification process, has data generated from both reference standards and their labeled stable isotope analogues, facilitated by METLIN-guided analysis of isotope-labeled microorganisms. The MS/MS data, coupled with the fragment similarity search function, expand the tool's capabilities into the identification of unknowns. Fragment similarity search is performed independent of the precursor mass, relying solely on the fragment ions to identify similar structures within the database. Stable isotope data also facilitate characterization by coupling the similarity search output with the isotopic m/ z shifts. Examples of both are demonstrated here with the characterization of four previously unknown metabolites. METLIN also now features in silico MS/MS data, which has been made possible through the creation of algorithms trained on METLIN's MS/MS data from both standards and their isotope analogues. With these informatic and experimental data features, METLIN is being designed to address the characterization of known and unknown molecules.


Subject(s)
Cell Extracts/analysis , Databases, Chemical/statistics & numerical data , Datasets as Topic/statistics & numerical data , Metabolomics/methods , Metabolomics/statistics & numerical data , Pichia/chemistry , Pichia/metabolism , Tandem Mass Spectrometry/statistics & numerical data
20.
J Proteome Res ; 16(2): 1014-1026, 2017 02 03.
Article in English | MEDLINE | ID: mdl-28052195

ABSTRACT

Tandem mass spectrometry based shotgun proteomics of distal gut microbiomes is exceedingly difficult due to the inherent complexity and taxonomic diversity of the samples. We introduce two new methodologies to improve metaproteomic studies of microbiome samples. These methods include the stable isotope labeling in mammals to permit protein quantitation across two mouse cohorts as well as the application of activity-based probes to enrich and analyze both host and microbial proteins with specific functionalities. We used these technologies to study the microbiota from the adoptive T cell transfer mouse model of inflammatory bowel disease (IBD) and compare these samples to an isogenic control, thereby limiting genetic and environmental variables that influence microbiome composition. The data generated highlight quantitative alterations in both host and microbial proteins due to intestinal inflammation and corroborates the observed phylogenetic changes in bacteria that accompany IBD in humans and mouse models. The combination of isotope labeling with shotgun proteomics resulted in the total identification of 4434 protein clusters expressed in the microbial proteomic environment, 276 of which demonstrated differential abundance between control and IBD mice. Notably, application of a novel cysteine-reactive probe uncovered several microbial proteases and hydrolases overrepresented in the IBD mice. Implementation of these methods demonstrated that substantial insights into the identity and dysregulation of host and microbial proteins altered in IBD can be accomplished and can be used in the interrogation of other microbiome-related diseases.


Subject(s)
Bacterial Proteins/isolation & purification , Gastrointestinal Microbiome/genetics , Inflammatory Bowel Diseases/microbiology , Metagenome , Proteome/isolation & purification , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Chromatography, Liquid , Disease Models, Animal , Feces/microbiology , Female , Gene Deletion , Gene Expression , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/pathology , Intestines/microbiology , Intestines/pathology , Isotope Labeling , Mice , Proteome/genetics , Proteome/metabolism , Tandem Mass Spectrometry
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