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1.
Proc Natl Acad Sci U S A ; 119(13): e2123566119, 2022 03 29.
Article in English | MEDLINE | ID: mdl-35320042

ABSTRACT

SignificanceMethanobactins (Mbns), copper-binding peptidic compounds produced by some bacteria, are candidate therapeutics for human diseases of copper overload. The paired oxazolone-thioamide bidentate ligands of methanobactins are generated from cysteine residues in a precursor peptide, MbnA, by the MbnBC enzyme complex. MbnBC activity depends on the presence of iron and oxygen, but the catalytically active form has not been identified. Here, we provide evidence that a dinuclear Fe(II)Fe(III) center in MbnB, which is the only representative of a >13,000-member protein family to be characterized, is responsible for this reaction. These findings expand the known roles of diiron enzymes in biology and set the stage for mechanistic understanding, and ultimately engineering, of the MbnBC biosynthetic complex.


Subject(s)
Cysteine , Oxazolone , Copper/metabolism , Ferric Compounds/chemistry , Humans , Imidazoles , Oligopeptides , Oxygen/metabolism , Thioamides
2.
Nucleic Acids Res ; 50(D1): D526-D533, 2022 01 07.
Article in English | MEDLINE | ID: mdl-34986596

ABSTRACT

The Human Proteoform Atlas (HPfA) is a web-based repository of experimentally verified human proteoforms on-line at http://human-proteoform-atlas.org and is a direct descendant of the Consortium of Top-Down Proteomics' (CTDP) Proteoform Atlas. Proteoforms are the specific forms of protein molecules expressed by our cells and include the unique combination of post-translational modifications (PTMs), alternative splicing and other sources of variation deriving from a specific gene. The HPfA uses a FAIR system to assign persistent identifiers to proteoforms which allows for redundancy calling and tracking from prior and future studies in the growing community of proteoform biology and measurement. The HPfA is organized around open ontologies and enables flexible classification of proteoforms. To achieve this, a public registry of experimentally verified proteoforms was also created. Submission of new proteoforms can be processed through email vianrtdphelp@northwestern.edu, and future iterations of these proteoform atlases will help to organize and assign function to proteoforms, their PTMs and their complexes in the years ahead.


Subject(s)
Alternative Splicing , Databases, Protein , Protein Processing, Post-Translational , Proteome/chemistry , Proto-Oncogene Proteins p21(ras)/chemistry , User-Computer Interface , Amino Acid Sequence , Atlases as Topic , Gene Ontology , Humans , Models, Molecular , Molecular Sequence Annotation , Polymorphism, Single Nucleotide , Protein Conformation , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proteome/classification , Proteome/genetics , Proteome/metabolism , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism
3.
Proteomics ; 22(11-12): e2100209, 2022 06.
Article in English | MEDLINE | ID: mdl-35286768

ABSTRACT

The effectiveness of any proteomics database search depends on the theoretical candidate information contained in the protein database. Unfortunately, candidate entries from protein databases such as UniProt rarely contain all the post-translational modifications (PTMs), disulfide bonds, or endogenous cleavages of interest to researchers. These omissions can limit discovery of novel and biologically important proteoforms. Conversely, searching for a specific proteoform becomes a computationally difficult task for heavily modified proteins. Both situations require updates to the database through user-annotated entries. Unfortunately, manually creating properly formatted UniProt Extensible Markup Language (XML) files is tedious and prone to errors. ProSight Annotator solves these issues by providing a graphical interface for adding user-defined features to UniProt-formatted XML files for better informed proteoform searches. It can be downloaded from http://prosightannotator.northwestern.edu.


Subject(s)
Language , Proteins , Databases, Protein , Protein Processing, Post-Translational , Proteins/chemistry , Proteomics , Software
4.
J Proteome Res ; 21(4): 1189-1195, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35290070

ABSTRACT

It is important for the proteomics community to have a standardized manner to represent all possible variations of a protein or peptide primary sequence, including natural, chemically induced, and artifactual modifications. The Human Proteome Organization Proteomics Standards Initiative in collaboration with several members of the Consortium for Top-Down Proteomics (CTDP) has developed a standard notation called ProForma 2.0, which is a substantial extension of the original ProForma notation developed by the CTDP. ProForma 2.0 aims to unify the representation of proteoforms and peptidoforms. ProForma 2.0 supports use cases needed for bottom-up and middle-/top-down proteomics approaches and allows the encoding of highly modified proteins and peptides using a human- and machine-readable string. ProForma 2.0 can be used to represent protein modifications in a specified or ambiguous location, designated by mass shifts, chemical formulas, or controlled vocabulary terms, including cross-links (natural and chemical) and atomic isotopes. Notational conventions are based on public controlled vocabularies and ontologies. The most up-to-date full specification document and information about software implementations are available at http://psidev.info/proforma.


Subject(s)
Proteome , Proteomics , Humans , Protein Processing, Post-Translational , Proteome/genetics , Reference Standards , Software
5.
J Proteome Res ; 21(1): 274-288, 2022 01 07.
Article in English | MEDLINE | ID: mdl-34878788

ABSTRACT

Methods of antibody detection are used to assess exposure or immunity to a pathogen. Here, we present Ig-MS, a novel serological readout that captures the immunoglobulin (Ig) repertoire at molecular resolution, including entire variable regions in Ig light and heavy chains. Ig-MS uses recent advances in protein mass spectrometry (MS) for multiparametric readout of antibodies, with new metrics like Ion Titer (IT) and Degree of Clonality (DoC) capturing the heterogeneity and relative abundance of individual clones without sequencing of B cells. We applied Ig-MS to plasma from subjects with severe and mild COVID-19 and immunized subjects after two vaccine doses, using the receptor-binding domain (RBD) of the spike protein of SARS-CoV-2 as the bait for antibody capture. Importantly, we report a new data type for human serology, that could use other antigens of interest to gauge immune responses to vaccination, pathogens, or autoimmune disorders.


Subject(s)
COVID-19 , SARS-CoV-2 , Antibodies, Neutralizing , Antibodies, Viral , Humans , Mass Spectrometry , Spike Glycoprotein, Coronavirus/genetics
6.
J Am Chem Soc ; 144(50): 23104-23114, 2022 12 21.
Article in English | MEDLINE | ID: mdl-36475650

ABSTRACT

G protein-coupled receptors (GPCRs) are the largest family of membrane receptors in humans. They mediate nearly all aspects of human physiology and thus are of high therapeutic interest. GPCR signaling is regulated in space and time by receptor phosphorylation. It is believed that different phosphorylation states are possible for a single receptor, and each encodes for unique signaling outcomes. Methods to determine the phosphorylation status of GPCRs are critical for understanding receptor physiology and signaling properties of GPCR ligands and therapeutics. However, common proteomic techniques have provided limited quantitative information regarding total receptor phosphorylation stoichiometry, relative abundances of isomeric modification states, and temporal dynamics of these parameters. Here, we report a novel middle-down proteomic strategy and parallel reaction monitoring (PRM) to quantify the phosphorylation states of the C-terminal tail of metabotropic glutamate receptor 2 (mGluR2). By this approach, we found that mGluR2 is subject to both basal and agonist-induced phosphorylation at up to four simultaneous sites with varying probability. Using a PRM tandem mass spectrometry methodology, we localized the positions and quantified the relative abundance of phosphorylations following treatment with an agonist. Our analysis showed that phosphorylation within specific regions of the C-terminal tail of mGluR2 is sensitive to receptor activation, and subsequent site-directed mutagenesis of these sites identified key regions which tune receptor sensitivity. This study demonstrates that middle-down purification followed by label-free quantification is a powerful, quantitative, and accessible tool for characterizing phosphorylation states of GPCRs and other challenging proteins.


Subject(s)
Receptors, G-Protein-Coupled , Signal Transduction , Humans , Receptors, G-Protein-Coupled/chemistry , Phosphorylation , Signal Transduction/physiology , Ligands , Proteomics , Mass Spectrometry , Carrier Proteins/metabolism
7.
Mol Cell Proteomics ; 19(2): 405-420, 2020 02.
Article in English | MEDLINE | ID: mdl-31888965

ABSTRACT

Top-down proteomics studies intact proteoform mixtures and offers important advantages over more common bottom-up proteomics technologies, as it avoids the protein inference problem. However, achieving complete molecular characterization of investigated proteoforms using existing technologies remains a fundamental challenge for top-down proteomics. Here, we benchmark the performance of ultraviolet photodissociation (UVPD) using 213 nm photons generated by a solid-state laser applied to the study of intact proteoforms from three organisms. Notably, the described UVPD setup applies multiple laser pulses to induce ion dissociation, and this feature can be used to optimize the fragmentation outcome based on the molecular weight of the analyzed biomolecule. When applied to complex proteoform mixtures in high-throughput top-down proteomics, 213 nm UVPD demonstrated a high degree of complementarity with the most employed fragmentation method in proteomics studies, higher-energy collisional dissociation (HCD). UVPD at 213 nm offered higher average proteoform sequence coverage and degree of proteoform characterization (including localization of post-translational modifications) than HCD. However, previous studies have shown limitations in applying database search strategies developed for HCD fragmentation to UVPD spectra which contains up to nine fragment ion types. We therefore performed an analysis of the different UVPD product ion type frequencies. From these data, we developed an ad hoc fragment matching strategy and determined the influence of each possible ion type on search outcomes. By paring down the number of ion types considered in high-throughput UVPD searches from all types down to the four most abundant, we were ultimately able to achieve deeper proteome characterization with UVPD. Lastly, our detailed product ion analysis also revealed UVPD cleavage propensities and determined the presence of a product ion produced specifically by 213 nm photons. All together, these observations could be used to better elucidate UVPD dissociation mechanisms and improve the utility of the technique for proteomic applications.


Subject(s)
Proteomics/methods , Ultraviolet Rays , Animals , Carbonic Anhydrases , Cells, Cultured , Chromatography, Liquid , Fibroblasts , Fungal Proteins , Humans , Mice , Myocytes, Cardiac , Myoglobin , Photons , Pseudomonas aeruginosa , Tandem Mass Spectrometry , Ubiquitin
8.
Angew Chem Int Ed Engl ; 61(29): e202200721, 2022 07 18.
Article in English | MEDLINE | ID: mdl-35446460

ABSTRACT

Unraveling the complexity of biological systems relies on the development of new approaches for spatially resolved proteoform-specific analysis of the proteome. Herein, we employ nanospray desorption electrospray ionization mass spectrometry imaging (nano-DESI MSI) for the proteoform-selective imaging of biological tissues. Nano-DESI generates multiply charged protein ions, which is advantageous for their structural characterization using tandem mass spectrometry (MS/MS) directly on the tissue. Proof-of-concept experiments demonstrate that nano-DESI MSI combined with on-tissue top-down proteomics is ideally suited for the proteoform-selective imaging of tissue sections. Using rat brain tissue as a model system, we provide the first evidence of differential proteoform expression in different regions of the brain.


Subject(s)
Spectrometry, Mass, Electrospray Ionization , Tandem Mass Spectrometry , Animals , Ions , Proteome/analysis , Proteomics/methods , Rats , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods
9.
Biochemistry ; 60(3): 161-169, 2021 01 26.
Article in English | MEDLINE | ID: mdl-33426883

ABSTRACT

Efforts to expand the scope of ribosome-mediated polymerization to incorporate noncanonical amino acids (ncAAs) into peptides and proteins hold promise for creating new classes of enzymes, therapeutics, and materials. Recently, the integrated synthesis, assembly, and translation (iSAT) system was established to construct functional ribosomes in cell-free systems. However, the iSAT system has not been shown to be compatible with genetic code expansion. Here, to address this gap, we develop an iSAT platform capable of manufacturing pure proteins with site-specifically incorporated ncAAs. We first establish an iSAT platform based on extracts from genomically recoded Escherichia coli lacking release factor 1 (RF-1). This permits complete reassignment of the amber codon translation function. Next, we optimize orthogonal translation system components to demonstrate the benefits of genomic RF-1 deletion on incorporation of ncAAs into proteins. Using our optimized platform, we demonstrate high-level, multi-site incorporation of p-acetyl-phenylalanine (pAcF) and p-azido-phenylalanine into superfolder green fluorescent protein (sfGFP). Mass spectrometry analysis confirms the high accuracy of incorporation for pAcF at one, two, and five amber sites in sfGFP. The iSAT system updated for ncAA incorporation sets the stage for investigating ribosomal mutations to better understand the fundamental basis of protein synthesis, manufacturing proteins with new properties, and engineering ribosomes for novel polymerization chemistries.


Subject(s)
Codon, Terminator , Escherichia coli/chemistry , Green Fluorescent Proteins/biosynthesis , Protein Biosynthesis , Ribosomes/chemistry , Amino Acids , Amino Acyl-tRNA Synthetases/chemistry , Cell-Free System/chemistry
10.
Biochemistry ; 60(38): 2845-2850, 2021 09 28.
Article in English | MEDLINE | ID: mdl-34510894

ABSTRACT

Methanobactins (Mbns) are ribosomally produced, post-translationally modified peptidic natural products that bind copper with high affinity. Methanotrophic bacteria use Mbns to acquire copper needed for enzymatic methane oxidation. Despite the presence of Mbn operons in a range of methanotroph and other bacterial genomes, few Mbns have been isolated and structurally characterized. Here we report the isolation of a novel Mbn from the methanotroph Methylosinus (Ms.) sp. LW3. Mass spectrometric and nuclear magnetic resonance spectroscopic data indicate that this Mbn, the largest characterized to date, consists of a 13-amino acid backbone modified to include pyrazinedione/oxazolone rings and neighboring thioamide groups derived from cysteine residues. The pyrazinedione ring is more stable to acid hydrolysis than the oxazolone ring and likely protects the Mbn from degradation. The structure corresponds exactly to that predicted on the basis of the Ms. sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.


Subject(s)
Copper/metabolism , Methylosinus/enzymology , Methylosinus/metabolism , Amino Acid Sequence/genetics , Bacterial Proteins/metabolism , Biological Products/metabolism , Chelating Agents/chemistry , Copper/chemistry , Gene Expression/genetics , Gene Expression Regulation, Bacterial/genetics , Genome, Bacterial/genetics , Imidazoles/metabolism , Methane/metabolism , Methylosinus/genetics , Methylosinus trichosporium/enzymology , Methylosinus trichosporium/genetics , Methylosinus trichosporium/metabolism , Oligopeptides/metabolism , Operon/genetics , Oxidation-Reduction , Peptides/metabolism
11.
Anal Chem ; 93(16): 6323-6328, 2021 04 27.
Article in English | MEDLINE | ID: mdl-33844503

ABSTRACT

Field asymmetric ion mobility spectrometry (FAIMS), when used in proteomics studies, provides superior selectivity and enables more proteins to be identified by providing additional gas-phase separation. Here, we tested the performance of cylindrical FAIMS for the identification and characterization of proteoforms by top-down mass spectrometry of heterogeneous protein mixtures. Combining FAIMS with chromatographic separation resulted in a 62% increase in protein identifications, an 8% increase in proteoform identifications, and an improvement in proteoform identification compared to samples analyzed without FAIMS. In addition, utilization of FAIMS resulted in the identification of proteins encoded by lower-abundance mRNA transcripts. These improvements were attributable, in part, to improved signal-to-noise for proteoforms with similar retention times. Additionally, our results show that the optimal compensation voltage of any given proteoform was correlated with the molecular weight of the analyte. Collectively these results suggest that the addition of FAIMS can enhance top-down proteomics in both discovery and targeted applications.


Subject(s)
Ion Mobility Spectrometry , Proteomics , Mass Spectrometry , Proteins
12.
Blood ; 133(11): 1171-1185, 2019 03 14.
Article in English | MEDLINE | ID: mdl-30587525

ABSTRACT

Aberrant activation of mTOR signaling in acute myeloid leukemia (AML) results in a survival advantage that promotes the malignant phenotype. To improve our understanding of factors that contribute to mammalian target of rapamycin (mTOR) signaling activation and identify novel therapeutic targets, we searched for unique interactors of mTOR complexes through proteomics analyses. We identify cyclin dependent kinase 9 (CDK9) as a novel binding partner of the mTOR complex scaffold protein, mLST8. Our studies demonstrate that CDK9 is present in distinct mTOR-like (CTOR) complexes in the cytoplasm and nucleus. In the nucleus, CDK9 binds to RAPTOR and mLST8, forming CTORC1, to promote transcription of genes important for leukemogenesis. In the cytoplasm, CDK9 binds to RICTOR, SIN1, and mLST8, forming CTORC2, and controls messenger RNA (mRNA) translation through phosphorylation of LARP1 and rpS6. Pharmacological targeting of CTORC complexes results in suppression of growth of primitive human AML progenitors in vitro and elicits strong antileukemic responses in AML xenografts in vivo.


Subject(s)
Carcinogenesis/drug effects , Cyclin-Dependent Kinase 9/antagonists & inhibitors , Leukemia, Myeloid, Acute/drug therapy , Mechanistic Target of Rapamycin Complex 1/metabolism , Mechanistic Target of Rapamycin Complex 2/metabolism , RNA, Messenger/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Antimetabolites, Antineoplastic/pharmacology , Apoptosis , Biomarkers, Tumor/metabolism , Carcinogenesis/metabolism , Carcinogenesis/pathology , Cell Proliferation , Cyclin-Dependent Kinase 9/genetics , Cyclin-Dependent Kinase 9/metabolism , Cytarabine/pharmacology , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Mice , Mice, Nude , Phosphorylation , Protein Biosynthesis , Proteome/analysis , RNA, Messenger/drug effects , RNA, Messenger/genetics , Signal Transduction , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
13.
Clin Chem Lab Med ; 59(4): 653-661, 2021 03 26.
Article in English | MEDLINE | ID: mdl-33079696

ABSTRACT

OBJECTIVES: Multiple myeloma (MM) is a malignant plasma cell neoplasm, requiring the integration of clinical examination, laboratory and radiological investigations for diagnosis. Detection and isotypic identification of the monoclonal protein(s) and measurement of other relevant biomarkers in serum and urine are pivotal analyses. However, occasionally this approach fails to characterize complex protein signatures. Here we describe the development and application of next generation mass spectrometry (MS) techniques, and a novel adaptation of immunofixation, to interrogate non-canonical monoclonal immunoproteins. METHODS: Immunoprecipitation immunofixation (IP-IFE) was performed on a Sebia Hydrasys Scan2. Middle-down de novo sequencing and native MS were performed with multiple instruments (21T FT-ICR, Q Exactive HF, Orbitrap Fusion Lumos, and Orbitrap Eclipse). Post-acquisition data analysis was performed using Xcalibur Qual Browser, ProSight Lite, and TDValidator. RESULTS: We adapted a novel variation of immunofixation electrophoresis (IFE) with an antibody-specific immunosubtraction step, providing insight into the clonal signature of gamma-zone monoclonal immunoglobulin (M-protein) species. We developed and applied advanced mass spectrometric techniques such as middle-down de novo sequencing to attain in-depth characterization of the primary sequence of an M-protein. Quaternary structures of M-proteins were elucidated by native MS, revealing a previously unprecedented non-covalently associated hetero-tetrameric immunoglobulin. CONCLUSIONS: Next generation proteomic solutions offer great potential for characterizing complex protein structures and may eventually replace current electrophoretic approaches for the identification and quantification of M-proteins. They can also contribute to greater understanding of MM pathogenesis, enabling classification of patients into new subtypes, improved risk stratification and the potential to inform decisions on future personalized treatment modalities.


Subject(s)
Multiple Myeloma , Myeloma Proteins , Proteomics/methods , Antibodies, Monoclonal , Humans , Immunoelectrophoresis , Mass Spectrometry , Multiple Myeloma/diagnosis
14.
Mol Cell Proteomics ; 18(4): 796-805, 2019 04.
Article in English | MEDLINE | ID: mdl-30647073

ABSTRACT

Within the last several years, top-down proteomics has emerged as a high throughput technique for protein and proteoform identification. This technique has the potential to identify and characterize thousands of proteoforms within a single study, but the absence of accurate false discovery rate (FDR) estimation could hinder the adoption and consistency of top-down proteomics in the future. In automated identification and characterization of proteoforms, FDR calculation strongly depends on the context of the search. The context includes MS data quality, the database being interrogated, the search engine, and the parameters of the search. Particular to top-down proteomics-there are four molecular levels of study: proteoform spectral match (PrSM), protein, isoform, and proteoform. Here, a context-dependent framework for calculating an accurate FDR at each level was designed, implemented, and validated against a manually curated training set with 546 confirmed proteoforms. We examined several search contexts and found that an FDR calculated at the PrSM level under-reported the true FDR at the protein level by an average of 24-fold. We present a new open-source tool, the TDCD_FDR_Calculator, which provides a scalable, context-dependent FDR calculation that can be applied post-search to enhance the quality of results in top-down proteomics from any search engine.


Subject(s)
Proteomics/methods , Algorithms , Databases, Protein , Humans , Protein Isoforms/metabolism , Reproducibility of Results
15.
Nat Chem Biol ; 14(3): 206-214, 2018 02 14.
Article in English | MEDLINE | ID: mdl-29443976

ABSTRACT

Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.


Subject(s)
Genome, Human , Protein Processing, Post-Translational , Proteins/chemistry , Proteome/chemistry , Proteomics/methods , Databases, Protein , Humans , Mass Spectrometry , Phenotype , Protein Biosynthesis , Protein Isoforms/chemistry , Ubiquitin/chemistry
16.
Malar J ; 19(1): 10, 2020 Jan 07.
Article in English | MEDLINE | ID: mdl-31910830

ABSTRACT

BACKGROUND: Immunization with attenuated malaria sporozoites protects humans from experimental malaria challenge by mosquito bite. Protection in humans is strongly correlated with the production of T cells targeting a heterogeneous population of pre-erythrocyte antigen proteoforms, including liver stage antigens. Currently, few T cell epitopes derived from Plasmodium falciparum, the major aetiologic agent of malaria in humans are known. METHODS: In this study both in vitro and in vivo malaria liver stage models were used to sequence host and pathogen proteoforms. Proteoforms from these diverse models were subjected to mild acid elution (of soluble forms), multi-dimensional fractionation, tandem mass spectrometry, and top-down bioinformatics analysis to identify proteoforms in their intact state. RESULTS: These results identify a group of host and malaria liver stage proteoforms that meet a 5% false discovery rate threshold. CONCLUSIONS: This work provides proof-of-concept for the validity of this mass spectrometry/bioinformatic approach for future studies seeking to reveal malaria liver stage antigens towards vaccine development.


Subject(s)
Liver/parasitology , Malaria, Falciparum/immunology , Plasmodium falciparum/immunology , Animals , Antigens, Protozoan/immunology , Disease Models, Animal , Epitopes, T-Lymphocyte , Female , Hepatocytes , Immunity, Cellular , Malaria Vaccines/immunology , Malaria, Falciparum/prevention & control , Mass Spectrometry , Mice , Proteomics , Serum Albumin, Human
17.
Proteomics ; 19(10): e1800361, 2019 05.
Article in English | MEDLINE | ID: mdl-31050378

ABSTRACT

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post-translational modifications. In top-down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top-down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.


Subject(s)
Amino Acids/analysis , Mass Spectrometry , Protein Processing, Post-Translational , Proteome/analysis , Proteomics/methods , Animals , Computational Biology , Electrophoresis, Capillary , Humans , Programming Languages , Reproducibility of Results , Software
18.
J Proteome Res ; 18(11): 3999-4012, 2019 11 01.
Article in English | MEDLINE | ID: mdl-31550894

ABSTRACT

Cocaine addiction afflicts nearly 1 million adults in the United States, and to date, there are no known treatments approved for this psychiatric condition. Women are particularly vulnerable to developing a cocaine use disorder and suffer from more serious cardiac consequences than men when using cocaine. Estrogen is one biological factor contributing to the increased risk for females to develop problematic cocaine use. Animal studies have demonstrated that estrogen (17ß-estradiol or E2) enhances the rewarding properties of cocaine. Although E2 affects the dopamine system, the molecular and cellular mechanisms of E2-enhanced cocaine reward have not been characterized. In this study, quantitative top-down proteomics was used to measure intact proteins in specific regions of the female mouse brain after mice were trained for cocaine-conditioned place preference, a behavioral test of cocaine reward. Several proteoform changes occurred in the ventral tegmental area after combined cocaine and E2 treatments, with the most numerous proteoform alterations on myelin basic protein, indicating possible changes in white matter structure. There were also changes in histone H4, protein phosphatase inhibitors, cholecystokinin, and calmodulin proteoforms. These observations provide insight into estrogen signaling in the brain and may guide new approaches to treating women with cocaine use disorder.


Subject(s)
Brain/drug effects , Cocaine/pharmacology , Estradiol/pharmacology , Proteome/metabolism , Proteomics/methods , Animals , Brain/metabolism , Conditioning, Classical/drug effects , Dopamine/metabolism , Dopamine Uptake Inhibitors/pharmacology , Estrogens/pharmacology , Female , Mice, Inbred C57BL , Ovariectomy , Reward , Ventral Tegmental Area/drug effects , Ventral Tegmental Area/metabolism
19.
Am J Transplant ; 19(9): 2421-2433, 2019 09.
Article in English | MEDLINE | ID: mdl-30947382

ABSTRACT

Reactivation of latent cytomegalovirus remains an important complication after transplant. Although immunosuppression (IS) has been implicated as a primary cause, we have previously shown that the implantation response of a kidney allograft can lead to early transcriptional activation of latent murine cytomegalovirus (MCMV) genes in an immune-competent host and to MCMV reactivation and dissemination to other organs in a genetically immune-deficient recipient. We now describe a model that allows us to separately analyze the impact of the implantation effect vs that of a clinically relevant IS regimen. Treatment with IS of latently infected mice alone does not induce viral reactivation, but transplant of latently infected allogeneic kidneys combined with IS facilitates MCMV reactivation in the graft and dissemination to other organs. The IS regimen effectively dampens allo-immune inflammatory pathways and depletes recipient anti-MCMV but does not affect ischemia-reperfusion injury pathways. MCMV reactivation similar to that seen in allogeneic transplants combined with also occurs after syngeneic transplants. Thus, our data strongly suggest that while ischemia-reperfusion injury of the implanted graft is sufficient and necessary to initiate transcriptional reactivation of latent MCMV ("first hit"), IS is permissive to the first hit and facilitates dissemination to other organs ("second hit").


Subject(s)
Cytomegalovirus Infections/complications , Kidney Transplantation/adverse effects , Muromegalovirus/physiology , Renal Insufficiency/surgery , Virus Activation , Animals , Disease Models, Animal , Gene Deletion , Histones/metabolism , Immunosuppression Therapy , Kidney/pathology , Mice , Mice, Inbred BALB C , Phenotype , Postoperative Complications/virology , Proteomics , Renal Insufficiency/complications , Reperfusion Injury , Transplantation, Homologous
20.
J Cell Sci ; 130(1): 111-118, 2017 01 01.
Article in English | MEDLINE | ID: mdl-27815408

ABSTRACT

EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions.


Subject(s)
Epidermis/metabolism , Keratinocytes/metabolism , Microfilament Proteins/metabolism , Proteomics , Receptor, EphA2/metabolism , Tight Junctions/metabolism , Carbon-Nitrogen Ligases/metabolism , Cells, Cultured , Escherichia coli Proteins/metabolism , Humans , Infant, Newborn , Male , Protein Binding , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Reproducibility of Results
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