Search

Samir Samman,1 Ben Crossett,2 Miles Somers,1 Kirstine J Bell,1 Nicole T Lai,1,3 David R Sullivan,3 Peter Petocz4 1Discipline of Nutrition and Metabolism, 2Discipline of Proteomics and Biotechnology, School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia; 3Department of Clinical Biochemistry, Royal Prince Alfred Hospital, Sydney, NSW, Australia; 4Department of Statistics, Macquarie University, Sydney, NSW, Australia Abstract: Amino acid (AA) status is determined by factors including nutrition, metabolic rate, and interactions between the metabolism of AA, carbohydrates, and lipids. Analysis of the plasma AA profile, together with markers of glucose and lipid metabolism, will shed light on metabolic regulation. The objectives of this study were to investigate the acute responses to the consumption of meals containing either pork (PM) or chicken (CM), and to identify relationships between plasma AA and markers of glycemic and lipemic control. A secondary aim was to explore AA predictors of plasma zinc concentrations. Ten healthy adults participated in a postprandial study on two separate occasions. In a randomized cross-over design, participants consumed PM or CM. The concentrations of 21 AA, glucose, insulin, triglycerides, nonesterified fatty acids, and zinc were determined over 5 hours postprandially. The meal composition did not influence glucose, insulin, triglyceride, nonesterified fatty acid, or zinc concentrations. Plasma histidine was higher following the consumption of PM (P=0.014), with consistently higher changes observed after 60 minutes (P

Abstract Background Pseudomonas aeruginosa is an opportunistic pathogen that is the major cause of morbidity and mortality in patients with cystic fibrosis (CF). While most CF patients are thought to acquire P. aeruginosa from the environment, person-person transmissible strains have been identified in CF clinics worldwide. The molecular basis for transmissibility and colonization of the CF lung remains poorly understood. Results A dual proteomics approach consisting of gel-based and gel-free comparisons were undertaken to analyse protein profiles in a transmissible, early (acute) isolate of the Australian epidemic strain 1 (AES-1R), the virulent burns/wound isolate PA14, and the poorly virulent, laboratory-associated strain PAO1. Over 1700 P. aeruginosa proteins were confidently identified. AES-1R protein profiles revealed elevated abundance of proteins associated with virulence and siderophore biosynthesis and acquisition, antibiotic resistance and lipopolysaccharide and fatty acid biosynthesis. The most abundant protein in AES-1R was confirmed as a previously hypothetical protein with sequence similarity to carbohydrate-binding proteins and database search revealed this gene is only found in the CF-associated strain PA2192. The link with CF infection may suggest that transmissible strains have acquired an ability to rapidly interact with host mucosal glycoproteins. Conclusions Our data suggest that AES-1R expresses higher levels of proteins, such as those involved in antibiotic resistance, iron acquisition and virulence that may provide a competitive advantage during early infection in the CF lung. Identification of novel proteins associated with transmissibility and acute infection may aid in deciphering new strategies for intervention to limit P. aeruginosa infections in CF patients.

Abstract Mycoplasma haemofelis is a pathogenic feline hemoplasma. Despite its importance, little is known about its metabolic pathways or mechanism of pathogenicity due to it being uncultivatable. The recently sequenced M. haemofelis str. Langford 1 genome was analysed and compared to those of other available hemoplasma genomes. Analysis showed that in hemoplasmas genes involved in carbohydrate metabolism are limited to enzymes of the glycolytic pathway, with glucose appearing to be the sole energy source. The majority of the pentose phosphate pathway enzymes that catalyze the de novo synthesis of ribonucleotides were absent, as were cell division protein FtsZ and chaperonins GroEL/ES. Uncharacterized protein paralogs containing putative surface expression motifs, comprised 62% of M. haemofelis and 19% of Mycoplasma suis genome coverage respectively, the majority of which were present in a small number of unstructured islands. Limited mass spectrometry and immunoblot data matched a number of characterized proteins and uncharacterized paralogs, confirming their expression and immunogenicity in vivo. These data have allowed further characterization of these important pathogens, including their limited metabolic capabilities, which may contribute to their uncultivatable status. A number of immunogenic proteins, and a potential mechanism for host immune system evasion, have been identified.

Abstract Background The pathogenesis of HIV-associated dementia (HAD) is poorly understood. To date, detailed proteomic fingerprinting directly from autopsied brain tissues of HAD and HIV non-dementia patients has not been performed. Result Here, we have analyzed total proteins from the frontal cortex of 9 HAD and 5 HIV non-dementia patients. Using 2-Dimensional differential in-gel electrophoresis (2-DIGE) to analyze the brain tissue proteome, 76 differentially expressed proteins (p < 0.05; fold change>1.25) were identified between HAD and HIV non-dementia patients, of which 36 protein spots (based on 3D appearance of spots on the images) were chosen for the mass spectrometry analysis. The large majority of identified proteins were represented in the energy metabolic (mitochondria) and signal transduction pathways. Furthermore, over 90% of the protein candidates are common to both HAD and other non-viral neurodegenerative disease, such as Alzheimer's disease. The data was further validated using specific antibodies to 4 proteins (CA2, GS, CKMT and CRMP2) by western blot (WB) in the same samples used for 2D-DIGE, with additional confirmation by immunohistochemitsry (IHC) using frontal lobe tissue from different HAD and HIV+ non-dementia patients. The validation for all 4 antibodies by WB and IHC was in concordance with the DIGE results, lending further credence to the current findings. Conclusion These results suggest not only convergent pathogenetic pathways for the two diseases but also the possibility of increased Alzheimer's disease (AD) susceptibility in HAD patients whose life expectancy has been significantly increased by highly active antiretroviral therapy.

Inositol tris/tetrakis phosphate kinases (IP 3-4 K) in the human fungal priority pathogens, Cryptococcus neoformans ( Cn Arg1) and Candida albicans ( Ca Ipk2), convey numerous virulence functions, yet it is not known whether the IP 3-4 K catalytic activity or a scaffolding role is responsible. We therefore generated a C. neoformans strain with a non-functional kinase, referred to as the dead-kinase (dk) Cn Arg1 strain (dkArg1). We verified that, although dk ARG1 cDNA cloned from this strain produced a protein with the expected molecular weight, dkArg1 was catalytically inactive with no IP 3-4 K activity. Using recombinant Cn Arg1 and Ca Ipk2 , we confirmed that, unlike the IP 3-4 K homologs in humans and Saccharomyces cerevisiae , Cn Arg1 and Ca Ipk2 do not phosphorylate the lipid-based substrate, phosphatidylinositol 4,5-bisphosphate, and therefore do not function as class I PI3Ks. Inositol polyphosphate profiling using capillary electrophoresis-electrospray ionization-mass spectrometry revealed that IP 3 conversion is blocked in the dkArg1 and ARG1 deletion ( Cnarg1 Δ) strains and that 1-IP 7 and a recently discovered isomer (4/6-IP 7 ) are made by wild-type C. neoformans . Importantly, the dkArg1 and Cnarg1 Δ strains had similar virulence defects, including suppressed growth at 37°C, melanization, capsule production, and phosphate starvation response, and were avirulent in an insect model, confirming that virulence is dependent on IP 3-4 K catalytic activity. Our data also implicate the dkArg1 scaffold in transcriptional regulation of arginine metabolism but via a different mechanism to S. cerevisiae since Cn Arg1 is dispensable for growth on different nitrogen sources. IP 3-4 K catalytic activity therefore plays a dominant role in fungal virulence, and IPK pathway function has diverged in fungal pathogens.IMPORTANCEThe World Health Organization has emphasized the urgent need for global action in tackling the high morbidity and mortality rates stemming from invasive fungal infections, which are exacerbated by the limited variety and compromised effectiveness of available drug classes. Fungal IP 3-4 K is a promising target for new therapy, as it is critical for promoting virulence of the human fungal priority pathogens, Cryptococcus neoformans and Candida albicans , and impacts numerous functions, including cell wall integrity. This contrasts to current therapies, which only target a single function. IP 3-4 K enzymes exert their effect through their inositol polyphosphate products or via the protein scaffold. Here, we confirm that the IP 3-4 K catalytic activity of Cn Arg1 promotes all virulence traits in C. neoformans that are attenuated by ARG1 deletion , reinforcing our ongoing efforts to find inositol polyphosphate effector proteins and to create inhibitors targeting the IP 3-4 K catalytic site, as a new antifungal drug class., Competing Interests: The authors declare no conflict of interest.

Objective: Chronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear., Design: Using an in vivo mouse model of cigarette smoke (CS)-induced COPD and faecal microbial transfer (FMT), we characterised the faecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in 16 patients with COPD using a randomised, double-blind, placebo-controlled pilot study of inulin supplementation., Results: FMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation, and transfer of CS-associated microbiota after antibiotic-induced microbiome depletion was sufficient to increase lung inflammation while suppressing colonic immunity in the absence of CS exposure. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in CS-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes., Conclusion: The gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Mouse monoclonal 12E8 antibody, which recognises conserved serine phosphorylated KXGS motifs in the microtubule binding domains of tau/tau-like microtubule associated proteins (MAPs), shows elevated binding in brain during normal embryonic development (mammals and birds) and at the early stages of human Alzheimer's disease (AD). It also labels ADF/cofilin-actin rods that form in neurites during exposure to stressors. We aimed to identify direct and indirect 12E8 binding proteins in postnatal mouse brain and embryonic chick brain by immunoprecipitation (IP), mass spectrometry and immunofluorescence. Tau and/or MAP2 were major direct 12E8-binding proteins detected in all IPs, and actin and/or tubulin were co-immunoprecipitated in most samples. Additional proteins were different in mouse versus chick brain IP. In mouse brain IPs, FSD1l and intermediate filament proteins - vimentin, α-internexin, neurofilament polypeptides - were prominent. Immunofluorescence and immunoblot using recombinant intermediate filament subunits, suggests an indirect interaction of these proteins with the 12E8 antibody. In chick brain IPs, subunits of eukaryotic translation initiation factor 3 (EIF3) were found, but no direct interaction between 12E8 and recombinant Eif3e protein was detected. Fluorescence microscopy in primary cultured chick neurons showed evidence of co-localisation of Eif3e and tubulin labelling, consistent with previous data demonstrating cytoskeletal organisation of the translation apparatus. Neither total tau or MAP2 immunolabelling accumulated at ADF/cofilin-actin rods generated in primary cultured chick neurons, and we were unable to narrow down the major antigen recognised by 12E8 antibody on ADF/cofilin-actin rods., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

The repair of double-strand DNA breaks (DSBs) by homologous recombination is crucial in the maintenance of genome integrity. While the key role of the Mre11-Rad50-Nbs1 (MRN) complex in repair is well known, hSSB1 (SOSSB and OBFC2B), one of the main components of the sensor of single-stranded DNA (SOSS) protein complex, has also been shown to rapidly localize to DSB breaks and promote repair. We have previously demonstrated that hSSB1 binds directly to Nbs1, a component of the MRN complex, in a DNA damage-independent manner. However, recruitment of the MRN complex has also been demonstrated by an interaction between Integrator Complex Subunit 3 (INTS3; also known as SOSSA), another member of the SOSS complex, and Nbs1. In this study, we utilize a combined approach of in silico, biochemical, and functional experiments to uncover the molecular details of INTS3 binding to Nbs1. We demonstrate that the forkhead-associated domain of Nbs1 interacts with INTS3 via phosphorylation-dependent binding to INTS3 at Threonine 592, with contributions from Serine 590. Based on these data, we propose a model of MRN recruitment to a DSB via INTS3., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å -1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å -1 , but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H 2 O and in D 2 O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å -1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set., (open access.)

(1) Background: MALDI imaging is a technique that still largely depends on time of flight (TOF)-based instrument such as the Bruker UltrafleXtreme. While capable of performing targeted MS/MS, these instruments are unable to perform fragmentation while imaging a tissue section necessitating the reliance of MS1 values for peptide level identifications. With this premise in mind, we have developed a hybrid bioinformatic/image-based method for the identification and validation of viral biomarkers. (2) Methods: Formalin-Fixed Paraffin-Embedded (FFPE) mouse samples were sectioned, mounted and prepared for mass spectrometry imaging using our well-established methods. Peptide identification was achieved by first extracting confident images corresponding to theoretical viral peptides. Next, those masses were used to perform a Peptide Mmass Fingerprint (PMF) searched against known viral FASTA sequences against a background mouse FASTA database. Finally, a correlational analysis was performed with imaging data to confirm pixel-by-pixel colocalization and intensity of viral peptides. (3) Results: 14 viral peptides were successfully identified with significant PMF Scores and a correlational result of >0.79 confirming the presence of the virus and distinguishing it from the background mouse proteins. (4) Conclusions: this novel approach leverages the power of mass spectrometry imaging and provides confident identifications for viral proteins without requiring MS/MS using simple MALDI Time Of Flight/Time Of Flight (TOF/TOF) instrumentation.

Diagnosis of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection has primarily been achieved using reverse transcriptase polymerase chain reaction (RT-PCR) for acute infection, and serology for prior infection. Assay with RT-PCR provides data on presence or absence of viral RNA, with no information on virus replication competence, infectivity, or virus characterisation. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is typically not used in clinical virology, despite its potential to provide supplemental data about the presence of viral proteins and thus the potential for replication-competent, transmissible virus. Using the SARS-CoV-2 as a model virus, we developed a fast 'bottom-up' proteomics workflow for discovery of target virus peptides using 'serum-free' culture conditions, providing high coverage of viral proteins without the need for protein or peptide fractionation techniques. This workflow was then applied to Coronaviruses OC43 and 229E, Influenza A/H1N1 and H3N2, Influenza B, and Respiratory Syncytial Viruses A and B. Finally, we created an LC-MS/MS method for targeted detection of the eight-virus panel in clinical specimens, successfully detecting peptides from the SARS-CoV-2 ORF9B and nucleoprotein in RT-PCR positive samples. The method provides specific detection of respiratory viruses from clinical samples containing moderate viral loads and is an important further step to the use of LC-MS/MS in diagnosis of viral infection., (© 2022. The Author(s).)

Rationale: Matrix-assisted laser desorption ionisation with mass spectrometry imaging (MSI) has seen rapid development in recent years and as such is becoming an important technique for the mapping of biomolecules from the surface of tissues. One key area of development is the optimisation of analyte extraction by using modified matrices or mixes of common ones., Methods: A series of serial sections were prepared for lipid MSI by either dry coating (sublimation) or by wet spray application of several matrices. These samples were then evaluated for analyte extraction, delocalisation and dynamic range., Results: We have shown that the spraying and sublimation methods of matrix application can be used complementarily. This creates large datasets, with each preparation method applied narrowly and then interpreted as a 'fraction' of the whole. Once combined, the dynamic range is significantly increased. We have dubbed this technique 'matrix phase fractionation'., Conclusions: We have found that, by utilising matrix phase fractionation for the detection of lipids in brain tissue, it is possible to create a significantly more comprehensive dataset than would otherwise be possible with traditional 'single-run' workflows., (© 2021 John Wiley & Sons Ltd.)

Plant transmembrane proteins (TMPs) are essential for normal cellular homeostasis, nutrient exchange, and responses to environmental cues. Commonly used bottom-up proteomic approaches fail to identify a broad coverage of peptide fragments derived from TMPs. Here, we used mass spectrometry (MS) to compare the effectiveness of two solubilization and protein cleavage methods to identify shoot-derived TMPs from the legume Medicago. We compared a urea solubilization, trypsin Lys-C (UR-TLC) cleavage method to a formic acid solubilization, cyanogen bromide and trypsin Lys-C (FA-CTLC) cleavage method. We assessed the effectiveness of these methods by (i) comparing total protein identifications, (ii) determining how many TMPs were identified, and (iii) defining how many peptides incorporate all, or part, of transmembrane domains (TMD) sequences. The results show that the FA-CTLC method identified nine-fold more TMDs, and enriched more hydrophobic TMPs than the UR-TLC method. FA-CTLC identified more TMPs, particularly transporters, whereas UR-TLC preferentially identified TMPs with one TMD, particularly signaling proteins. The results suggest that combining plant membrane purification techniques with both the FA-CTLC and UR-TLC methods will achieve a more complete identification and coverage of TMPs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Lee, Carroll, Crossett, Connolly, Batarseh and Djordjevic.)

In the human-pathogenic fungus Cryptococcus neoformans , the inositol polyphosphate signaling pathway is critical for virulence. We recently demonstrated the key role of the inositol pyrophosphate IP 7 (isomer 5-PP-IP 5 ) in driving fungal virulence; however, the mechanism of action remains elusive. Using genetic and biochemical approaches, and mouse infection models, we show that IP 7 synthesized by Kcs1 regulates fungal virulence by binding to a conserved lysine surface cluster in the SPX domain of Pho81. Pho81 is the cyclin-dependent kinase (CDK) inhibitor of the phosphate signaling (PHO) pathway. We also provide novel mechanistic insight into the role of IP 7 in PHO pathway regulation by demonstrating that IP 7 functions as an intermolecular "glue" to stabilize Pho81 association with Pho85/Pho80 and, hence, promote PHO pathway activation and phosphate acquisition. Blocking IP 7 -Pho81 interaction using site-directed mutagenesis led to a dramatic loss of fungal virulence in a mouse infection model, and the effect was similar to that observed following PHO81 gene deletion, highlighting the key importance of Pho81 in fungal virulence. Furthermore, our findings provide additional evidence of evolutionary divergence in PHO pathway regulation in fungi by demonstrating that IP 7 isomers have evolved different roles in PHO pathway control in C. neoformans and nonpathogenic yeast. IMPORTANCE Invasive fungal diseases pose a serious threat to human health globally with >1.5 million deaths occurring annually, 180,000 of which are attributable to the AIDS-related pathogen, Cryptococcus neoformans Here, we demonstrate that interaction of the inositol pyrophosphate, IP 7 , with the CDK inhibitor protein, Pho81, is instrumental in promoting fungal virulence. IP 7 -Pho81 interaction stabilizes Pho81 association with other CDK complex components to promote PHO pathway activation and phosphate acquisition. Our data demonstrating that blocking IP 7 -Pho81 interaction or preventing Pho81 production leads to a dramatic loss in fungal virulence, coupled with Pho81 having no homologue in humans, highlights Pho81 function as a potential target for the development of urgently needed antifungal drugs., (Copyright © 2020 Desmarini et al.)

Background: PromarkerD is a novel proteomics derived blood test for predicting diabetic kidney disease (DKD). The test is based on an algorithm that combines the measurement of three plasma protein biomarkers (CD5L, APOA4, and IBP3) with three clinical variables (age, HDL-cholesterol, and eGFR). The initial format of the assay used immunodepletion of plasma samples followed by targeted mass spectrometry (MRM-LCMS). The aim of this study was to convert the existing assay into an immunoaffinity approach compatible with higher throughput and robust clinical application., Methods: A newly optimised immunoaffinity-based assay was developed in a 96 well format with MRM measurements made using a low-flow LCMS method. The stability, reproducibility and precision of the assay was evaluated. A direct comparison between the immunoaffinity method and the original immunodepletion method was conducted on a 100-person cohort. Subsequently, an inter-lab study was performed of the optimised immunoaffinity method in two independent laboratories., Results: Processing of plasma samples was greatly simplified by switching to an immunoaffinity bead capture method, coupled to a faster and more robust microflow LCMS system. Processing time was reduced from seven to two days and the chromatography reduced from 90 to 8 min. Biomarker stability by temperature and time difference treatments passed acceptance criteria. Intra/Inter-day test reproducibility and precision were within 11% CV for all biomarkers. PromarkerD test results from the new immunoaffinity method demonstrated excellent correlation (R = 0.96) to the original immunodepletion method. The immunoaffinity assay was successfully transferred to a second laboratory (R = 0.98) demonstrating the robustness of the methodology and ease of method transfer., Conclusions: An immunoaffinity capture targeted mass spectrometry assay was developed and optimised. It showed statistically comparable results to those obtained from the original immunodepletion method and was also able to provide comparable results when deployed to an independent laboratory. Taking a research grade assay and optimising to a clinical grade workflow provides insights into the future of multiplex biomarker measurement with an immunoaffinity mass spectrometry foundation. In the current format the PromarkerD immunoaffinity assay has the potential to make a significant impact on prediction of diabetic kidney disease with consequent benefit to patients., Competing Interests: Competing interestsSB, JI, TC, ST, KP, RL are employees of Proteomics International, with SB and RL holding shares in the company. SP, OC and HE are employees of Atturos. Proteomics International is a beneficiary of patent PCT/AU2011/001212 that relates to biomarkers described in this manuscript., (© The Author(s) 2020.)

Cancer has no borders: Generation and analysis of molecular data across multiple centers worldwide is necessary to gain statistically significant clinical insights for the benefit of patients. Here we conceived and standardized a proteotype data generation and analysis workflow enabling distributed data generation and evaluated the quantitative data generated across laboratories of the international Cancer Moonshot consortium. Using harmonized mass spectrometry (MS) instrument platforms and standardized data acquisition procedures, we demonstrate robust, sensitive, and reproducible data generation across eleven international sites on seven consecutive days in a 24/7 operation mode. The data presented from the high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) workflow shows that coordinated proteotype data acquisition is feasible from clinical specimens using such standardized strategies. This work paves the way for the distributed multi-omic digitization of large clinical specimen cohorts across multiple sites as a prerequisite for turning molecular precision medicine into reality.

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases that are considered to be on the same disease spectrum because of overlapping genetic, pathological and clinical traits. Changes in serum proteins in FTD and ALS are poorly understood, and currently no definitive biomarkers exist for diagnosing or monitoring disease progression for either disease. Here we applied quantitative discovery proteomics to analyze protein changes in FTD (N = 72) and ALS (N = 28) patient serum compared to controls (N = 22). Twenty three proteins were significantly altered in FTD compared to controls (increased-APOL1, C3, CTSH, EIF5A, MYH2, S100A8, SUSD5, WDR1; decreased-C1S, C7, CILP2, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, IGHV1, ITIH2, PROS1, SHBG, UMOD, VASN) and 14 proteins were significantly altered in ALS compared to controls (increased-APOL1, CKM, CTSH, IGHG1, IGKC, MYH2; decreased-C7, COMP, CRTAC1, EFEMP1, FBLN1, GSN, HSPG2, SHBG). There was substantial overlap in the proteins that were altered in FTD and ALS. These results were validated using western blotting. Gene ontology tools were used to assess functional pathways potentially dysregulated in the two diseases, and calcium ion binding and innate immunity pathways were altered in both diseases. When put together, these results suggest significant overlap in pathophysiological peripheral changes in FTD and ALS. This study represents the first proteomics side-by-side comparison of serum changes in FTD and ALS, providing new insights into under-recognized perturbed pathways and an avenue for biomarker development for FTD and ALS.

Purpose: Abnormalities in lipid metabolism are implicated in age-related macular degeneration (AMD), but the pathways involved remain unclear. We assessed whether acylcarnitine concentrations, a marker of lipid and mitochondrial metabolism, differed between patients with AMD and controls., Methods: In this cross-sectional case-control study, cases (n = 81) had neovascular AMD and controls (n = 79) had cataract with no other ocular pathology. Participants were recruited from eye clinics in Western Sydney, Australia, between 2016 and 2018. Plasma blood samples were collected and liquid chromatography mass spectrometry analyses performed to identify acylcarnitine concentrations. Acylcarnitine levels were adjusted for age, gender and smoking in multivariable models. Confirmation of key acylcarnitine identities was conducted using high mass accuracy liquid chromatography-tandem mass spectrometry., Results: After multivariable adjustment, C2-carnitine (acetylcarnitine) levels were significantly lower in patients with neovascular AMD compared to controls (0.810 ± 0.053 (standard error) compared to 1.060 ± 0.053), p = 0.002). C18:2-DC carnitine (a dicarboxylic acylcarnitine with a 18 carbon side chain and 2 double bonds), levels were significantly higher in patients with neovascular AMD compared to controls (1.244 ± 0.046 compared to 1.013 ± 0.046), p = 0.001). Other acylcarnitines examined were not significantly different between cases and controls., Conclusions: Reduced plasma levels of C2-carnitine (acetylcarnitine) and increased plasma levels of C18:2-DC carnitine were observed in patients with neovascular AMD compared to controls. These findings suggest mitochondrial dysfunction could be involved in the pathogenesis of neovascular AMD.

Disulfide bonds play a key role in stabilizing proteins by cross-linking secondary structures. Whilst many disulfides are effectively unreactive, it is increasingly clear that some disulfides are redox active, participate in enzymatic reactions and/or regulate protein function by allosteric mechanisms. Previously (Karimi et al., Sci. Rep. 2016, 6, 38752) we have shown that some disulfides react rapidly with biological oxidants due to favourable interactions with available lone-pairs of electrons. Here we present data from kinetic, mechanistic and product studies for HOCl-mediated oxidation of a protected nine-amino acid model peptide containing a N- to C-terminal disulfide bond. This peptide reacts with HOCl with k 2 1.8 × 10 6  M -1  s -1 , similar to other highly-reactive disulfide-containing compounds. With low oxidant excesses, oxidation yields multiple oxidation products from the disulfide, with reaction predominating at the N-terminal Cys to give sulfenic, sulfinic and sulfonic acids, and disulfide bond cleavage. Limited oxidation occurs, with higher oxidant excesses, at Trp and His residues to give mono- and di- (for Trp) oxygenated products. Site-specific backbone cleavage also occurs between Arg and Trp, probably via initial side-chain modification. Treatment of the previously-oxidised peptide with thiols (GSH, N-Ac-Cys), results in adduction of the thiol to the oxidised peptide, with this occurring at the original disulfide bond. This gives an open-chain peptide, and a new mixed disulfide containing GSH or N-Ac-Cys as determined by mass spectrometry. Disulfide bond oxidation may therefore markedly alter the structure, activity and function of disulfide-containing proteins, and provides a potential mechanism for protein glutathionylation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The application of proteomic liquid chromatography mass spectrometry (LC-MS) for identifying proteins and peptides associated with human disease is rapidly growing in clinical diagnostics. However, the ability to accurately and consistently detect disease-associated peptides remains clinically uncertain. Variability in diagnostic testing occurs in part due to the absence of appropriate reference testing materials and standardised clinical guidelines for proteomic testing. In addition, multiple proteomic testing pipelines have not been fully assessed through external quality assurance (EQA). This trial was therefore devised to evaluate the performance of a small number of mass spectrometry (MS) testing facilities to (i) evaluate the EQA material for potential usage in a proteomic quality assurance program, and to (ii) identify key problem areas associated with human peptide testing. Five laboratories were sent six peptide reference testing samples formulated to contain a total of 35 peptides in differing ratios of light (natural) to heavy (labelled) peptides. Proficiency assessment of laboratory data used a modified approach to similarity and dissimilarity testing that was based on Bray-Curtis and Sorensen indices. Proficiency EQA concordant consensus values could not be derived from the assessed data since none of the laboratories correctly identified all reference testing peptides in all samples. However, the produced data may be reflective of specific inter-laboratory differences for detecting multiple peptides since no two testing pipelines used were the same for any laboratory. In addition, laboratory feedback indicated that peptide filtering of the reference material was a common key problem area prior to analysis. These data highlight the importance of an EQA programme for identifying underlying testing issues so that improvements can be made and confidence for clinical diagnostic analysis can be attained.

Introduction: German shepherd dogs (GSDs) are a popular breed affected by numerous disorders. Few studies have explored genetic variations that influence canine blood metabolite levels., Objectives: To investigate genetic variants affecting the natural metabolite variation in GSDs., Methods: A total of 82 healthy GSDs were genotyped on the Illumina CanineHD Beadchip, assaying 173,650 markers. For each dog, 74 metabolites were measured through liquid and gas chromatography mass spectrometry (LC-MS and GC-MS) and were used as phenotypes for genome-wide association analyses (GWAS). Sliding window and homozygosity analyses were conducted to fine-map regions of interest, and to identify haplotypes and gene dosage effects., Results: Summary statistics for 74 metabolites in this population of GSDs are reported. Forty-one metabolites had significant associations at a false discovery rate of 0.05. Two associations were located around genes which encode for enzymes for the relevant metabolites: 4-hydroxyproline was significantly associated to D-amino acid oxidase (DAO), and threonine to L-threonine 3-dehydrogenase (LOC477365). Three of the top ten haplotypes associated to 4-hydroxyproline included at least one SNP on DAO. These haplotypes occurred only in dogs with the highest 15 measurements of 4-hydroxyproline, ranging in frequency from 16.67 to 20%. None of the dogs were homozygous for these haplotypes. The top two haplotypes associated to threonine included SNPs on LOC477365 and were also overrepresented in dogs with the highest 15 measurements of threonine. These haplotypes occurred at a frequency of 90%, with 80% of these dogs homozygous for the haplotypes. In dogs with the lowest 15 measurements of threonine, the haplotypes occurred at a frequency of 26.67% and 0% homozygosity., Conclusion: DAO and LOC477365 were identified as candidate genes affecting the natural plasma concentration of 4-hydroxyproline and threonine, respectively. Further investigations are needed to validate the effects of the variants on these genes.

Limiting contamination of liquid chromatography/tandem mass spectrometry (LC-MS) systems and reducing the downtime associated with maintenance and cleaning is essential for productivity. We developed a simple device that creates a gas curtain barrier to prevent ions from entering the MS inlet. The gas can be quickly and easily applied when certain contaminant ions are known to elute. We show that the device can prevent the buildup of contaminants on the heated transfer capillary following >100 injections of a crude tissue lysate and improves peptide identifications. The device may provide a promising approach toward improving instrument robustness.

The increasing role played by liquid chromatography-mass spectrometry (LC-MS)-based proteomics in biological discovery has led to a growing need for quality control (QC) on the LC-MS systems. While numerous quality control tools have been developed to track the performance of LC-MS systems based on a pre-defined set of performance factors (e.g., mass error, retention time), the precise influence and contribution of the performance factors and their generalization property to different biological samples are not as well characterized. Here, a web-based application (QCMAP) is developed for interactive diagnosis and prediction of the performance of LC-MS systems across different biological sample types. Leveraging on a standardized HeLa cell sample run as QC within a multi-user facility, predictive models are trained on a panel of commonly used performance factors to pinpoint the precise conditions to a (un)satisfactory performance in three LC-MS systems. It is demonstrated that the learned model can be applied to predict LC-MS system performance for brain samples generated from an independent study. By compiling these predictive models into our web-application, QCMAP allows users to benchmark the performance of their LC-MS systems using their own samples and identify key factors for instrument optimization. QCMAP is freely available from: http://shiny.maths.usyd.edu.au/QCMAP/., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.