Middle-Down Mass Spectrometry Reveals Activity-Modifying Phosphorylation Barcode in a Class C G Protein-Coupled Receptor.

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.

Proteoform-Selective Imaging of Tissues Using Mass Spectrometry.

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.

A mixed-valent Fe(II)Fe(III) species converts cysteine to an oxazolone/thioamide pair in methanobactin biosynthesis.

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.

ProSight Annotator: Complete control and customization of protein entries in UniProt XML files.

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.

Proteomics Standards Initiative's ProForma 2.0: Unifying the Encoding of Proteoforms and Peptidoforms.

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.

The Human Proteoform Atlas: a FAIR community resource for experimentally derived proteoforms.

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.

The Blood Proteoform Atlas: A reference map of proteoforms in human hematopoietic cells.

Human biology is tightly linked to proteins, yet most measurements do not precisely determine alternatively spliced sequences or posttranslational modifications. Here, we present the primary structures of ~30,000 unique proteoforms, nearly 10 times more than in previous studies, expressed from 1690 human genes across 21 cell types and plasma from human blood and bone marrow. The results, compiled in the Blood Proteoform Atlas (BPA), indicate that proteoforms better describe protein-level biology and are more specific indicators of differentiation than their corresponding proteins, which are more broadly expressed across cell types. We demonstrate the potential for clinical application, by interrogating the BPA in the context of liver transplantation and identifying cell and proteoform signatures that distinguish normal graft function from acute rejection and other causes of graft dysfunction.

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

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.

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

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.

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

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.

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

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.

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

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.

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.

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.

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

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.

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

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.

Chemical composition of material extractives influences microbial growth and dynamics on wetted wood materials.

The impact of material chemical composition on microbial growth on building materials remains relatively poorly understood. We investigate the influence of the chemical composition of material extractives on microbial growth and community dynamics on 30 different wood species that were naturally inoculated, wetted, and held at high humidity for several weeks. Microbial growth was assessed by visual assessment and molecular sequencing. Unwetted material powders and microbial swab samples were analyzed using reverse phase liquid chromatography with tandem mass spectrometry. Different wood species demonstrated varying susceptibility to microbial growth after 3 weeks and visible coverage and fungal qPCR concentrations were correlated (R2 = 0.55). Aspergillaceae was most abundant across all samples; Meruliaceae was more prevalent on 8 materials with the highest visible microbial growth. A larger and more diverse set of compounds was detected from the wood shavings compared to the microbial swabs, indicating a complex and heterogeneous chemical composition within wood types. Several individual compounds putatively identified in wood samples showed statistically significant, near-monotonic associations with microbial growth, including C11H16O4, C18H34O4, and C6H15NO. A pilot experiment confirmed the inhibitory effects of dosing a sample of wood materials with varying concentrations of liquid C6H15NO (assuming it presented as Diethylethanolamine).

Heterologous Expression of the Unusual Terreazepine Biosynthetic Gene Cluster Reveals a Promising Approach for Identifying New Chemical Scaffolds.

Advances in genome sequencing have revitalized natural product discovery efforts, revealing the untapped biosynthetic potential of fungi. While the volume of genomic data continues to expand, discovery efforts are slowed due to the time-consuming nature of experiments required to characterize new molecules. To direct efforts toward uncharacterized biosynthetic gene clusters most likely to encode novel chemical scaffolds, we took advantage of comparative metabolomics and heterologous gene expression using fungal artificial chromosomes (FACs). By linking mass spectral profiles with structural clues provided by FAC-encoded gene clusters, we targeted a compound originating from an unusual gene cluster containing an indoleamine 2,3-dioxygenase (IDO). With this approach, we isolate and characterize R and S forms of the new molecule terreazepine, which contains a novel chemical scaffold resulting from cyclization of the IDO-supplied kynurenine. The discovery of terreazepine illustrates that FAC-based approaches targeting unusual biosynthetic machinery provide a promising avenue forward for targeted discovery of novel scaffolds and their biosynthetic enzymes, and it also represents another example of a biosynthetic gene cluster "repurposing" a primary metabolic enzyme to diversify its secondary metabolite arsenal.IMPORTANCE Here, we provide evidence that Aspergillus terreus encodes a biosynthetic gene cluster containing a repurposed indoleamine 2,3-dioxygenase (IDO) dedicated to secondary metabolite synthesis. The discovery of this neofunctionalized IDO not only enabled discovery of a new compound with an unusual chemical scaffold but also provided insight into the numerous strategies fungi employ for diversifying and protecting themselves against secondary metabolites. The observations in this study set the stage for further in-depth studies into the function of duplicated IDOs present in fungal biosynthetic gene clusters and presents a strategy for accessing the biosynthetic potential of gene clusters containing duplicated primary metabolic genes.

NMT1 and NMT2 are lysine myristoyltransferases regulating the ARF6 GTPase cycle.

Lysine fatty acylation in mammalian cells was discovered nearly three decades ago, yet the enzymes catalyzing it remain unknown. Unexpectedly, we find that human N-terminal glycine myristoyltransferases (NMT) 1 and 2 can efficiently myristoylate specific lysine residues. They modify ADP-ribosylation factor 6 (ARF6) on lysine 3 allowing it to remain on membranes during the GTPase cycle. We demonstrate that the NAD+-dependent deacylase SIRT2 removes the myristoyl group, and our evidence suggests that NMT prefers the GTP-bound while SIRT2 prefers the GDP-bound ARF6. This allows the lysine myrisotylation-demyristoylation cycle to couple to and promote the GTPase cycle of ARF6. Our study provides an explanation for the puzzling dissimilarity of ARF6 to other ARFs and suggests the existence of other substrates regulated by this previously unknown function of NMT. Furthermore, we identified a NMT/SIRT2-ARF6 regulatory axis, which may offer new ways to treat human diseases.

Thorough Performance Evaluation of 213 nm Ultraviolet Photodissociation for Top-down Proteomics.

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.