Fast and deep phosphoproteome analysis with the Orbitrap Astral mass spectrometer.

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method, we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology is benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We apply this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detect 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of phosphorylation events relevant to mitochondrial and brain biology.

RNA sequestration in P-bodies sustains myeloid leukaemia.

Post-transcriptional mechanisms are fundamental safeguards of progenitor cell identity and are often dysregulated in cancer. Here, we identified regulators of P-bodies as crucial vulnerabilities in acute myeloid leukaemia (AML) through genome-wide CRISPR screens in normal and malignant haematopoietic progenitors. We found that leukaemia cells harbour aberrantly elevated numbers of P-bodies and show that P-body assembly is crucial for initiation and maintenance of AML. Notably, P-body loss had little effect upon homoeostatic haematopoiesis but impacted regenerative haematopoiesis. Molecular characterization of P-bodies purified from human AML cells unveiled their critical role in sequestering messenger RNAs encoding potent tumour suppressors from the translational machinery. P-body dissolution promoted translation of these mRNAs, which in turn rewired gene expression and chromatin architecture in leukaemia cells. Collectively, our findings highlight the contrasting and unique roles of RNA sequestration in P-bodies during tissue homoeostasis and oncogenesis. These insights open potential avenues for understanding myeloid leukaemia and future therapeutic interventions.

Succinate dehydrogenase-complex II regulates skeletal muscle cellular respiration and contractility but not muscle mass in genetically induced pulmonary emphysema.

Reduced skeletal muscle mass and oxidative capacity coexist in patients with pulmonary emphysema and are independently associated with higher mortality. If reduced cellular respiration contributes to muscle atrophy in that setting remains unknown. Using a mouse with genetically induced pulmonary emphysema that recapitulates muscle dysfunction, we found that reduced activity of succinate dehydrogenase (SDH) is a hallmark of its myopathic changes. We generated an inducible, muscle-specific SDH knockout mouse that demonstrates lower mitochondrial oxygen consumption, myofiber contractility, and exercise endurance. Respirometry analyses show that in vitro complex I respiration is unaffected by loss of SDH subunit C in muscle mitochondria, which is consistent with the pulmonary emphysema animal data. SDH knockout initially causes succinate accumulation associated with a down-regulated transcriptome but modest proteome effects. Muscle mass, myofiber type composition, and overall body mass constituents remain unaltered in the transgenic mice. Thus, while SDH regulates myofiber respiration in experimental pulmonary emphysema, it does not control muscle mass or other body constituents.

Boosting the Sensitivity of Quantitative Single-Cell Proteomics with Infrared-Tandem Mass Tags.

Single-cell proteomics is a powerful approach to precisely profile protein landscapes within individual cells toward a comprehensive understanding of proteomic functions and tissue and cellular states. The inherent challenges associated with limited starting material demand heightened analytical sensitivity. Just as advances in sample preparation maximize the amount of material that makes it from the cell to the mass spectrometer, we strive to maximize the number of ions that make it from ion source to the detector. In isobaric tagging experiments, limited reporter ion generation limits quantitative accuracy and precision. The combination of infrared photoactivation and ion parking circumvents the m/z dependence inherent in HCD, maximizing reporter generation and avoiding unintended degradation of TMT reporter molecules in infrared-tandem mass tags (IR-TMT). The method was applied to single-cell human proteomes using 18-plex TMTpro, resulting in 4-5-fold increases in reporter signal compared to conventional SPS-MS3 approaches. IR-TMT enables faster duty cycles, higher throughput, and increased peptide identification and quantification. Comparative experiments showcase 4-5-fold lower injection times for IR-TMT, providing superior sensitivity without compromising accuracy. In all, IR-TMT enhances the dynamic range of proteomic experiments and is compatible with gas-phase fractionation and real-time searching, promising increased gains in the study of cellular heterogeneity.

Lysosomal storage disease proteo/lipidomic profiling using nMOST links ferritinophagy with mitochondrial iron deficiencies in cells lacking NPC2.

Lysosomal storage diseases (LSDs) comprised ~50 monogenic diseases characterized by the accumulation of cellular material in lysosomes and associated defects in lysosomal function, but systematic molecular phenotyping is lacking. Here, we develop a nanoflow-based multi-omic single-shot technology (nMOST) workflow allowing simultaneously quantify HeLa cell proteomes and lipidomes from more than two dozen LSD mutants, revealing diverse molecular phenotypes. Defects in delivery of ferritin and its autophagic receptor NCOA4 to lysosomes (ferritinophagy) were pronounced in NPC2-/- cells, which correlated with increased lyso-phosphatidylcholine species and multi-lamellar membrane structures visualized by cryo-electron-tomography. Ferritinophagy defects correlated with loss of mitochondrial cristae, MICOS-complex components, and electron transport chain complexes rich in iron-sulfur cluster proteins. Strikingly, mitochondrial defects were alleviated when iron was provided through the transferrin system. This resource reveals how defects in lysosomal function can impact mitochondrial homeostasis in trans and highlights nMOST as a discovery tool for illuminating molecular phenotypes across LSDs.

Chemical Proteomics Strategies for Analyzing Protein Lipidation Reveal the Bacterial O-Mycoloylome.

Protein lipidation dynamically controls protein localization and function within cellular membranes. A unique form of protein O-fatty acylation in Corynebacterium, termed protein O-mycoloylation, involves the attachment of mycolic acids─unusually large and hydrophobic fatty acids─to serine residues of proteins in these organisms' outer mycomembrane. However, as with other forms of protein lipidation, the scope and functional consequences of protein O-mycoloylation are challenging to investigate due to the inherent difficulties of enriching and analyzing lipidated peptides. To facilitate the analysis of protein lipidation and enable the comprehensive profiling and site mapping of protein O-mycoloylation, we developed a chemical proteomics strategy integrating metabolic labeling, click chemistry, cleavable linkers, and a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing LC separation and complementary fragmentation methods tailored to the analysis of lipophilic, MS-labile O-acylated peptides. Using these tools in the model organism Corynebacterium glutamicum, we identified approximately 30 candidate O-mycoloylated proteins, including porins, mycoloyltransferases, secreted hydrolases, and other proteins with cell envelope-related functions─consistent with a role for O-mycoloylation in targeting proteins to the mycomembrane. Site mapping revealed that many of the proteins contained multiple spatially proximal modification sites, which occurred predominantly at serine residues surrounded by conformationally flexible peptide motifs. Overall, this study (i) discloses the putative protein O-mycoloylome for the first time, (ii) yields new insights into the undercharacterized proteome of the mycomembrane, which is a hallmark of important pathogens (e.g., Corynebacterium diphtheriae, Mycobacterium tuberculosis), and (iii) provides generally applicable chemical strategies for the proteomic analysis of protein lipidation.

The One Hour Human Proteome.

We describe deep analysis of the human proteome in less than 1 h. We achieve this expedited proteome characterization by leveraging state-of-the-art sample preparation, chromatographic separations, and data analysis tools, and by using the new Orbitrap Astral mass spectrometer equipped with a quadrupole mass filter, a high-field Orbitrap mass analyzer, and an asymmetric track lossless (Astral) mass analyzer. The system offers high tandem mass spectrometry acquisition speed of 200 Hz and detects hundreds of peptide sequences per second within data-independent acquisition or data-dependent acquisition modes of operation. The fast-switching capabilities of the new quadrupole complement the sensitivity and fast ion scanning of the Astral analyzer to enable narrow-bin data-independent analysis methods. Over a 30-min active chromatographic method consuming a total analysis time of 56 min, the Q-Orbitrap-Astral hybrid MS collects an average of 4319 MS1 scans and 438,062 tandem mass spectrometry scans per run, producing 235,916 peptide sequences (1% false discovery rate). On average, each 30-min analysis achieved detection of 10,411 protein groups (1% false discovery rate). We conclude, with these results and alongside other recent reports, that the 1-h human proteome is within reach.

LipiDex 2 Integrates MSn Tree-Based Fragmentation Methods and Quality Control Modules to Improve Discovery Lipidomics.

As lipidomics experiments increase in scale and complexity, data processing tools must support workflows for new liquid chromatography-mass spectrometry (LC-MS) methods while simultaneously supporting quality controls to maximize the confidence in lipid identifications. LipiDex 2 improves lipidomics data processing algorithms from LipiDex 1 and introduces new tools for spectral matching and peak annotation functions, with improvements in speed and user-friendliness. In silico spectral library generation now supports tandem mass spectral (MSn) tree-based fragmentation methods, and the LipiDex 2 workflow fully integrates the fragmentation logic into the data processing steps to enable lipid identification at the appropriate level of structural resolution. Finally, LipiDex 2 features new modules for automated quality control checks that also allow users to visualize data quality in a data dashboard user interface.

An alphacoronavirus polymerase structure reveals conserved replication factor functions.

Coronaviruses are a diverse subfamily of viruses containing pathogens of humans and animals. This subfamily of viruses replicates their RNA genomes using a core polymerase complex composed of viral non-structural proteins: nsp7, nsp8 and nsp12. Most of our understanding of coronavirus molecular biology comes from betacoronaviruses like SARS-CoV and SARS-CoV-2, the latter of which is the causative agent of COVID-19. In contrast, members of the alphacoronavirus genus are relatively understudied despite their importance in human and animal health. Here we have used cryo-electron microscopy to determine structures of the alphacoronavirus porcine epidemic diarrhea virus (PEDV) core polymerase complex bound to RNA. One structure shows an unexpected nsp8 stoichiometry despite remaining bound to RNA. Biochemical analysis shows that the N-terminal extension of one nsp8 is not required for in vitro RNA synthesis for alpha- and betacoronaviruses. Our work demonstrates the importance of studying diverse coronaviruses in revealing aspects of coronavirus replication and identifying areas of conservation to be targeted by antiviral drugs.

Scd1 and monounsaturated lipids are required for autophagy and survival of adipocytes.

OBJECTIVE: Exposure of adipocytes to 'cool' temperatures often found in the periphery of the body induces expression of Stearoyl-CoA Desaturase-1 (Scd1), an enzyme that converts saturated fatty acids to monounsaturated fatty acids. The goal of this study is to further investigate the roles of Scd in adipocytes. METHOD: In this study, we employed Scd1 knockout cells and mouse models, along with pharmacological Scd1 inhibition to dissect the enzyme's function in adipocyte physiology. RESULTS: Our study reveals that production of monounsaturated lipids by Scd1 is necessary for fusion of autophagosomes to lysosomes and that with a Scd1-deficiency, autophagosomes accumulate. In addition, Scd1-deficiency impairs lysosomal and autolysosomal acidification resulting in vacuole accumulation and eventual cell death. Blocking autophagosome formation or supplementation with monounsaturated fatty acids maintains vitality of Scd1-deficient adipocytes. CONCLUSION: This study demonstrates the indispensable role of Scd1 in adipocyte survival, with its inhibition in vivo triggering autophagy-dependent cell death and its depletion in vivo leading to the loss of bone marrow adipocytes.

Phosphorothioate RNA Analysis by NETD Tandem Mass Spectrometry.

Therapeutic RNAs are routinely modified during their synthesis to ensure proper drug uptake, stability, and efficacy. Phosphorothioate (PS) RNA, molecules in which one or more backbone phosphates are modified with a sulfur atom in place of standard nonbridging oxygen, is one of the most common modifications because of ease of synthesis and pharmacokinetic benefits. Quality assessment of RNA synthesis, including modification incorporation, is essential for drug selectivity and performance, and the synthetic nature of the PS linkage incorporation often reveals impurities. Here, we present a comprehensive analysis of PS RNA via tandem mass spectrometry (MS). We show that activated ion-negative electron transfer dissociation MS/MS is especially useful in diagnosing PS incorporation, producing diagnostic a- and z-type ions at PS linkage sites, beyond the standard d- and w-type ions. Analysis using resonant and beam-type collision-based activation reveals that, overall, more intense sequence ions and base-loss ions result when a PS modification is present. Furthermore, we report increased detection of b- and x-type product ions at sites of PS incorporation, in addition to the standard c- and y-type ions. This work reveals that the gas-phase chemical stability afforded by sulfur alters RNA dissociation and necessitates inclusion of additional product ions for MS/MS of PS RNA.

Positioning appliance for an implant-retained prosthesis for a patient with surgically and radiation-induced microstomia: A clinical report.

When treating a patient with microstomia, prosthesis design can become challenging and often requires modifications. The patient's ability to insert and remove the appliance may also be a problem. This clinical report describes the fabrication of a positioning appliance for a patient with microstomia. The appliance used the maxillary teeth as a guide to orient the prosthesis appropriately and engage the mandibular LOCATOR abutments.

Lipid membrane remodeling and metabolic response during isobutanol and ethanol exposure in Zymomonas mobilis.

BACKGROUND: Recent engineering efforts have targeted the ethanologenic bacterium Zymomonas mobilis for isobutanol production. However, significant hurdles remain due this organism's vulnerability to isobutanol toxicity, adversely affecting its growth and productivity. The limited understanding of the physiological impacts of isobutanol on Z. mobilis constrains our ability to overcome these production barriers. RESULTS: We utilized a systems-level approach comprising LC-MS/MS-based lipidomics, metabolomics, and shotgun proteomics, to investigate how exposure to ethanol and isobutanol impact the lipid membrane composition and overall physiology of Z. mobilis. Our analysis revealed significant and distinct alterations in membrane phospholipid and fatty acid composition resulting from ethanol and isobutanol exposure. Notably, ethanol exposure increased membrane cyclopropane fatty acid content and expression of cyclopropane fatty acid (CFA) synthase. Surprisingly, isobutanol decreased cyclopropane fatty acid content despite robust upregulation of CFA synthase. Overexpression of the native Z. mobilis' CFA synthase increased cyclopropane fatty acid content in all phospholipid classes and was associated with a significant improvement in growth rates in the presence of added ethanol and isobutanol. Heterologous expression of CFA synthase from Clostridium acetobutylicum resulted in a near complete replacement of unsaturated fatty acids with cyclopropane fatty acids, affecting all lipid classes. However, this did not translate to improved growth rates under isobutanol exposure. Correlating with its greater susceptibility to isobutanol, Z. mobilis exhibited more pronounced alterations in its proteome, metabolome, and overall cell morphology-including cell swelling and formation of intracellular protein aggregates -when exposed to isobutanol compared to ethanol. Isobutanol triggered a broad stress response marked by the upregulation of heat shock proteins, efflux transporters, DNA repair systems, and the downregulation of cell motility proteins. Isobutanol also elicited widespread dysregulation of Z. mobilis' primary metabolism evidenced by increased levels of nucleotide degradation intermediates and the depletion of biosynthetic and glycolytic intermediates. CONCLUSIONS: This study provides a comprehensive, systems-level evaluation of the impact of ethanol and isobutanol exposure on the lipid membrane composition and overall physiology of Z. mobilis. These findings will guide engineering of Z. mobilis towards the creation of isobutanol-tolerant strains that can serve as robust platforms for the industrial production of isobutanol from lignocellulosic sugars.

High-content phenotypic analysis of a C. elegans recombinant inbred population identifies genetic and molecular regulators of lifespan.

Lifespan is influenced by complex interactions between genetic and environmental factors. Studying those factors in model organisms of a single genetic background limits their translational value for humans. Here, we mapped lifespan determinants in 85 genetically diverse C. elegans recombinant intercross advanced inbred lines (RIAILs). We assessed molecular profiles - transcriptome, proteome, and lipidome - and life-history traits, including lifespan, development, growth dynamics, and reproduction. RIAILs exhibited large variations in lifespan, which positively correlated with developmental time. Among the top candidates obtained from multi-omics data integration and QTL mapping, we validated known and novel longevity modulators, including rict-1, gfm-1 and mltn-1. We translated their relevance to humans using UK Biobank data and showed that variants in RICTOR and GFM1 are associated with an elevated risk of age-related heart disease, dementia, diabetes, kidney, and liver diseases. We organized our dataset as a resource (https://lisp-lms.shinyapps.io/RIAILs/) that allows interactive explorations for new longevity targets.

Fast and Deep Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer.

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence and structural context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

Heme biosynthesis regulates BCAA catabolism and thermogenesis in brown adipose tissue.

With age, people tend to accumulate body fat and reduce energy expenditure 1 . Brown (BAT) and beige adipose tissue dissipate heat and increase energy expenditure via the activity of the uncoupling protein UCP1 and other thermogenic futile cycles 2,3 . The activity of brown and beige depots inversely correlates with BMI and age 4-11 , suggesting that promoting thermogenesis may be an effective approach for combating age-related metabolic disease 12-15 . Heme is an enzyme cofactor and signaling molecule that we recently showed to regulate BAT function 16 . Here, we show that heme biosynthesis is the primary contributor to intracellular heme levels in brown adipocytes. Inhibition of heme biosynthesis leads to mitochondrial dysfunction and reduction in UCP1. Although supplementing heme can restore mitochondrial function in heme-synthesis-deficient cells, the downregulation of UCP1 persists due to the accumulation of the heme precursors, particularly propionyl-CoA, which is a product of branched-chain amino acids (BCAA) catabolism. Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis 17 . However, BCAAs' contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux 18 . Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.

SCD1 and monounsaturated lipids are required for autophagy and survival of adipocytes.

Exposure of adipocytes to 'cool' temperatures often found in the periphery of the body induces expression of Stearoyl-CoA Desaturase-1 (SCD1), an enzyme that converts saturated fatty acids to monounsaturated fatty acids. In this study, we employed Scd1 knockout cells and mouse models, along with pharmacological SCD1 inhibition, to investigate further the roles of SCD1 in adipocytes. Our study reveals that production of monounsaturated lipids by SCD1 is necessary for fusion of autophagosomes to lysosomes and that with a SCD1-deficiency, autophagosomes accumulate. In addition, SCD1-deficiency impairs lysosomal and autolysosomal acidification resulting in vacuole accumulation and eventual cell death. Blocking autophagosome formation or supplementation with monounsaturated fatty acids maintains vitality of SCD1-deficient adipocytes. Taken together, our results demonstrate that in vitro inhibition of SCD1 in adipocytes leads to autophagy-dependent cell death, and in vivo depletion leads to loss of bone marrow adipocytes.

Modulation of hepatic transcription factor EB activity during cold exposure uncovers direct regulation of bis(monoacylglycero)phosphate lipids by Pla2g15.

Cold exposure is an environmental stress that elicits a rapid metabolic shift in endotherms and is required for survival. The liver provides metabolic flexibility through its ability to rewire lipid metabolism to respond to an increased demand in energy for thermogenesis. We leveraged cold exposure to identify novel lipids contributing to energy homeostasis and found that lysosomal bis(monoacylglycero)phosphate (BMP) lipids were significantly increased in the liver during acute cold exposure. BMP lipid changes occurred independently of lysosomal abundance but were dependent on the lysosomal transcriptional regulator transcription factor EB (TFEB). Knockdown of TFEB in hepatocytes decreased BMP lipid levels. Through molecular biology and biochemical assays, we found that TFEB regulates lipid catabolism during cold exposure and that TFEB knockdown mice were cold intolerant. To identify how TFEB regulates BMP lipid levels, we used a combinatorial approach to identify TFEB target Pla2g15 , a lysosomal phospholipase, as capable of degrading BMP lipids in in vitro liposome assays. Knockdown of Pla2g15 in hepatocytes led to a decrease in BMP lipid species. Together, our studies uncover a required role of TFEB in mediating lipid liver remodeling during cold exposure and identified Pla2g15 as an enzyme that regulates BMP lipid catabolism.

A transcriptional network governing ceramide homeostasis establishes a cytokine-dependent developmental process.

Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.