Defining mitochondrial protein functions through deep multiomic profiling.

Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.

Argonaut: A Web Platform for Collaborative Multi-omic Data Visualization and Exploration.

Researchers now generate large multi-omic datasets using increasingly mature mass spectrometry techniques at an astounding pace, facing new challenges of "Big Data" dissemination, visualization, and exploration. Conveniently, web-based data portals accommodate the complexity of multi-omic experiments and the many experts involved. However, developing these tailored companion resources requires programming expertise and knowledge of web server architecture-a substantial burden for most. Here, we describe Argonaut, a simple, code-free, and user-friendly platform for creating customizable, interactive data-hosting websites. Argonaut carries out real-time statistical analyses of the data, which it organizes into easily sharable projects. Collaborating researchers worldwide can explore the results, visualized through popular plots, and modify them to streamline data interpretation. Increasing the pace and ease of access to multi-omic data, Argonaut aims to propel discovery of new biological insights. We showcase the capabilities of this tool using a published multi-omics dataset on the large mitochondrial protease deletion collection.

Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy.

Macroautophagy/autophagy is suppressed by MTOR (mechanistic target of rapamycin kinase) and is an anticancer target under active investigation. Yet, MTOR-regulated autophagy remains incompletely mapped. We used proteomic profiling to identify proteins in the MTOR-autophagy axis. Wild-type (WT) mouse cell lines and cell lines lacking individual autophagy genes (Atg5 or Ulk1/Ulk2) were treated with an MTOR inhibitor to induce autophagy and cultured in media with either glucose or galactose. Mass spectrometry proteome profiling revealed an elevation of known autophagy proteins and candidates for new autophagy components, including CALCOCO1 (calcium binding and coiled-coil domain protein 1). We show that CALCOCO1 physically interacts with MAP1LC3C, a key protein in the machinery of autophagy. Genetic deletion of CALCOCO1 disrupted autophagy of the endoplasmic reticulum (reticulophagy). Together, these results reveal a role for CALCOCO1 in MTOR-regulated selective autophagy. More generally, the resource generated by this work provides a foundation for establishing links between the MTOR-autophagy axis and proteins not previously linked to this pathway. Abbreviations: ATG: autophagy-related; CALCOCO1: calcium binding and coiled-coil domain protein 1; CALCOCO2/NDP52: calcium binding and coiled-coil domain protein 2; CLIR: MAP1LC3C-interacting region; CQ: chloroquine; KO: knockout; LIR: MAP1LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MEF: mouse embryonic fibroblast; MLN: MLN0128 ATP-competitive MTOR kinase inhibitor; MTOR: mechanistic target of rapamycin kinase; reticulophagy: selective autophagy of the endoplasmic reticulum; TAX1BP1/CALCOCO3: TAX1 binding protein 1; ULK: unc 51-like autophagy activating kinase; WT: wild-type.

Interactive Peptide Spectral Annotator: A Versatile Web-based Tool for Proteomic Applications.

Here we present IPSA, an innovative web-based spectrum annotator that visualizes and characterizes peptide tandem mass spectra. A tool for the scientific community, IPSA can visualize peptides collected using a wide variety of experimental and instrumental configurations. Annotated spectra are customizable via a selection of interactive features and can be exported as editable scalable vector graphics to aid in the production of publication-quality figures. Single spectra can be analyzed through provided web forms, whereas data for multiple peptide spectral matches can be uploaded using the Proteomics Standards Initiative file formats mzTab, mzIdentML, and mzML. Alternatively, peptide identifications and spectral data can be provided using generic file formats. IPSA provides supports for annotating spectra collecting using negative-mode ionization and facilitates the characterization of experimental MS/MS performance through the optional export of fragment ion statistics from one to many peptide spectral matches. This resource is made freely accessible at http://interactivepeptidespectralannotator.com, whereas the source code and user guides are available at https://github.com/coongroup/IPSA for private hosting or custom implementations.

Islet proteomics reveals genetic variation in dopamine production resulting in altered insulin secretion.

The mouse is a critical model in diabetes research, but most research in mice has been limited to a small number of mouse strains and limited genetic variation. Using the eight founder strains and both sexes of the Collaborative Cross (C57BL/6J (B6), A/J, 129S1/SvImJ (129), NOD/ShiLtJ (NOD), NZO/HILtJ (NZO), PWK/PhJ (PWK), WSB/EiJ (WSB), and CAST/EiJ (CAST)), we investigated the genetic dependence of diabetes-related metabolic phenotypes and insulin secretion. We found that strain background is associated with an extraordinary range in body weight, plasma glucose, insulin, triglycerides, and insulin secretion. Our whole-islet proteomic analysis of the eight mouse strains demonstrates that genetic background exerts a strong influence on the islet proteome that can be linked to the differences in diabetes-related metabolic phenotypes and insulin secretion. We computed protein modules consisting of highly correlated proteins that enrich for biological pathways and provide a searchable database of the islet protein expression profiles. To validate the data resource, we identified tyrosine hydroxylase (Th), a key enzyme in catecholamine synthesis, as a protein that is highly expressed in ß-cells of PWK and CAST islets. We show that CAST islets synthesize elevated levels of dopamine, which suppresses insulin secretion. Prior studies, using only the B6 strain, concluded that adult mouse islets do not synthesize l-3,4-dihydroxyphenylalanine (l-DOPA), the product of Th and precursor of dopamine. Thus, the choice of the CAST strain, guided by our islet proteomic survey, was crucial for these discoveries. In summary, we provide a valuable data resource to the research community, and show that proteomic analysis identified a strain-specific pathway by which dopamine synthesized in ß-cells inhibits insulin secretion.

Improved Precursor Characterization for Data-Dependent Mass Spectrometry.

Modern ion trap mass spectrometers are capable of collecting up to 60 tandem MS (MS/MS) scans per second, in theory providing acquisition speeds that can sample every eluting peptide precursor presented to the MS system. In practice, however, the precursor sampling capacity enabled by these ultrafast acquisition rates is often underutilized due to a host of reasons (e.g., long injection times and wide analyzer mass ranges). One often overlooked reason for this underutilization is that the instrument exhausts all the peptide features it identifies as suitable for MS/MS fragmentation. Highly abundant features can prevent annotation of lower abundance precursor ions that occupy similar mass-to-charge (m/z) space, which ultimately inhibits the acquisition of an MS/MS event. Here, we present an advanced peak determination (APD) algorithm that uses an iterative approach to annotate densely populated m/z regions to increase the number of peptides sampled during data-dependent LC-MS/MS analyses. The APD algorithm enables nearly full utilization of the sampling capacity of a quadrupole-Orbitrap-linear ion trap MS system, which yields up to a 40% increase in unique peptide identifications from whole cell HeLa lysates (approximately 53 000 in a 90 min LC-MS/MS analysis). The APD algorithm maintains improved peptide and protein identifications across several modes of proteomic data acquisition, including varying gradient lengths, different degrees of prefractionation, peptides derived from multiple proteases, and phosphoproteomic analyses. Additionally, the use of APD increases the number of peptides characterized per protein, providing improved protein quantification. In all, the APD algorithm increases the number of detectable peptide features, which maximizes utilization of the high MS/MS capacities and significantly improves sampling depth and identifications in proteomic experiments.

Multi-omics Reveal Specific Targets of the RNA-Binding Protein Puf3p and Its Orchestration of Mitochondrial Biogenesis.

Coenzyme Q (CoQ) is a redox-active lipid required for mitochondrial oxidative phosphorylation (OxPhos). How CoQ biosynthesis is coordinated with the biogenesis of OxPhos protein complexes is unclear. Here, we show that the Saccharomyces cerevisiae RNA-binding protein (RBP) Puf3p regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a multi-omic strategy to identify mRNAs that not only bind Puf3p but also are regulated by Puf3p in vivo. The CoQ biosynthesis enzyme Coq5p is a critical Puf3p target: Puf3p regulates the abundance of Coq5p and prevents its detrimental hyperaccumulation, thereby enabling efficient CoQ production. More broadly, Puf3p represses a specific set of proteins involved in mitochondrial protein import, translation, and OxPhos complex assembly (pathways essential to prime mitochondrial biogenesis). Our data reveal a mechanism for post-transcriptionally coordinating CoQ production with OxPhos biogenesis, and they demonstrate the power of multi-omics for defining genuine targets of RBPs.

Multi-omic Mitoprotease Profiling Defines a Role for Oct1p in Coenzyme Q Production.

Mitoproteases are becoming recognized as key regulators of diverse mitochondrial functions, although their direct substrates are often difficult to discern. Through multi-omic profiling of diverse Saccharomyces cerevisiae mitoprotease deletion strains, we predicted numerous associations between mitoproteases and distinct mitochondrial processes. These include a strong association between the mitochondrial matrix octapeptidase Oct1p and coenzyme Q (CoQ) biosynthesis-a pathway essential for mitochondrial respiration. Through Edman sequencing and in vitro and in vivo biochemistry, we demonstrated that Oct1p directly processes the N terminus of the CoQ-related methyltransferase, Coq5p, which markedly improves its stability. A single mutation to the Oct1p recognition motif in Coq5p disrupted its processing in vivo, leading to CoQ deficiency and respiratory incompetence. This work defines the Oct1p processing of Coq5p as an essential post-translational event for proper CoQ production. Additionally, our data visualization tool enables efficient exploration of mitoprotease profiles that can serve as the basis for future mechanistic investigations.

Gender Diversity in a STEM Subfield - Analysis of a Large Scientific Society and Its Annual Conferences.

Speaking engagements, serving as session chairs, and receiving awards at national meetings are essential stepping stones towards professional success for scientific researchers. Studies of gender parity in meetings of national scientific societies repeatedly uncover bias in speaker selection, engendering underrepresentation of women among featured presenters. To continue this dialogue, we analyzed membership data and annual conference programs of a large scientific society (>7000 members annually) in a male-rich (~70% males), technology-oriented STEM subfield. We detected a pronounced skew towards males among invited keynote lecturers, plenary speakers, and recipients of the society's Senior Investigator award (15%, 13%, and 8% females, respectively). However, the proportion of females among Mid-Career and Young Investigator award recipients and oral session chairs resembled the current gender distribution of the general membership. Female members were more likely to present at the conferences and equally likely to apply and be accepted for oral presentations as their male counterparts. The gender of a session chair had no effect on the gender distribution of selected applicants. Interestingly, we identified several research subareas that were naturally enriched (i.e., not influenced by unequal selection of presenters) for either female or male participants, illustrating within a single subfield the gender divide along biology-technology line typical of all STEM disciplines. Two female-enriched topics experienced a rapid growth in popularity within the examined period, more than doubling the number of associated researchers. Collectively, these findings contribute to the contemporary discourse on gender in science and hopefully will propel positive changes within this and other societies. Graphical abstract ᅟ.

Global mapping of CARM1 substrates defines enzyme specificity and substrate recognition.

Protein arginine methyltransferases (PRMTs) introduce arginine methylation, a post-translational modification with the increasingly eminent role in normal physiology and disease. PRMT4 or coactivator-associated arginine methyltransferase 1 (CARM1) is a propitious target for cancer therapy; however, few CARM1 substrates are known, and its mechanism of substrate recognition is poorly understood. Here we employed a quantitative mass spectrometry approach to globally profile CARM1 substrates in breast cancer cell lines. We identified >130 CARM1 protein substrates and validated in vitro >90% of sites they encompass. Bioinformatics analyses reveal enrichment of proline-containing motifs, in which both methylation sites and their proximal sequences are frequently targeted by somatic mutations in cancer. Finally, we demonstrate that the N-terminus of CARM1 is involved in substrate recognition and nearly indispensable for substrate methylation. We propose that development of CARM1-specific inhibitors should focus on its N-terminus and predict that other PRMTs may employ similar mechanism for substrate recognition.

Mitochondrial protein functions elucidated by multi-omic mass spectrometry profiling.

Mitochondrial dysfunction is associated with many human diseases, including cancer and neurodegeneration, that are often linked to proteins and pathways that are not well-characterized. To begin defining the functions of such poorly characterized proteins, we used mass spectrometry to map the proteomes, lipidomes, and metabolomes of 174 yeast strains, each lacking a single gene related to mitochondrial biology. 144 of these genes have human homologs, 60 of which are associated with disease and 39 of which are uncharacterized. We present a multi-omic data analysis and visualization tool that we use to find covariance networks that can predict molecular functions, correlations between profiles of related gene deletions, gene-specific perturbations that reflect protein functions, and a global respiration deficiency response. Using this multi-omic approach, we link seven proteins including Hfd1p and its human homolog ALDH3A1 to mitochondrial coenzyme Q (CoQ) biosynthesis, an essential pathway disrupted in many human diseases. This Resource should provide molecular insights into mitochondrial protein functions.

A proteomic atlas of the legume Medicago truncatula and its nitrogen-fixing endosymbiont Sinorhizobium meliloti.

Legumes are essential components of agricultural systems because they enrich the soil in nitrogen and require little environmentally deleterious fertilizers. A complex symbiotic association between legumes and nitrogen-fixing soil bacteria called rhizobia culminates in the development of root nodules, where rhizobia fix atmospheric nitrogen and transfer it to their plant host. Here we describe a quantitative proteomic atlas of the model legume Medicago truncatula and its rhizobial symbiont Sinorhizobium meliloti, which includes more than 23,000 proteins, 20,000 phosphorylation sites, and 700 lysine acetylation sites. Our analysis provides insight into mechanisms regulating symbiosis. We identify a calmodulin-binding protein as a key regulator in the host and assign putative roles and targets to host factors (bioactive peptides) that control gene expression in the symbiont. Further mining of this proteomic resource may enable engineering of crops and their microbial partners to increase agricultural productivity and sustainability.

Cerebellar Ataxia and Coenzyme Q Deficiency through Loss of Unorthodox Kinase Activity.

The UbiB protein kinase-like (PKL) family is widespread, comprising one-quarter of microbial PKLs and five human homologs, yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. Although COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates, functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.

NeuCode Labeling in Nematodes: Proteomic and Phosphoproteomic Impact of Ascaroside Treatment in Caenorhabditis elegans.

The nematode Caenorhabditis elegans is an important model organism for biomedical research. We previously described NeuCode stable isotope labeling by amino acids in cell culture (SILAC), a method for accurate proteome quantification with potential for multiplexing beyond the limits of traditional stable isotope labeling by amino acids in cell culture. Here we apply NeuCode SILAC to profile the proteomic and phosphoproteomic response of C. elegans to two potent members of the ascaroside family of nematode pheromones. By consuming labeled E. coli as part of their diet, C. elegans nematodes quickly and easily incorporate the NeuCode heavy lysine isotopologues by the young adult stage. Using this approach, we report, at high confidence, one of the largest proteomic and phosphoproteomic data sets to date in C. elegans: 6596 proteins at a false discovery rate ≤ 1% and 6620 phosphorylation isoforms with localization probability ≥75%. Our data reveal a post-translational signature of pheromone sensing that includes many conserved proteins implicated in longevity and response to stress.

A role for the mevalonate pathway in early plant symbiotic signaling.

Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system.

High-resolution filtering for improved small molecule identification via GC/MS.

Gas chromatography/mass spectrometry (GC/MS) has long been considered one of the premiere analytical tools for small molecule analysis. Recently, a number of GC/MS systems equipped with high-resolution mass analyzers have been introduced. These systems provide analysts with a new dimension of information, accurate mass measurement to the third or fourth decimal place; however, existing data processing tools do not capitalize on this information. Beyond that, GC/MS spectral reference libraries, which have been curated over the last several decades, contain almost exclusively unit resolution MS spectra making integration of accurate mass data dubious. Here we present an informatic approach, called high-resolution filtering (HRF), which bridges this gap. During HRF, high-resolution mass spectra are assigned putative identifications through traditional spectral matching at unit resolution. Once candidate identities have been assigned, all unique combinations of atoms from these candidate precursors are generated and matched to m/z peaks using narrow mass tolerances. The total amount of measured signal that is annotated is used as a metric of plausibility for the presumed identification. Here we demonstrate that the HRF approach is both feasible and highly specific toward correct identifications.

The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD).

The field of proteomics almost uniformly relies on peptide cation analysis, leading to an underrepresentation of acidic portions of proteomes, including relevant acidic posttranslational modifications. Despite the many benefits negative mode proteomics can offer, peptide anion analysis remains in its infancy due mainly to challenges with high-pH reversed-phase separations and a lack of robust fragmentation methods suitable for peptide anion characterization. Here, we report the first implementation of activated ion negative electron transfer dissociation (AI-NETD) on the chromatographic timescale, generating 7,601 unique peptide identifications from Saccharomyces cerevisiae in single-shot nLC-MS/MS analyses of tryptic peptides-a greater than 5-fold increase over previous results with NETD alone. These improvements translate to identification of 1,106 proteins, making this work the first negative mode study to identify more than 1,000 proteins in any system. We then compare the performance of AI-NETD for analysis of peptides generated by five proteases (trypsin, LysC, GluC, chymotrypsin, and AspN) for negative mode analyses, identifying as many as 5,356 peptides (1,045 proteins) with LysC and 4,213 peptides (857 proteins) with GluC in yeast-characterizing 1,359 proteins in total. Finally, we present the first deep-sequencing approach for negative mode proteomics, leveraging offline low-pH reversed-phase fractionation prior to online high-pH separations and peptide fragmentation with AI-NETD. With this platform, we identified 3,467 proteins in yeast with trypsin alone and characterized a total of 3,730 proteins using multiple proteases, or nearly 83% of the expressed yeast proteome. This work represents the most extensive negative mode proteomics study to date, establishing AI-NETD as a robust tool for large-scale peptide anion characterization and making the negative mode approach a more viable platform for future proteomic studies.

A calibration routine for efficient ETD in large-scale proteomics.

Electron transfer dissociation (ETD) has been broadly adopted and is now available on a variety of commercial mass spectrometers. Unlike collisional activation techniques, optimal performance of ETD requires considerable user knowledge and input. ETD reaction duration is one key parameter that can greatly influence spectral quality and overall experiment outcome. We describe a calibration routine that determines the correct number of reagent anions necessary to reach a defined ETD reaction rate. Implementation of this automated calibration routine on two hybrid Orbitrap platforms illustrate considerable advantages, namely, increased product ion yield with concomitant reduction in scan rates netting up to 75% more unique peptide identifications in a shotgun experiment. Graphical Abstract ᅟ.

Structural Characterization of Methylenedianiline Regioisomers by Ion Mobility-Mass Spectrometry, Tandem Mass Spectrometry, and Computational Strategies. 2. Electrospray Spectra of 3-Ring and 4-Ring Isomers.

Building on results from our previous study of 2-ring methylenedianiline (MDA), a combined mass spectrometry approach utilizing ion mobility-mass spectrometry (IM-MS) and tandem mass spectrometry (MS/MS) coupled with computational methods enables the structural characterization of purified 3-ring and 4-ring MDA regioisomers in this current study. The preferred site of protonation for the 3-ring and 4-ring MDA was determined to be on the amino groups. Additionally, the location of the protonated amine along the MDA multimer was found to influence the gas phase stability of these molecules. Fragmentation mechanisms similar to the 2-ring MDA species were observed for both the 3-ring and 4-ring MDA. The structural characterization of 3-ring and 4-ring MDA isomers using modern MS techniques may aid polyurethane synthesis by the characterization of industrial grade MDA, multimeric MDA species, and methylene diphenyl diisocyanate (MDI) mixtures.

Structural characterization of methylenedianiline regioisomers by ion mobility-mass spectrometry, tandem mass spectrometry, and computational strategies: I. Electrospray spectra of 2-ring isomers.

Purified methylenedianiline (MDA) regioisomers were structurally characterized and differentiated using tandem mass spectrometry (MS/MS), ion mobility-mass spectrometry (IM-MS), and IM-MS/MS in conjunction with computational methods. It was determined that protonation sites on the isomers can vary depending on the position of amino groups, and the resulting protonation sites play a role in the gas-phase stability of the isomer. We also observed differences in the relative distributions of protonated conformations depending on experimental conditions and instrumentation, which is consistent with previous studies on aniline in the gas phase. This work demonstrates the utility of a multifaceted approach for the study of isobaric species and elucidates why previous MDA studies may have been unable to detect and/or differentiate certain isomers. Such analysis may prove useful in the characterization of larger MDA multimeric species, industrial MDA mixtures, and methylene diphenyl diisocyanate (MDI) mixtures used in polyurethane synthesis.