Online Bipolar Dual Spray for the Charge State Reduction and Characterization of Complex Synthetic Polymers.

Charge reduction mass spectrometry (CR/MS) hyphenated to liquid chromatography (LC) couples liquid-phase compound separation and mass spectral decompression to resolve and characterize multicomponent systems. LC/CR/MS has proven to be effective for complex mixture analysis, particularly synthetic polymers. A newer charge manipulation approach called bipolar dual spray has previously been demonstrated to reduce the observed charge state distribution of ammoniated polyethene glycol. In this approach, two electrospray emitters, in close proximity and of opposite polarity, fuse droplets from their electrospray plumes, which allows the subsequent chemistry. In this work, we investigate the ability of bipolar dual spray to reduce the charge of synthetic polyols, thereby simplifying complex mixture analysis and generating new compositional information only available through the coupling of charge reduction with LC/MS analysis. This work also represents the first demonstration of online charge reduction via dual spray. Polyethylene glycol (PEG) 7.2K subjected to LC/MS with dual spray reduced the average charge state from 8.2+ to 4.4+. LC/MS with dual spray was also applied to the characterization of an end-group-modified PEG 10K (i.e., aminated) containing several reaction impurities. This approach allowed for the identification of low-level starting material, tosylated PEG, and PEG mono(amine), where both LC/MS and direct infusion dual spray did not detect the impurities. Overall, the results demonstrated that bipolar dual spray can be incorporated into an LC/MS analysis and affords the ability to reduce the charge state distribution of PEG cations, decompress the m/z axis, lower spectra complexity, and enable/simplify data interpretation.

Improved Structural Characterization of Glycerophospholipids and Sphingomyelins with Real-Time Library Searching.

In mass spectrometry-based lipidomics, complex lipid mixtures undergo chromatographic separation, are ionized, and are detected using tandem MS (MSn) to simultaneously quantify and structurally characterize eluting species. The reported structural granularity of these identified lipids is strongly reliant on the analytical techniques leveraged in a study. For example, lipid identifications from traditional collisionally activated data-dependent acquisition experiments are often reported at either species level or molecular species level. Structural resolution of reported lipid identifications is routinely enhanced by integrating both positive and negative mode analyses, requiring two separate runs or polarity switching during a single analysis. MS3+ can further elucidate lipid structure, but the lengthened MS duty cycle can negatively impact analysis depth. Recently, functionality has been introduced on several Orbitrap Tribrid mass spectrometry platforms to identify eluting molecular species on-the-fly. These real-time identifications can be leveraged to trigger downstream MSn to improve structural characterization with lessened impacts on analysis depth. Here, we describe a novel lipidomics real-time library search (RTLS) approach, which utilizes the lipid class of real-time identifications to trigger class-targeted MSn and to improve the structural characterization of phosphotidylcholines, phosphotidylethanolamines, phosphotidylinositols, phosphotidylglycerols, phosphotidylserine, and sphingomyelins in the positive ion mode. Our class-based RTLS method demonstrates improved selectivity compared to the current methodology of triggering MSn in the presence of characteristic ions or neutral losses.

Development of Two-Dimensional Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for the Separation of a Five-Component Submicrometer Particle Mixture.

Submicrometer colloidal particles are widely applied in a variety of industrial products. While precise size and surface charge control is crucial to the stability and functionality of these materials, a tool to determine these properties with sufficient resolution, detection sensitivity, and robustness is still not available. The recently reported offline coupling of asymmetrical flow field-flow fractionation and capillary electrophoresis (AF4 × CE) shows success in improving the separation resolution for nanoparticles; however, challenges remain for sensitive multiple-component submicrometer particle analysis because of wide size and mobility distributions. We here report offline coupling of an AF4 method and a CE method, which utilized the online reversed electrode polarity stacking mode, to successfully characterize a five-component, submicrometer particle mixture. The mixture was successfully separated and detected with an improved inter- and intracomponent resolution. Therefore, our developed platform holds great potential for industrial applications involving multiple-component particle mixtures.

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.

Maximizing MS/MS Acquisition for Lipidomics Using Capillary Separation and Orbitrap Tribrid Mass Spectrometer.

Liquid chromatography-mass spectrometry (LC-MS) is a typical strategy for lipidomics analysis. Although capillary LC-MS is a common analytical technique for proteomics analysis, its application to lipidomics has been limited. In this study, we aim at improving lipid identifications achieved in a single LC-MS analysis by a 3-fold approach: capillary LC and nanoelectrospray for enhanced ionization, ion trap for higher sensitivity tandem MS, and parallelization of mass analyzers for increased speed of acquisition on an Orbitrap hybrid system. By applying the methods to a complex lipid mixture of human plasma, we identified and performed relative quantification on over 1500 lipids within a 60 min capillary LC-MS analysis.

Social and Economic Costs of Attention-Deficit/Hyperactivity Disorder Across the Lifespan.

OBJECTIVE: To determine the financial and non-financial costs of Attention-Deficit/Hyperactivity Disorder (ADHD) across the lifespan. METHOD: The population costs of ADHD in Australia were estimated for the financial year 2018 to 2019 using a prevalence approach to cost estimation across all ages. Financial (healthcare, productivity, education and justice systems, and deadweight losses) and non-financial costs were measured (Disability Adjusted Life Years (DALYs)). RESULTS: The total social and economic cost of ADHD in 2018 to 2019 were US$12.76 billion (range US$8.40 billion to US$17.44 billion, with per person costs of US$15,664 per year). Productivity costs made up 81% of the total financial costs, followed by deadweight losses (11%), and health system costs (4%). Loss in terms of wellbeing was significant (US$5.31 billion). CONCLUSION: There is a need to raise public awareness of the considerable socioeconomic impact and burden of ADHD in order to drive investment and policy decisions that improve identification and treatment of ADHD.

UbiB proteins regulate cellular CoQ distribution in Saccharomyces cerevisiae.

Beyond its role in mitochondrial bioenergetics, Coenzyme Q (CoQ, ubiquinone) serves as a key membrane-embedded antioxidant throughout the cell. However, how CoQ is mobilized from its site of synthesis on the inner mitochondrial membrane to other sites of action remains a longstanding mystery. Here, using a combination of Saccharomyces cerevisiae genetics, biochemical fractionation, and lipid profiling, we identify two highly conserved but poorly characterized mitochondrial proteins, Ypl109c (Cqd1) and Ylr253w (Cqd2), that reciprocally affect this process. Loss of Cqd1 skews cellular CoQ distribution away from mitochondria, resulting in markedly enhanced resistance to oxidative stress caused by exogenous polyunsaturated fatty acids, whereas loss of Cqd2 promotes the opposite effects. The activities of both proteins rely on their atypical kinase/ATPase domains, which they share with Coq8-an essential auxiliary protein for CoQ biosynthesis. Overall, our results reveal protein machinery central to CoQ trafficking in yeast and lend insights into the broader interplay between mitochondria and the rest of the cell.

A large-scale genome-lipid association map guides lipid identification.

Despite the crucial roles of lipids in metabolism, we are still at the early stages of comprehensively annotating lipid species and their genetic basis. Mass spectrometry-based discovery lipidomics offers the potential to globally survey lipids and their relative abundances in various biological samples. To discover the genetics of lipid features obtained through high-resolution liquid chromatography-tandem mass spectrometry, we analysed liver and plasma from 384 diversity outbred mice, and quantified 3,283 molecular features. These features were mapped to 5,622 lipid quantitative trait loci and compiled into a public web resource termed LipidGenie. The data are cross-referenced to the human genome and offer a bridge between genetic associations in humans and mice. Harnessing this resource, we used genome-lipid association data as an additional aid to identify a number of lipids, for example gangliosides through their association with B4galnt1, and found evidence for a group of sex-specific phosphatidylcholines through their shared locus. Finally, LipidGenie's ability to query either mass or gene-centric terms suggests acyl-chain-specific functions for proteins of the ABHD family.

Accelerating Lipidomic Method Development through in Silico Simulation.

Judicious selection of mass spectrometry (MS) acquisition parameters is essential for effectively profiling the broad diversity and dynamic range of biomolecules. Typically, acquisition parameters are individually optimized to maximally characterize analytes from each new sample matrix. This time-consuming process often ignores the synergistic relationship between MS method parameters, producing suboptimal results. Here we detail the creation of an algorithm which accurately simulates LC-MS/MS lipidomic data acquisition performance for a benchtop quadrupole-Orbitrap MS system. By coupling this simulation tool with a genetic algorithm for constrained parameter optimization, we demonstrate the efficient identification of LC-MS/MS method parameter sets individually suited for specific sample matrices. Finally, we utilize the in silico simulation to examine how continued developments in MS acquisition speed and sensitivity will further increase the power of MS lipidomics as a vital tool for impactful biochemical analysis.

Biorelevant dissolution testing of a weak base: Interlaboratory reproducibility and investigation of parameters controlling in vitro precipitation.

Following a previous study which aimed to determine the interlaboratory reproducibility of biorelevant dissolution testing in the USP 2 apparatus for commercial formulations of two weak acids (ibuprofen and zafirlukast), this study attempts to determine the interlaboratory reproducibility using a similar protocol for a commercially available formulation of a weak base, indinavir. Fourteen partners including twelve industrial and two academic partners participated in this study. To ensure uniformity, all partners were provided with a standardized protocol to perform (i) a single medium dissolution test in fasted state simulated gastric and intestinal fluids (FaSSGF and FaSSIF, respectively) and (ii) a two-stage dissolution experiment simulating gastrointestinal transfer. Optionally, partners could run a single-stage dissolution test in fed state simulated intestinal fluid (FeSSIF). For each dissolution test, one Crixivan® capsule (containing 400 mg indinavir as its sulfate salt) was added as dose of interest. For the single medium dissolution test in FaSSIF, all partners observed rapid release of indinavir resulting in supersaturated concentrations, followed by precipitation to equilibrium solubility. The degree and period of supersaturation varied among the participating laboratories. Average dissolution profiles in FeSSIF appeared to be highly reproducible with dissolved concentrations remaining lower than the thermodynamic solubility of indinavir in FeSSIF. For the two-stage dissolution test, most partners observed supersaturated concentrations in the intestinal compartment; two partners observed no supersaturation due to immediate precipitation. Given the fact that a high interlaboratory but low intralaboratory variability was observed when supersaturation/precipitation occurred, an undefined factor was hypothesized as a potential cause of the variability in precipitation. Hence, the impact of several experimental factors on the supersaturation and precipitation behavior of indinavir was investigated in a next step. The investigation indicated that variability is likely attributable to a combination of factors, especially, the time elapsed between sampling and dilution of the sample with the mobile phase. Therefore, when designing a test in which supersaturation and precipitation is anticipated, stringent control of the test methodology, especially regarding sampling and dilution, is needed.

Mapping Lipid Fragmentation for Tailored Mass Spectral Libraries.

Libraries of simulated lipid fragmentation spectra enable the identification of hundreds of unique lipids from complex lipid extracts, even when the corresponding lipid reference standards do not exist. Often, these in silico libraries are generated through expert annotation of spectra to extract and model fragmentation rules common to a given lipid class. Although useful for a given sample source or instrumental platform, the time-consuming nature of this approach renders it impractical for the growing array of dissociation techniques and instrument platforms. Here, we introduce Library Forge, a unique algorithm capable of deriving lipid fragment mass-to-charge (m/z) and intensity patterns directly from high-resolution experimental spectra with minimal user input. Library Forge exploits the modular construction of lipids to generate m/z transformed spectra in silico which reveal the underlying fragmentation pathways common to a given lipid class. By learning these fragmentation patterns directly from observed spectra, the algorithm increases lipid spectral matching confidence while reducing spectral library development time from days to minutes. We embed the algorithm within the preexisting lipid analysis architecture of LipiDex to integrate automated and robust library generation within a comprehensive LC-MS/MS lipidomics workflow. Graphical Abstract.

An Isoprene Lipid-Binding Protein Promotes Eukaryotic Coenzyme Q Biosynthesis.

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. In eukaryotes, CoQ precursors-among nature's most hydrophobic molecules-must somehow be presented to a series of enzymes peripherally associated with the mitochondrial inner membrane. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. We show that COQ9 repurposes the bacterial TetR fold to bind aromatic isoprenes with high specificity, including CoQ intermediates that likely reside entirely within the bilayer. We reveal a process by which COQ9 associates with cardiolipin-rich membranes and warps the membrane surface to access this cargo. Finally, we identify a molecular interface between COQ9 and the hydroxylase COQ7, motivating a model whereby COQ9 presents intermediates directly to CoQ enzymes. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

Effects of methylphenidate on cognition and behaviour in children with neurofibromatosis type 1: a study protocol for a randomised placebo-controlled crossover trial.

INTRODUCTION: Dopamine dysregulation has been identified as a key modulator of behavioural impairment in neurofibromatosis type 1 (NF1) and a potential therapeutic target. Preclinical research demonstrates reduced dopamine in the brains of genetically engineered NF1 mouse strains is associated with reduced spatial-learning and attentional dysfunction. Methylphenidate, a stimulant medication that increases dopaminergic and noradrenergic neurotransmission, rescued the behavioural and dopamine abnormalities. Although preliminary clinical trials have demonstrated that methylphenidate is effective in treating attention deficit hyperactivity disorder (ADHD) symptoms in children with NF1, its therapeutic effect on cognitive performance is unclear. The primary aim of this clinical trial is to assess the efficacy of methylphenidate for reducing attention deficits, spatial working memory impairments and ADHD symptoms in children with NF1. METHODS AND ANALYSIS: A randomised, double-blind, placebo-controlled trial of methylphenidate with a two period crossover design. Thirty-six participants with NF1 aged 7-16 years will be randomised to one of two treatment sequences: 6 weeks of methylphenidate followed by 6 weeks of placebo or; 6 weeks of placebo followed by 6 weeks of methylphenidate. Neurocognitive and behavioural outcomes as well as neuroimaging measures will be completed at baseline and repeated at the end of each treatment condition (week 6, week 12). Primary outcome measures are omission errors on the Conners Continuous Performance Test-II (attention), between-search errors on the Spatial Working Memory task from the Cambridge Neuropsychological Test Automated Battery (spatial working memory) and the Inattentive and Hyperactivity/Impulsivity Symptom Scales on the Conners 3-Parent. Secondary outcomes will examine the effect of methylphenidate on executive functions, attention, visuospatial skills, behaviour, fine-motor skills, language, social skills and quality of life. ETHICS AND DISSEMINATION: This trial has hospital ethics approval and the results will be disseminated through peer-reviewed publications and international conferences. TRIAL REGISTRATION NUMBER: ACTRN12611000765921.

LipiDex: An Integrated Software Package for High-Confidence Lipid Identification.

State-of-the-art proteomics software routinely quantifies thousands of peptides per experiment with minimal need for manual validation or processing of data. For the emerging field of discovery lipidomics via liquid chromatography-tandem mass spectrometry (LC-MS/MS), comparably mature informatics tools do not exist. Here, we introduce LipiDex, a freely available software suite that unifies and automates all stages of lipid identification, reducing hands-on processing time from hours to minutes for even the most expansive datasets. LipiDex utilizes flexible in silico fragmentation templates and lipid-optimized MS/MS spectral matching routines to confidently identify and track hundreds of lipid species and unknown compounds from diverse sample matrices. Unique spectral and chromatographic peak purity algorithms accurately quantify co-isolation and co-elution of isobaric lipids, generating identifications that match the structural resolution afforded by the LC-MS/MS experiment. During final data filtering, ionization artifacts are removed to significantly reduce dataset redundancy. LipiDex interfaces with several LC-MS/MS software packages, enabling robust lipid identification to be readily incorporated into pre-existing data workflows.

Caloric Restriction Engages Hepatic RNA Processing Mechanisms in Rhesus Monkeys.

Caloric restriction (CR) extends lifespan and delays the onset of age-related disorders in diverse species. Metabolic regulatory pathways have been implicated in the mechanisms of CR, but the molecular details have not been elucidated. Here, we show that CR engages RNA processing of genes associated with a highly integrated reprogramming of hepatic metabolism. We conducted molecular profiling of liver biopsies collected from adult male rhesus monkeys (Macaca mulatta) at baseline and after 2 years on control or CR (30% restricted) diet. Quantitation of over 20,000 molecules from the hepatic transcriptome, proteome, and metabolome indicated that metabolism and RNA processing are major features of the response to CR. Predictive models identified lipid, branched-chain amino acid, and short-chain carbon metabolic pathways, with alternate transcript use for over half of the genes in the CR network. We conclude that RNA-based mechanisms are central to the CR response and integral in metabolic reprogramming.

Conserved Lipid and Small-Molecule Modulation of COQ8 Reveals Regulation of the Ancient Kinase-like UbiB Family.

Human COQ8A (ADCK3) and Saccharomyces cerevisiae Coq8p (collectively COQ8) are UbiB family proteins essential for mitochondrial coenzyme Q (CoQ) biosynthesis. However, the biochemical activity of COQ8 and its direct role in CoQ production remain unclear, in part due to lack of known endogenous regulators of COQ8 function and of effective small molecules for probing its activity in vivo. Here, we demonstrate that COQ8 possesses evolutionarily conserved ATPase activity that is activated by binding to membranes containing cardiolipin and by phenolic compounds that resemble CoQ pathway intermediates. We further create an analog-sensitive version of Coq8p and reveal that acute chemical inhibition of its endogenous activity in yeast is sufficient to cause respiratory deficiency concomitant with CoQ depletion. Collectively, this work defines lipid and small-molecule modulators of an ancient family of atypical kinase-like proteins and establishes a chemical genetic system for further exploring the mechanistic role of COQ8 in CoQ biosynthesis.

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.

Validation of Dissolution Testing with Biorelevant Media: An OrBiTo Study.

Dissolution testing with biorelevant media has become widespread in the pharmaceutical industry as a means of better understanding how drugs and formulations behave in the gastrointestinal tract. Until now, however, there have been few attempts to gauge the reproducibility of results obtained with these methods. The aim of this study was to determine the interlaboratory reproducibility of biorelevant dissolution testing, using the paddle apparatus (USP 2). Thirteen industrial and three academic laboratories participated in this study. All laboratories were provided with standard protocols for running the tests: dissolution in FaSSGF to simulate release in the stomach, dissolution in a single intestinal medium, FaSSIF, to simulate release in the small intestine, and a "transfer" (two-stage) protocol to simulate the concentration profile when conditions are changed from the gastric to the intestinal environment. The test products chosen were commercially available ibuprofen tablets and zafirlukast tablets. The biorelevant dissolution tests showed a high degree of reproducibility among the participating laboratories, even though several different batches of the commercially available medium preparation powder were used. Likewise, results were almost identicalbetween the commercial biorelevant media and those produced in-house. Comparing results to previous ring studies, including those performed with USP calibrator tablets or commercially available pharmaceutical products in a single medium, the results for the biorelevant studies were highly reproducible on an interlaboratory basis. Interlaboratory reproducibility with the two-stage test was also acceptable, although the variability was somewhat greater than with the single medium tests. Biorelevant dissolution testing is highly reproducible among laboratories and can be relied upon for cross-laboratory comparisons.

Ptc7p Dephosphorylates Select Mitochondrial Proteins to Enhance Metabolic Function.

Proper maintenance of mitochondrial activity is essential for metabolic homeostasis. Widespread phosphorylation of mitochondrial proteins may be an important element of this process; yet, little is known about which enzymes control mitochondrial phosphorylation or which phosphosites have functional impact. We investigate these issues by disrupting Ptc7p, a conserved but largely uncharacterized mitochondrial matrix PP2C-type phosphatase. Loss of Ptc7p causes respiratory growth defects concomitant with elevated phosphorylation of select matrix proteins. Among these, Δptc7 yeast exhibit an increase in phosphorylation of Cit1p, the canonical citrate synthase of the tricarboxylic acid (TCA) cycle, that diminishes its activity. We find that phosphorylation of S462 can eliminate Cit1p enzymatic activity likely by disrupting its proper dimerization, and that Ptc7p-driven dephosphorylation rescues Cit1p activity. Collectively, our work connects Ptc7p to an essential TCA cycle function and to additional phosphorylation events that may affect mitochondrial activity inadvertently or in a regulatory manner.