Publications by authors named "Neil L Kelleher"

334 Publications

Elevated N-Linked Glycosylation of IgG V Regions in Myasthenia Gravis Disease Subtypes.

J Immunol 2021 Oct 20;207(8):2005-2014. Epub 2021 Sep 20.

Department of Neurology, Yale University School of Medicine, New Haven, CT;

Elevated N-linked glycosylation of IgG V regions (IgG-V) is an emerging molecular phenotype associated with autoimmune disorders. To test the broader specificity of elevated IgG-V, we studied patients with distinct subtypes of myasthenia gravis (MG), a B cell-mediated autoimmune disease. Our experimental design focused on examining the B cell repertoire and total IgG. It specifically included adaptive immune receptor repertoire sequencing to quantify and characterize N-linked glycosylation sites in the circulating BCR repertoire, proteomics to examine glycosylation patterns of the total circulating IgG, and an exploration of human-derived recombinant autoantibodies, which were studied with mass spectrometry and Ag binding assays to respectively confirm occupation of glycosylation sites and determine whether they alter binding. We found that the frequency of IgG-V motifs was increased in the total BCR repertoire of patients with MG when compared with healthy donors. The elevated frequency was attributed to both biased V gene segment usage and somatic hypermutation. IgG-V could be observed in the total circulating IgG in a subset of patients with MG. Autoantigen binding, by four patient-derived MG autoantigen-specific mAbs with experimentally confirmed presence of IgG-V, was not altered by the glycosylation. Our findings extend prior work on patterns of Ig V region N-linked glycosylation in autoimmunity to MG subtypes.
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http://dx.doi.org/10.4049/jimmunol.2100225DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8492536PMC
October 2021

Reassembling protein complexes after controlled disassembly by top-down mass spectrometry in native mode.

Int J Mass Spectrom 2021 Jul 27;465. Epub 2021 Mar 27.

Departments of Chemistry, Chemical and Biological Engineering, and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, 2170 Tech Dr., Silverman Hall, 60208, Evanston, IL, USA.

The combined use of electrospray ionization run in so-called "native mode" with top-down mass spectrometry (nTDMS) is enhancing both structural biology and discovery proteomics by providing three levels of information in a single experiment: the intact mass of a protein or complex, the masses of its subunits and non-covalent cofactors, and fragment ion masses from direct dissociation of subunits that capture the primary sequence and combinations of diverse post-translational modifications (PTMs). While intact mass data are readily deconvoluted using well-known software options, the analysis of fragmentation data that result from a tandem MS experiment - essential for proteoform characterization - is not yet standardized. In this tutorial, we offer a decision-tree for the analysis of nTDMS experiments on protein complexes and diverse bioassemblies. We include an overview of strategies to navigate this type of analysis, provide example data sets, and highlight software for the hypothesis-driven interrogation of fragment ions for localization of PTMs, metals, and cofactors on native proteoforms. Throughout we have emphasized the key features (deconvolution, search mode, validation, other) that the reader can consider when deciding upon their specific experimental and data processing design using both open-access and commercial software.
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http://dx.doi.org/10.1016/j.ijms.2021.116591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8445521PMC
July 2021

Characterization of a Copper-Chelating Natural Product from the Methanotroph sp. LW3.

Biochemistry 2021 Sep 12;60(38):2845-2850. Epub 2021 Sep 12.

Departments of Molecular Biosciences and Chemistry, Northwestern University, Evanston, Illinois 60208, United States.

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 (.) 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 . sp. LW3 Mbn operon content, providing support for the proposed role of an uncharacterized biosynthetic enzyme, MbnF, and expanding the diversity of known Mbns.
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http://dx.doi.org/10.1021/acs.biochem.1c00443DOI Listing
September 2021

SPT5 stabilization of promoter-proximal RNA polymerase II.

Mol Cell 2021 Aug 31. Epub 2021 Aug 31.

Simpson Querrey Institute for Epigenetics, Department of Biochemistry and Molecular Genetics Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA. Electronic address:

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.
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http://dx.doi.org/10.1016/j.molcel.2021.08.006DOI Listing
August 2021

Spectrum of Apolipoprotein AI and Apolipoprotein AII Proteoforms and Their Associations With Indices of Cardiometabolic Health: The CARDIA Study.

J Am Heart Assoc 2021 Sep 2;10(17):e019890. Epub 2021 Sep 2.

Department of Chemistry Chemistry of Life Processes Institute and Proteomics Center of Excellence Northwestern University Evanston IL.

Background ApoAI (apolipoproteins AI) and apoAII (apolipoprotein AII) are structural and functional proteins of high-density lipoproteins (HDL) which undergo post-translational modifications at specific residues, creating distinct proteoforms. While specific post-translational modifications have been reported to alter apolipoprotein function, the full spectrum of apoAI and apoAII proteoforms and their associations with cardiometabolic phenotype remains unknown. Herein, we comprehensively characterize apoAI and apoAII proteoforms detectable in serum and their post-translational modifications and quantify their associations with cardiometabolic health indices. Methods and Results Using top-down proteomics (mass-spectrometric analysis of intact proteins), we analyzed paired serum samples from 150 CARDIA (Coronary Artery Risk Development in Young Adults) study participants from year 20 and 25 exams. Measuring 15 apoAI and 9 apoAII proteoforms, 6 of which carried novel post-translational modifications, we quantified associations between percent proteoform abundance and key cardiometabolic indices. Canonical (unmodified) apoAI had inverse associations with HDL cholesterol and HDL-cholesterol efflux, and positive associations with obesity indices (body mass index, waist circumference), and triglycerides, whereas glycated apoAI showed positive associations with serum glucose and diabetes mellitus. Fatty-acid‒modified ApoAI proteoforms had positive associations with HDL cholesterol and efflux, and inverse associations with obesity indices and triglycerides. Truncated and dimerized proteoforms of apoAII were associated with HDL cholesterol (positively) and obesity indices (inversely). Several proteoforms had no significant associations with phenotype. Conclusions Associations between apoAI and AII and cardiometabolic indices are proteoform-specific. These results provide "proof-of-concept" that precise chemical characterization of human apolipoproteins will yield improved insights into the complex pathways through which proteins signify and mediate health and disease.
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http://dx.doi.org/10.1161/JAHA.120.019890DOI Listing
September 2021

Next-generation Serology by Mass Spectrometry: Readout of the SARS-CoV-2 Antibody Repertoire.

medRxiv 2021 Jul 7. Epub 2021 Jul 7.

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 multi-parametric 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 apply Ig-MS to plasma from subjects with severe & mild COVID-19, 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, with compatibility to any recombinant antigen to gauge our immune responses to vaccination, pathogens, or autoimmune disorders.
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http://dx.doi.org/10.1101/2021.07.06.21259226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8282107PMC
July 2021

Oncogenic KRAS creates an aspartate metabolism signature in colorectal cancer cells.

FEBS J 2021 Jul 6. Epub 2021 Jul 6.

Department of Molecular Biosciences and Chemistry, Northwestern University, Evanston, IL, USA.

Oncogenic mutations in the KRAS gene are found in 30-50% of colorectal cancers (CRC), and recent findings have demonstrated independent and nonredundant roles for wild-type and mutant KRAS alleles in governing signaling and metabolism. Here, we quantify proteomic changes manifested by KRAS mutation and KRAS allele loss in isogenic cell lines. We show that the expression of KRAS upregulates aspartate metabolizing proteins including PCK1, PCK2, ASNS, and ASS1. Furthermore, differential expression analyses of transcript-level data from CRC tumors identified the upregulation of urea cycle enzymes in CRC. We find that expression of ASS1 supports colorectal cancer cell proliferation and promotes tumor formation in vitro. We show that loss of ASS1 can be rescued with high levels of several metabolites.
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http://dx.doi.org/10.1111/febs.16111DOI Listing
July 2021

The emerging landscape of single-molecule protein sequencing technologies.

Nat Methods 2021 06 7;18(6):604-617. Epub 2021 Jun 7.

Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, the Netherlands.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.
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http://dx.doi.org/10.1038/s41592-021-01143-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8223677PMC
June 2021

Turnover and Inactivation Mechanisms for ()-3-Amino-4,4-difluorocyclopent-1-enecarboxylic Acid, a Selective Mechanism-Based Inactivator of Human Ornithine Aminotransferase.

J Am Chem Soc 2021 Jun 7;143(23):8689-8703. Epub 2021 Jun 7.

Department of Chemistry, Center for Molecular Innovation and Drug Discovery, and Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois 60208, United States.

The inhibition of human ornithine δ-aminotransferase (OAT) is a potential therapeutic approach to treat hepatocellular carcinoma. In this work, ()-3-amino-4,4-difluorocyclopent-1-enecarboxylic acid (SS-1-148, ) was identified as a potent mechanism-based inactivator of OAT while showing excellent selectivity over other related aminotransferases (e.g., GABA-AT). An integrated mechanistic study was performed to investigate the turnover and inactivation mechanisms of . A monofluorinated ketone () was identified as the primary metabolite of in OAT. By soaking OAT holoenzyme crystals with , a precursor to was successfully captured. This -diamine intermediate, covalently bound to Lys292, observed for the first time in OAT/ligand crystals, validates the turnover mechanism proposed for . Co-crystallization yielded OAT in complex with and revealed a novel noncovalent inactivation mechanism in OAT. Native protein mass spectrometry was utilized for the first time in a study of an aminotransferase inactivator to validate the noncovalent interactions between the ligand and the enzyme; a covalently bonded complex was also identified as a minor form observed in the denaturing intact protein mass spectrum. Spectral and stopped-flow kinetic experiments supported a lysine-assisted E2 fluoride ion elimination, which has never been observed experimentally in other studies of related aminotransferase inactivators. This elimination generated the second external aldimine directly from the initial external aldimine, rather than the typical E1cB elimination mechanism, forming a quinonoid transient state between the two external aldimines. The use of native protein mass spectrometry, X-ray crystallography employing both soaking and co-crystallization methods, and stopped-flow kinetics allowed for the detailed elucidation of unusual turnover and inactivation pathways.
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http://dx.doi.org/10.1021/jacs.1c02456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8367020PMC
June 2021

New Interface for Faster Proteoform Analysis: Immunoprecipitation Coupled with SampleStream-Mass Spectrometry.

J Am Soc Mass Spectrom 2021 Jul 27;32(7):1659-1670. Epub 2021 May 27.

Department of Chemistry, Chemistry of Life Processes Institute and Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States.

Different proteoform products of the same gene can exhibit differing associations with health and disease, and their patterns of modifications may offer more precise markers of phenotypic differences between individuals. However, currently employed protein-biomarker discovery and quantification tools, such as bottom-up proteomics and ELISAs, are mostly proteoform-unaware. Moreover, the current throughput for proteoform-level analyses by liquid chromatography mass spectrometry (LCMS) for quantitative top-down proteomics is incompatible with population-level biomarker surveys requiring robust, faster proteoform analysis. To this end, we developed immunoprecipitation coupled to SampleStream mass spectrometry (IP-SampleStream-MS) as a high-throughput, automated technique for the targeted quantification of proteoforms. We applied IP-SampleStream-MS to serum samples of 25 individuals to assess the proteoform abundances of apolipoproteins A-I (ApoA-I) and C-III (ApoC-III). The results for ApoA-I were compared to those of LCMS for these individuals, with IP-SampleStream-MS showing a >7-fold higher throughput with >50% better analytical variation. Proteoform abundances measured by IP-SampleStream-MS correlated strongly to LCMS-based values ( = 0.6-0.9) and produced convergent proteoform-to-phenotype associations, namely, the abundance of canonical ApoA-I was associated with lower HDL-C ( = 0.5) and glycated ApoA-I with higher fasting glucose ( = 0.6). We also observed proteoform-to-phenotype associations for ApoC-III, 22 glycoproteoforms of which were characterized in this study. The abundance of ApoC-III modified by a single -acetyl hexosamine (HexNAc) was associated with indices of obesity, such as BMI, weight, and waist circumference ( ∼ 0.7). These data show IP-SampleStream-MS to be a robust, scalable workflow for high-throughput associations of proteoforms to phenotypes.
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http://dx.doi.org/10.1021/jasms.1c00026DOI Listing
July 2021

Remarkable and Unexpected Mechanism for ()-3-Amino-4-(difluoromethylenyl)cyclohex-1-ene-1-carboxylic Acid as a Selective Inactivator of Human Ornithine Aminotransferase.

J Am Chem Soc 2021 Jun 20;143(21):8193-8207. Epub 2021 May 20.

Department of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, and Center for Developmental Therapeutics, Northwestern University, Evanston, Illinois 60208, United States.

Human ornithine aminotransferase (OAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that was recently found to play an important role in the metabolic reprogramming of hepatocellular carcinoma (HCC) via the proline and glutamine metabolic pathways. The selective inhibition of OAT by compound exhibited potent antitumor activity. Inspired by the discovery of the aminotransferase inactivator (1,3)-3-amino-4-(difluoromethylene)cyclopentane-1-carboxylic acid (), we rationally designed, synthesized, and evaluated a series of six-membered-ring analogs. Among them, was identified as a new selective OAT inactivator, which demonstrated a potency 22× greater than that of . Three different types of protein mass spectrometry approaches and two crystallographic approaches were employed to identify the structure of OAT- and the formation of a remarkable final adduct () in the active site. These spectral studies reveal an enzyme complex heretofore not observed in a PLP-dependent enzyme, which has covalent bonds to two nearby residues. Crystal soaking experiments and molecular dynamics simulations were carried out to identify the structure of the active-site intermediate and elucidate the order of the two covalent bonds that formed, leading to . The initial covalent reaction of the activated warhead occurs with *Thr322 from the second subunit, followed by a subsequent nucleophilic attack by the catalytic residue Lys292. The turnover mechanism of by OAT was supported by a mass spectrometric analysis of metabolites and fluoride ion release experiments. This novel mechanism for OAT with will contribute to the further rational design of selective inactivators and an understanding of potential inactivation mechanisms by aminotransferases.
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http://dx.doi.org/10.1021/jacs.1c03572DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8369387PMC
June 2021

Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry.

Methods Mol Biol 2021 ;2262:47-64

Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA.

The characterization of biologically relevant post-translational modifications (PTMs) on KRAS4B has historically been carried out through methodologies such as immunoblotting with PTM-specific antibodies or peptide-based proteomic methods. While these methods have the potential to identify a given PTM on KRAS4B, they are incapable of characterizing or distinguishing the different molecular forms or proteoforms of KRAS4B from those of related RAS isoforms. We present a method that combines immunoprecipitation of KRAS4B with top-down mass spectrometry (IP-TDMS), thus enabling the precise characterization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.
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http://dx.doi.org/10.1007/978-1-0716-1190-6_3DOI Listing
June 2021

An interpreted atlas of biosynthetic gene clusters from 1,000 fungal genomes.

Proc Natl Acad Sci U S A 2021 May;118(19)

Department of Molecular Biosciences, Northwestern University, Evanston, IL 60208;

Fungi are prolific producers of natural products, compounds which have had a large societal impact as pharmaceuticals, mycotoxins, and agrochemicals. Despite the availability of over 1,000 fungal genomes and several decades of compound discovery efforts from fungi, the biosynthetic gene clusters (BGCs) encoded by these genomes and the associated chemical space have yet to be analyzed systematically. Here, we provide detailed annotation and analyses of fungal biosynthetic and chemical space to enable genome mining and discovery of fungal natural products. Using 1,037 genomes from species across the fungal kingdom (e.g., Ascomycota, Basidiomycota, and non-Dikarya taxa), 36,399 predicted BGCs were organized into a network of 12,067 gene cluster families (GCFs). Anchoring these GCFs with reference BGCs enabled automated annotation of 2,026 BGCs with predicted metabolite scaffolds. We performed parallel analyses of the chemical repertoire of fungi, organizing 15,213 fungal compounds into 2,945 molecular families (MFs). The taxonomic landscape of fungal GCFs is largely species specific, though select families such as the equisetin GCF are present across vast phylogenetic distances with parallel diversifications in the GCF and MF. We compare these fungal datasets with a set of 5,453 bacterial genomes and their BGCs and 9,382 bacterial compounds, revealing dramatic differences between bacterial and fungal biosynthetic logic and chemical space. These genomics and cheminformatics analyses reveal the large extent to which fungal and bacterial sources represent distinct compound reservoirs. With a >10-fold increase in the number of interpreted strains and annotated BGCs, this work better regularizes the biosynthetic potential of fungi for rational compound discovery.
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http://dx.doi.org/10.1073/pnas.2020230118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8126772PMC
May 2021

Deeper Protein Identification Using Field Asymmetric Ion Mobility Spectrometry in Top-Down Proteomics.

Anal Chem 2021 04 12;93(16):6323-6328. Epub 2021 Apr 12.

Northwestern University, National Resource for Translational and Developmental Proteomics, Evanston, Illinois 60208, United States.

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.
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http://dx.doi.org/10.1021/acs.analchem.1c00402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8130575PMC
April 2021

Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis-Top-Down Mass Spectrometry.

Anal Chem 2021 03 22;93(12):5151-5160. Epub 2021 Mar 22.

Departments of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States.

We report a novel platform [native capillary zone electrophoresis-top-down mass spectrometry (nCZE-TDMS)] for the separation and characterization of whole nucleosomes, their histone subunits, and post-translational modifications (PTMs). As the repeating unit of chromatin, mononucleosomes (Nucs) are an ∼200 kDa complex of DNA and histone proteins involved in the regulation of key cellular processes central to human health and disease. Unraveling the covalent modification landscape of histones and their defined stoichiometries within Nucs helps to explain epigenetic regulatory mechanisms. In nCZE-TDMS, online Nuc separation is followed by a three-tier tandem MS approach that measures the intact mass of Nucs, ejects and detects the constituent histones, and fragments to sequence the histone. The new platform was optimized with synthetic Nucs to significantly reduce both sample requirements and cost compared to direct infusion. Limits of detection were in the low-attomole range, with linearity of over ∼3 orders of magnitude. The nCZE-TDMS platform was applied to endogenous Nucs from two cell lines distinguished by overexpression or knockout of histone methyltransferase NSD2/MMSET, where analysis of constituent histones revealed changes in histone abundances over the course of the CZE separation. We are confident the nCZE-TDMS platform will help advance nucleosome-level research in the fields of chromatin and epigenetics.
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http://dx.doi.org/10.1021/acs.analchem.0c04975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040852PMC
March 2021

Decoding the protein composition of whole nucleosomes with Nuc-MS.

Nat Methods 2021 03 15;18(3):303-308. Epub 2021 Feb 15.

Department of Chemistry, Northwestern University, Evanston, IL, USA.

Current proteomic approaches disassemble and digest nucleosome particles, blurring readouts of the 'histone code'. To preserve nucleosome-level information, we developed Nuc-MS, which displays the landscape of histone variants and their post-translational modifications (PTMs) in a single mass spectrum. Combined with immunoprecipitation, Nuc-MS quantified nucleosome co-occupancy of histone H3.3 with variant H2A.Z (sixfold over bulk) and the co-occurrence of oncogenic H3.3K27M with euchromatic marks (for example, a >15-fold enrichment of dimethylated H3K79me2). Nuc-MS is highly concordant with chromatin immunoprecipitation-sequencing (ChIP-seq) and offers a new readout of nucleosome-level biology.
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http://dx.doi.org/10.1038/s41592-020-01052-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954958PMC
March 2021

The serine hydroxymethyltransferase-2 (SHMT2) initiates lymphoma development through epigenetic tumor suppressor silencing.

Nat Cancer 2020 22;1:653-664. Epub 2020 Jun 22.

Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Cancer cells adapt their metabolic activities to support growth and proliferation. However, increased activity of metabolic enzymes is not usually considered an initiating event in the malignant process. Here, we investigate the possible role of the enzyme serine hydroxymethyltransferase-2 (SHMT2) in lymphoma initiation. localizes to the most frequent region of copy number gains at chromosome 12q14.1 in lymphoma. Elevated expression of cooperates with in lymphoma development; loss or inhibition of impairs lymphoma cell survival. SHMT2 catalyzes the conversion of serine to glycine and produces an activated one-carbon unit that can be used to support -adenosyl methionine synthesis. SHMT2 induces changes in DNA and histone methylation patterns leading to promoter silencing of previously uncharacterized mutational genes, such as and Together, our findings reveal that amplification of in cooperation with is sufficient in the initiation of lymphomagenesis through epigenetic tumor suppressor silencing.
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http://dx.doi.org/10.1038/s43018-020-0080-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872152PMC
June 2020

Standard procedures for native CZE-MS of proteins and protein complexes up to 800 kDa.

Electrophoresis 2021 05 27;42(9-10):1050-1059. Epub 2021 Jan 27.

Department of Chemistry and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois, USA.

Native mass spectrometry (nMS) is a rapidly growing method for the characterization of large proteins and protein complexes, preserving "native" non-covalent inter- and intramolecular interactions. Direct infusion of purified analytes into a mass spectrometer represents the standard approach for conducting nMS experiments. Alternatively, CZE can be performed under native conditions, providing high separation performance while consuming trace amounts of sample material. Here, we provide standard operating procedures for acquiring high-quality data using CZE in native mode coupled online to various Orbitrap mass spectrometers via a commercial sheathless interface, covering a wide range of analytes from 30-800 kDa. Using a standard protein mix, the influence of various CZE method parameters were evaluated, such as BGE/conductive liquid composition and separation voltage. Additionally, a universal approach for the optimization of fragmentation settings in the context of protein subunit and metalloenzyme characterization is discussed in detail for model analytes. A short section is dedicated to troubleshooting of the nCZE-MS setup. This study is aimed to help normalize nCZE-MS practices to enhance the CE community and provide a resource for the production of reproducible and high-quality data.
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http://dx.doi.org/10.1002/elps.202000317DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8122066PMC
May 2021

-Constructed Ribosomes Enable Multi-site Incorporation of Noncanonical Amino Acids into Proteins.

Biochemistry 2021 01 11;60(3):161-169. Epub 2021 Jan 11.

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 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 -acetyl-phenylalanine (pAcF) and -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.
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http://dx.doi.org/10.1021/acs.biochem.0c00829DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055168PMC
January 2021

Isotopic Resolution of Protein Complexes up to 466 kDa Using Individual Ion Mass Spectrometry.

Anal Chem 2021 02 15;93(5):2723-2727. Epub 2020 Dec 15.

Departments of Chemical and Biological Engineering, Chemistry, and Molecular Biosciences, the Chemistry of Life Processes Institute, and the Proteomics Center of Excellence, Northwestern University, Evanston, Illinois 60208, United States.

Native mass spectrometry involves transferring large biomolecular complexes into the gas phase, enabling the characterization of their composition and stoichiometry. However, the overlap in distributions created by residual solvation, ionic adducts, and post-translational modifications creates a high degree of complexity that typically goes unresolved at masses above ∼150 kDa. Therefore, native mass spectrometry would greatly benefit from higher resolution approaches for intact proteins and their complexes. By recording mass spectra of individual ions via charge detection mass spectrometry, we report isotopic resolution for pyruvate kinase (232 kDa) and β-galactosidase (466 kDa), extending the limits of isotopic resolution for high mass and high / by >2.5-fold and >1.6-fold, respectively.
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http://dx.doi.org/10.1021/acs.analchem.0c03282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878367PMC
February 2021

Histone H1 loss drives lymphoma by disrupting 3D chromatin architecture.

Nature 2021 01 9;589(7841):299-305. Epub 2020 Dec 9.

Division of Hematology and Medical Oncology, Sanford I. Weill Department of Medicine, Weill Cornell Medicine, New York, NY, USA.

Linker histone H1 proteins bind to nucleosomes and facilitate chromatin compaction, although their biological functions are poorly understood. Mutations in the genes that encode H1 isoforms B-E (H1B, H1C, H1D and H1E; also known as H1-5, H1-2, H1-3 and H1-4, respectively) are highly recurrent in B cell lymphomas, but the pathogenic relevance of these mutations to cancer and the mechanisms that are involved are unknown. Here we show that lymphoma-associated H1 alleles are genetic driver mutations in lymphomas. Disruption of H1 function results in a profound architectural remodelling of the genome, which is characterized by large-scale yet focal shifts of chromatin from a compacted to a relaxed state. This decompaction drives distinct changes in epigenetic states, primarily owing to a gain of histone H3 dimethylation at lysine 36 (H3K36me2) and/or loss of repressive H3 trimethylation at lysine 27 (H3K27me3). These changes unlock the expression of stem cell genes that are normally silenced during early development. In mice, loss of H1c and H1e (also known as H1f2 and H1f4, respectively) conferred germinal centre B cells with enhanced fitness and self-renewal properties, ultimately leading to aggressive lymphomas with an increased repopulating potential. Collectively, our data indicate that H1 proteins are normally required to sequester early developmental genes into architecturally inaccessible genomic compartments. We also establish H1 as a bona fide tumour suppressor and show that mutations in H1 drive malignant transformation primarily through three-dimensional genome reorganization, which leads to epigenetic reprogramming and derepression of developmentally silenced genes.
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http://dx.doi.org/10.1038/s41586-020-3017-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855728PMC
January 2021

Histone tail analysis reveals H3K36me2 and H4K16ac as epigenetic signatures of diffuse intrinsic pontine glioma.

J Exp Clin Cancer Res 2020 Nov 25;39(1):261. Epub 2020 Nov 25.

Department of Neurological Surgery, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

Background: Diffuse intrinsic pontine glioma (DIPG) is an aggressive pediatric brainstem tumor. Most DIPGs harbor a histone H3 mutation, which alters histone post-translational modification (PTM) states and transcription. Here, we employed quantitative proteomic analysis to elucidate the impact of the H3.3K27M mutation, as well as radiation and bromodomain inhibition (BRDi) with JQ1, on DIPG PTM profiles.

Methods: We performed targeted mass spectrometry on H3.3K27M mutant and wild-type tissues (n = 12) and cell lines (n = 7).

Results: We found 29.2 and 26.4% of total H3.3K27 peptides were H3.3K27M in mutant DIPG tumor cell lines and tissue specimens, respectively. Significant differences in modification states were observed in H3.3K27M specimens, including at H3K27, H3K36, and H4K16. In addition, H3.3K27me1 and H4K16ac were the most significantly distinct modifications in H3.3K27M mutant tumors, relative to wild-type. Further, H3.3K36me2 was the most abundant co-occurring modification on the H3.3K27M mutant peptide in DIPG tissue, while H4K16ac was the most acetylated residue. Radiation treatment caused changes in PTM abundance in vitro, including increased H3K9me3. JQ1 treatment resulted in increased mono- and di-methylation of H3.1K27, H3.3K27, H3.3K36 and H4K20 in vitro.

Conclusion: Taken together, our findings provide insight into the effects of the H3K27M mutation on histone modification states and response to treatment, and suggest that H3K36me2 and H4K16ac may represent unique tumor epigenetic signatures for targeted DIPG therapy.
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http://dx.doi.org/10.1186/s13046-020-01773-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687710PMC
November 2020

Streptomyces buecherae sp. nov., an actinomycete isolated from multiple bat species.

Antonie Van Leeuwenhoek 2020 Dec 16;113(12):2213-2221. Epub 2020 Nov 16.

Department of Biology, University of New Mexico, Albuquerque, NM, 87131, USA.

A putative novel clade within the genus Streptomyces was discovered following antifungal screening against Pseudogymnoascus destructans, the causative agent of white-nose syndrome, and described using multi-locus sequencing analysis. Swabs from both the cave myotis bat (Myotis velifer) and the Brazilian free-tailed bat (Tadarida brasiliensis) in southern New Mexico bore isolates AC536, AC541 and AC563, which were characterised using phylogenetic, morphological, and phenotypic analyses. Multi-locus sequence analysis positions AC541 with neighbors Streptomyces rubidus (NRRL B-24619), Streptomyces guanduensis (NRRL B-24617), and Streptomyces yeochonensis (NRRL B-24245). A complete genome of the type strain was assembled to determine its taxonomy and secondary metabolite potential. ANI comparisons between all closely related types strains are shown to be well below the 95-96% species delineation. DNA-DNA relatedness between AC541 and its nearest neighbors ranged between 23.7 and 24.1% confirming novelty. Approximately 1.49 Mb or 17.76% of the whole genome is devoted to natural product biosynthesis. The DNA G + C content of the genomic DNA of the type strain is 73.13 mol %. Micromorphology depicts ovoid spores with smooth surfaces in flexuous chains. Strains presented an ivory to yellow hue on most ISP media except inorganic salts-starch agar (ISP4) and can grow on D-glucose, mannitol, and D-fructose, but exhibited little to no growth on L-arabinose, sucrose, D-xylose, inositol, L-rhamnose, D-raffinose, and cellulose. This clade possesses the capability to grow from 10 to 45 °C and 12.5% (w/v) NaCl. There was strain growth variation in pH, but all isolates thrive at alkaline levels. Based on our polyphasic study of AC541, the strain warrants the assignment to a novel species, for which the name Streptomyces buecherae sp. nov. is proposed. The type strain is AC541 (= JCM 34263, = ATCC TSD201).
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http://dx.doi.org/10.1007/s10482-020-01493-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830772PMC
December 2020

Genome Mining and Metabolomics Uncover a Rare d-Capreomycidine Containing Natural Product and Its Biosynthetic Gene Cluster.

ACS Chem Biol 2020 11 5;15(11):3013-3020. Epub 2020 Nov 5.

Department of Chemistry, Northwestern University, Evanston, Illinois 60208, United States.

We report the metabolomics-driven genome mining of a new cyclic-guanidino incorporating non-ribosomal peptide synthetase (NRPS) gene cluster and full structure elucidation of its associated hexapeptide product, faulknamycin. Structural studies unveiled that this natural product contained the previously unknown (,)-stereoisomer of capreomycidine, d-capreomycidine. Furthermore, heterologous expression of the identified gene cluster successfully reproduces faulknamycin production without an observed homologue of VioD, the pyridoxal phosphate (PLP)-dependent enzyme found in all previous l-capreomycidine biosynthesis. An alternative NRPS-dependent pathway for d-capreomycidine biosynthesis is proposed.
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http://dx.doi.org/10.1021/acschembio.0c00663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7830813PMC
November 2020

Targeted detection and quantitation of histone modifications from 1,000 cells.

PLoS One 2020 26;15(10):e0240829. Epub 2020 Oct 26.

Departments of Chemistry, Molecular Biosciences, and the Proteomics Center of Excellence, Northwestern University, Evanston, IL, United States of America.

Histone post-translational modifications (PTMs) create a powerful regulatory mechanism for maintaining chromosomal integrity in cells. Histone acetylation and methylation, the most widely studied histone PTMs, act in concert with chromatin-associated proteins to control access to genetic information during transcription. Alterations in cellular histone PTMs have been linked to disease states and have crucial biomarker and therapeutic potential. Traditional bottom-up mass spectrometry of histones requires large numbers of cells, typically one million or more. However, for some cell subtype-specific studies, it is difficult or impossible to obtain such large numbers of cells and quantification of rare histone PTMs is often unachievable. An established targeted LC-MS/MS method was used to quantify the abundance of histone PTMs from cell lines and primary human specimens. Sample preparation was modified by omitting nuclear isolation and reducing the rounds of histone derivatization to improve detection of histone peptides down to 1,000 cells. In the current study, we developed and validated a quantitative LC-MS/MS approach tailored for a targeted histone assay of 75 histone peptides with as few as 10,000 cells. Furthermore, we were able to detect and quantify 61 histone peptides from just 1,000 primary human stem cells. Detection of 37 histone peptides was possible from 1,000 acute myeloid leukemia patient cells. We anticipate that this revised method can be used in many applications where achieving large cell numbers is challenging, including rare human cell populations.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0240829PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588077PMC
December 2020

Development of novel methods for non-canonical myeloma protein analysis with an innovative adaptation of immunofixation electrophoresis, native top-down mass spectrometry, and middle-down de novo sequencing.

Clin Chem Lab Med 2020 Oct 20. Epub 2020 Oct 20.

Proteomics Center of Excellence & Departments of Chemistry and Molecular Biology,Northwestern University, Evanston, IL, USA.

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.
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http://dx.doi.org/10.1515/cclm-2020-1072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8055720PMC
October 2020

Mechanism-Based Design of 3-Amino-4-Halocyclopentenecarboxylic Acids as Inactivators of GABA Aminotransferase.

ACS Med Chem Lett 2020 Oct 18;11(10):1949-1955. Epub 2020 Feb 18.

Department of Chemistry, Chemistry of Life Processes Institute, Center for Molecular Innovation and Drug Discovery, and Center for Developmental Therapeutics and Department of Molecular Biosciences, Northwestern University, Evanston, Illinois 60208, United States.

Aminotransferases are pyridoxal 5'-phosphate-dependent enzymes that catalyze reversible transamination reactions between an amino acid and an α-keto acid, playing a critical role in cellular nitrogen metabolism. It is evident that γ-aminobutyric acid aminotransferase (GABA-AT), which balances the levels of inhibitory and excitatory neurotransmitters, has emerged as a promising therapeutic target for epilepsy and cocaine addiction based on mechanism-based inactivators (MBIs). In this work, we established an integrated approach using computational simulation, organic synthesis, biochemical evaluation, and mass spectrometry to facilitate our design and mechanistic studies of MBIs, which led to the identification of a new cyclopentene-based analogue (), 25-times more efficient as an inactivator of GABA-AT compared to the parent compound (1,3,4)-3-amino-4-fluorocyclopentane carboxylic acid (, ).
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http://dx.doi.org/10.1021/acsmedchemlett.9b00672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7549257PMC
October 2020

In the fungus where it happens: History and future propelling Aspergillus nidulans as the archetype of natural products research.

Fungal Genet Biol 2020 11 6;144:103477. Epub 2020 Oct 6.

Department of Medical Microbiology and Immunology, University of Wisconsin- Madison, Madison, WI, United States; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, United States. Electronic address:

In 1990 the first fungal secondary metabolite biosynthetic gene was cloned in Aspergillus nidulans. Thirty years later, >30 biosynthetic gene clusters (BGCs) have been linked to specific natural products in this one fungal species. While impressive, over half of the BGCs in A. nidulans remain uncharacterized and their compounds structurally and functionally unknown. Here, we provide a comprehensive review of past advances that have enabled A. nidulans to rise to its current status as a natural product powerhouse focusing on the discovery and annotation of secondary metabolite clusters. From genome sequencing, heterologous expression, and metabolomics to CRISPR and epigenetic manipulations, we present a guided tour through the evolution of technologies developed and utilized in the last 30 years. These insights provide perspective to future efforts to fully unlock the biosynthetic potential of A. nidulans and, by extension, the potential of other filamentous fungi.
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http://dx.doi.org/10.1016/j.fgb.2020.103477DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726033PMC
November 2020
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