Orbitrap Fusion Tribrid Mass Spectrometer Research Articles

Impact of a teat disinfectant based on Lactococcus cremoris on the cow milk proteome

BackgroundDairy cow milking practices require cleaning and disinfection of the teat skin before and after milking to ensure the safety and quality of milk and prevent intramammary infections. Antimicrobial proteins of natural origin can be valuable alternatives to traditional disinfectants. In a recent field trial, we demonstrated that a teat dip based on a nisin A-producing Lactococcus cremoris (L) had comparable efficacy to conventional iodophor dip (C) in preventing dairy cow mastitis. Here, we present the differential shotgun proteomics investigation of the milk collected during the trial.MethodsFour groups of quarter milk samples with low (LSCC) and high somatic cell count (HSCC) collected at the beginning (T0) and end (TF) of the trial were analyzed for a total of 28 LSCC (14 LSCC T0 and 14 LSCC TF) and 12 HSCC (6 HSCC T0 and 6 HSCC TF) samples. Milk proteins were digested into peptides, separated by nanoHPLC, and analyzed by tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Tribrid mass spectrometer. The proteins were identified with MaxQuant and interaction networks of the differential proteins were investigated with STRING. The proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD045030.ResultsIn healthy milk (LSCC), we detected 90 and 80 differential proteins at T0 and TF, respectively. At TF, the Lactococcus group showed higher levels of antimicrobial proteins. In mastitis milk (HSCC), we detected 88 and 106 differential proteins at T0 and TF, respectively. In the Lactococcus group, 14 proteins with antimicrobial and immune defense functions were enriched at TF vs. 4 proteins at T0. Cathelicidins were among the most relevant enriched proteins. Western immunoblotting validation confirmed the differential abundance.ConclusionsAt T0, the proteomic differences observed in healthy milk of the two groups were most likely dependent on physiological variation. On the other hand, antimicrobial and immune defense functions were higher in the milk of cows with mammary gland inflammation of the Lactococcus-treated group. Among other factors, the immunostimulatory action of nisin A might be considered as a contributor.

Toward a More Comprehensive Approach for Dissolved Organic Matter Chemical Characterization Using an Orbitrap Fusion Tribrid Mass Spectrometer Coupled with Ion and Liquid Chromatography Techniques.

Dissolved organic matter (DOM) represents one of the largest active organic carbon pools in the global carbon cycle. Although extensively studied, only <10% of DOM has been chemically characterized into individual dissolved compounds due to its molecular complexity. This study introduced a more comprehensive DOM characterization method by coupling both ion chromatography (IC) and liquid chromatography (LC) with high mass accuracy and resolution mass spectrometry. We presented a new on-the-fly mass calibration of the Orbitrap technique by utilizing the "lock mass" function in the Orbitrap Fusion Tribrid mass spectrometer (OT-FTMS), which assures high mass accuracy at every scan by a postcolumn introduction of internal labeled standards. With both IC and LC, tested unlabeled standards of amino acids, small peptides, and organic acids were consistently below 1.0 ppm mass error, giving the OT-FTMS the potential of reaching mass accuracy of the Fourier-transform ion cyclotron resonance mass spectrometer. In addition to mass accuracy, a pooled quality control sample (QC) was used to increase reproducibility by applying systematic error removal using random forest (SERRF). Using an untargeted mass spectrometry approach, estuarine DOM samples were analyzed by OT-FTMS coupled to IC in negative mode and LC in positive mode detection to cover a wide range of highly cationic to highly anionic molecules. As a proof of concept, we focused on elucidating the structures of three distinct DOM compound classes with varied acidities and basicities. In UPLC-OT-FTMS, a total of 915 compounds were detected. We putatively elucidated 44 small peptides and 33 deaminated peptides of these compounds. With IC-OT-FTMS, a total of 1432 compounds were detected. We putatively elucidated 20 peptides, 268 deaminated peptides, and 188 organic acids. Except for five compounds, all putatively elucidated compounds were uniquely detected in their corresponding chromatography technique. These results highlight the need for combining these two techniques to provide a more comprehensive method for DOM characterization. Application of the combined IC and LC techniques is not limited to DOM chemical characterization. It can analyze other complex compound mixtures, such as metabolites, and anthropogenic pollutants, such as pesticides and endocrine-disrupting chemicals, in environmental and biological samples.

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1μl of 100nmol/l amino acids comparable to a mere 1 [Formula: see text] 104-105 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the occurrence of both the Wood-Ljungdahl pathway and an incomplete reductive tricarboxylic acid cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acids, genomic information, and public databases. Among them, the presence of alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS can be considered the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to any kind of isolated microbe.

#6225 MICROPLASTICS: FIRST EXTENDED PROTEOMIC ANALYSIS ON KIDNEY TUBULAR CELLS

Abstract Background and Aims Several studies have shown the ubiquitous presence of microplastics (plastic fragments between 5 mm and 1 μm in diameter) and nanoplastics (&amp;lt;1 μm) in the environment and their toxicity. Furthermore, microplastics (MPs) absorb a lot of environmental pollutants, such as bisphenol A (BPA), and release them into tissues increasing their toxicity; this is the so called “Trojan Horse” effect. Recent studies have also proved their presence in human blood and human tissues of healthy people, such as placenta and lung, and in cirrhotic liver. Most frequent biological effects of MPs are inflammation, oxidative stress and alteration of metabolic pathways. In this study proteomic analysis was performed to evaluate the toxicity of polyethylene (PE) and bisphenol-A (BPA) MPs on renal tubular cells (HK2) in vitro. This is the first extended proteomic study on human cells. Method HK-2 cultures were exposed for 5-24-48 hours to BPA, PE Microspheres (PE-MP) and MP combined with BPA. Then it was performed a proteomic analysis by mass spectrometry (MS). MS data were obtain by Orbitrap Fusion Tribrid mass spectrometer (ThermoScientific). Analysis of data were performed using unsupervised hierarchical clustering using multidimensional scaling, non-linear support vector machine (SVM) learning, and partial least squares discriminant analysis. In SVM learning, a fourfold cross-validation approach was applied to estimate the prediction and classification accuracy. Results The proteomic analysis showed a clear differentiation of the HK2 proteome based on conditioning and identified a “core” of proteins, significant at ANOVA and above the 95th percentile for “fold increase” and significant at T-test compared with controls, highly discriminatory between groups. Finally, among these, a final set of 6 proteins was selected to be validated for distinguishing features: Nephronectin, GDF15, Vasorin, IGFBP7, Midkine, Tissue factor-F3. Nephronectin is a structural membrane's protein involved in cellular adhesion. GDF15, tissue factor F3 and midkine are markers of stress conditions, including inflammation and oxidative stress. IGFBP7 is a biomarker of acute kidney damage. Vasorin is a transmembrane glycoprotein that protects against apoptosis and fibrosis. Conclusion MP and BPA significantly modify the protein expression in renal tubular cells. These findings highlight the urgent need for additional research into the toxic effects of plastic debris on human kidneys and the eventual link to kidney diseases.

Integrated proteomics and phosphoproteomics profiling reveals the cardioprotective mechanism of bioactive compounds derived from Salvia miltiorrhiza Burge

BackgroundNatural products are an important source for discovering novel drugs due to their various pharmacological activities. Salvia miltiorrhiza Burge (Danshen) has been shown to have promising therapeutic potential in the management of heart diseases, making it a candidate for cardiovascular drug discovery. Currently, there is limited quantitative analysis of the phosphorylation levels of Danshen-derived natural products on a proteome-wide, which may bias the study of their mechanisms of action. PurposeThis study aimed to evaluate the global signaling perturbation induced by Danshen-derived bioactive compounds and their potential relationship with myocardial ischemia/reperfusion (IR) injury therapy. Study designWe employed quantitative proteome and phosphoproteome analysis to identify dysregulated signaling in IR injury hearts from mice. We compared changes induced by Danshen-derived compounds based on IR-associated phospho-events, using an integrative approach that maps relative abundance of proteins and phosphorylation sites. MethodsIsobaric chemical tandem mass tags (TMT) labeled multiplexing strategy was used to generate unbiased quantitative proteomics and phosphoproteomics data. Highly accurate and precise TMT quantitation was performed using the Orbitrap Fusion Tribrid Mass Spectrometer with synchronous precursor selection MS3 detection mode. Mass spectrometric raw files were analyzed with MaxQuant (2.0.1.0) and statistical and bioinformatics analysis was conducted with Perseus (1.6.15). ResultsWe quantified 3661 proteins and over 11,000 phosphosites in impaired heart tissue of the IR mice model, expanding our knowledge of signaling pathways and other biological processes disrupted in IR injury. Next, 1548 and 5545 differently expressed proteins and phosphosites were identified by quantifying the proteome and phosphoproteome of H9c2 cells treated by five Danshen bioactive compounds respectively. Results revealed the vast differences in abilities of five Danshen-derived bioactive compounds to regulate phosphorylation modifications in cardiomyocytes, with dihydrotanshinone I (DHT) showing potential for protecting against IR injury by modulating the AMPK/mTOR signaling pathway. ConclusionsThis study provides a new strategy for analyzing drug/natural product-regulated phosphorylation modification levels on a proteome-wide scale, leading to a better understanding of cell signaling pathways and downstream phenotypic responses.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: Classic Hodgkin lymphoma (cHL) is a highly curable disease, even in advanced stages. It is controversial whether bone involvement has a negative effect on overall- and progression-free survival in patients treated with intensive chemotherapy. cHL is characterized by a unique tumor microenvironment (TME) consisting of few, scattered neoplastic Hodgkin and Reed-Sternberg (HRS) cells, embedded in an abundant background of reactive immune and stromal cells. When cHL disseminates, the new disease sites also contain both HRS cells and TME cells. Whether cases that present with bone lesions, harbour specific TME features is unknown. We investigated protein expression in lymph node biopsies from cHL patients with and without skeletal involvement at diagnosis, using nano liquid chromatography—tandem mass spectrometry (nLC-MS/MS), to identify potential markers of skeletal disease. Methods: Protein expression patterns in pre-therapeutic formalin-fixed, paraffin-embedded lymphoma samples from 69 cHL patients diagnosed at Aarhus University Hospital, Denmark between 2009 and 2018 were analysed by nLC-MS/MS. Patients were grouped according to diagnostic 18F-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET)/computed tomography (CT). FDG PET/CT scans were reviewed specifically for bone involvement. The study cohort included 50 patients with nodal cHL (n-cHL) and no skeletal involvement, and 19 patients with both skeletal and nodal disease (s-cHL). Results: We identified 2,298 proteins; comparison of the protein profiles between the s-cHL and n-cHL groups revealed 220 unique proteins significantly differentially expressed (p < 0.05) and with a fold change of at least 25%. Of these, 117 proteins were upregulated (fold change 1.25–3.94), and 103 were downregulated (fold change 0.44–0.80) in s-cHL. In hierarchal clustering based on an even higher significance threshold (p < 0.001), i.e., 25 significantly differentially expressed proteins, two clusters were observed: (i) a skeletal-group comprising 12 s-cHL and 4 n-cHL samples; and (ii) a nodal-group of 46 n-cHL and 7 s-cHL samples. Of particular interest among the differentially expressed proteins, we identified isocitrate dehydrogenase (IDH1) and WD repeat- and FYVE domain-containing protein 4 (WDFY4). This pattern of protein expression suggests disturbance in cytoplasmic NADPH production, antigen processing/presentation, and B-cell survival in cHL patients with skeletal involvement compared to those without. Conclusion: Our data show differential protein expression in cHL lymphoma tissues that correlate with the presence or absence of concomitant bone involvement at diagnosis. This indicates that certain biological pathways are affected among those presenting with disease disseminated to the skeletal system. The research was funded by: Department of Clinical Medicine, Aarhus University; Health Research Foundation of Central Denmark Region; Brødrene Hartmanns Foundation; Carl and Ellen Hertz Foundation; the Danish Lymphoma Group; Kong Christian den Tiendes Foundation; Torben and Alice Frimodts Foundation; Aase and Ejnar Danielsens Foundation; Foundation of 1870; Dagmar Marshalls Foundation; Merchant Einar Willumsen’s Memorial Foundation; Eva and Henry Frænkel’s Memorial Foundation; Master Carpenter Jørgen Holm and wife Elisa f. Hansen’s Memorial Grant. The Orbitrap Fusion Tribrid mass spectrometer was funded by A. P. Møller og Hustru Chastine Mc-Kinney Møllers Fond til Almene Formaal. Keywords: Genomics, Epigenomics, and Other -Omics, Hodgkin lymphoma, Microenvironment No conflicts of interests pertinent to the abstract.

Label-Free Proteomics of Oral Mucosa Tissue to Identify Potential Biomarkers That Can Flag Predilection of Precancerous Lesions to Oral Cell Carcinoma: A Preliminary Study.

Oral squamous cell carcinomas are mostly preceded by precancerous lesions such as leukoplakia and erythroplakia. Our study is aimed at identifying potential biomarker proteins in precancerous lesions of leukoplakia and erythroplakia that can flag their transformation to oral cancer. Four biological replicate samples from clinical phenotypes of healthy control, leukoplakia, erythroplakia, and oral carcinoma were annotated based on clinical screening and histopathological evaluation of buccal mucosa tissue. Differentially expressed proteins were delineated using a label-free quantitative proteomic experiment done on an Orbitrap Fusion Tribrid mass spectrometer in three technical replicate sets of samples. Raw files were processed using MaxQuant version 2.0.1.0, and downstream analysis was done via Perseus version 1.6.15.0. Validation included functional annotation based on biological processes and pathways using the ClueGO plug-in of Cytoscape. Hierarchical clustering and principal component analysis were performed using the ClustVis tool. Across control, leukoplakia, and cancer, L-lactate dehydrogenase A chain, plectin, and WD repeat-containing protein 1 were upregulated, whereas thioredoxin 1 and spectrin alpha chain, nonerythrocytic 1 were downregulated. Across control, erythroplakia, and cancer, L-lactate dehydrogenase A chain was upregulated whereas aldehyde dehydrogenase 2, peroxiredoxin 1, heat shock 70 kDa protein 1B, and spectrin alpha chain, nonerythrocytic 1 were downregulated. We found that proteins involved in leukoplakia were associated with alteration in cytoskeletal disruption and glycolysis, while in erythroplakia, they were associated with alteration in response to oxidative stress and glycolysis across phenotypes. Hierarchical clustering subgrouped half of precancerous samples under the main branch of the control and the remaining half under carcinoma. Similarly, principal component analysis identified segregated clusters of control, precancerous lesions, and cancer, but erythroplakia phenotypes, in particular, overlapped more with the cancer cluster. Qualitative and quantitative protein signatures across control, precancer, and cancer phenotypes explain possible functional outcomes that dictate malignant transformation to oral carcinoma.

Rapid separation and identification of 96 main constituents in Huanglian Jiedu decoction via ultra-high performance liquid chromatography-Orbitrap Fusion Tribrid mass spectrometer.

Huanglian Jiedu decoction is a widely used traditional Chinese medicine with a broad spectrum of therapeutic effects, including heat clearing, detoxification, and attenuation of inflammation. However, the composition of Huanglian Jiedu decoction is still unclear due to its complexity and limitations of analytical methods. In this study, we established a fast and reliable analytical method based on ultra-performance LC-Orbitrap Fusion Tribrid mass spectrometer for high-speed separation and structural identification of multiple compounds in Huanglian Jiedu decoction. The analysis was carried out using a Hypersil GOLD C18 column (2.1 × 100 mm, 1.9μm) with gradient elution coupled to a high-definition mass spectrometer system operating in both positive and negative ESI modes. According to the chromatographic retention time, precise molecular weight, fragment ion peaks, and published data, the main chromatographic peaks were attributed to specific molecules whose chemical structures were determined. In total, 96 components were identified, including 34 flavonoids and their glycosides, 23 alkaloids, 18 organic acids, 13 terpenoids, and 8 miscellaneous compounds. This study revealed the detailed chemical composition of Huanglian Jiedu decoction, which is of great importance for quality control and further pharmacological and mechanistic studies.

An insight in proteome profiling of Tuta absoluta larvae after entomopathogenic fungal infection

Tuta absoluta (L.) (Lepidoptera: Gelechiidae), a major pest of solanaceous plant species, causes serious losses in the agriculture sector around the globe. For better pest management, entomopathogenic fungi such as Beauveria bassiana and Purpureocillium lilacinum, play an efficient role in suppressing the pest population. The present study was carried out to analyse the effects post fungal infections through proteome profiling using an Orbitrap Fusion Tribrid mass spectrometer. A total of 2,201 proteins were identified from the fourth instar larvae of T. absoluta, of which 442 and 423 proteins were significantly dysregulated upon infection with P. lilacinum and B. bassiana respectively. The potential proteins related to immune systems as well as detoxification processes showed significant alterations after post fungal infection. Studies on T. absoluta proteomics and genomics as well as the consequences of entomopathogenic fungal infection on the immune response of this insect could provide an initial framework for exploring more fungus-host interactions for the development of better strategies for integrated pest management.

Phosphoprotein network analysis of corneal epithelium of keratoconus patients.

Keratoconus (KC) is non-inflammatory, bilateral progressive corneal ectasia, and a disease of established biomechanical instability. The etiology of KC is believed to be multifactorial. Although previous studies gained insight into the understanding of the disease, little is known thus far on global protein phosphorylation changes in keratoconus. We performed phosphoproteome analysis of corneal epithelium from control (N=5) and KC patients. Tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography (IMAC) were used for the phosphopeptides enrichment and quantitation. Enriched peptides were analyzed on Orbitrap Fusion Tribrid mass spectrometer. This leads to the identification of 2939 unique phosphopeptides derived from 1270 proteins. We observed significant differential phosphorylation of 591 phosphopeptides corresponding to 375 proteins. Our results provide first phosphoproteomic signature of the keratoconus disease and identified dysregulated signaling pathways that can be targeted for therapy in future studies.

Comparison of Different Mass Spectrometry Workflows for the Proteomic Analysis of Tear Fluid.

The tear film is a multi-layer fluid that covers the corneal and conjunctival epithelia of the eye and provides lubrication, nutrients, and protection from the outside environment. Tear fluid contains a high concentration of proteins and has thus been recognized as a potential source of biomarkers for ocular disorders due to its proximity to disease sites on the ocular surface and the non-invasive nature of its collection. This is particularly true in the case of dry eye disease, which directly impacts the tear film and its components. Proteomic analysis of tear fluid is challenging mainly due to the wide dynamic range of proteins and the small sample volumes. However, recent advancements in mass spectrometry have revolutionized the field of proteomics enabling unprecedented depth, speed, and accuracy, even with small sample volumes. In this study using the Orbitrap Fusion Tribrid mass spectrometer, we compared four different mass spectrometry workflows for the proteomic analysis of tear fluid collected via Schirmer strips. We were able to establish a method of in-strip protein digestion that identified >3000 proteins in human tear samples from 11 healthy subjects. Our method offers a significant improvement in the number of proteins identified compared to previously reported methods without pooling samples.

Characterisation of the Tumour Proteome in Primary Extramedullary Multiple Myeloma Identifies Key Proteins Associated with Transendothelial Migration

Introduction: Extramedullary multiple myeloma (EMM) refers to the spread of clonal plasma cells to tissues extending outside of the bone marrow microenvironment. EMM is present at the time of diagnosis in 6-10% of patients, however, this increases to 13-26% in patients with disease progression and relapse. Cancer cells are suspected to spread to new tissues and organs via the circulatory system as a result of molecular changes that allow malignant cells to escape the bone marrow (BM). For example, downregulation of CXCR4 (C-X-C Motif Chemokine Receptor 4), an important factor in cellular homing to the BM, has frequently been reported to be linked to the EMM phenotype. In addition, the majority of patients presenting with EMM have highly complex cytogenetic abnormalities and high-risk cytogenetic markers such as t(14;16). As EMM is an indicator of a more aggressive disease, more intensive treatment, including combination chemotherapy, is often recommended. Many of the underlying molecular mechanisms accompanying EMM are yet to be characterised. Our mass-spectrometry (MS)-based proteomic study provides insight into the unique molecular mechanisms associated with EMM, identifying key proteins linked to the progression of medullary multiple myeloma (MM) to EMM.Methods: Label-free liquid chromatography mass spectrometric analysis of age and gender matched medullary MM (n=8) and EMM (n=9) bone-marrow derived mononuclear cells (MNCs) was carried out using a Thermo Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). Proteome Discoverer 2.2 using Sequest HT (Thermo Fisher Scientific) and a percolator were employed for the identification of peptides and proteins. For protein identification, the following search parameters were used: (i) 0.02 Da for MS/MS mass tolerance, (ii) 10 ppm for peptide mass tolerance, (iii) variable modification settings for methionine oxidation, (iv) fixed modification settings in relation to carbamido-methylation and (v) tolerance for up to two missed cleavages. Peptide probability was set to high confidence. Datasets were imported into Progenesis QI (version 2.0) software for further analysis. Data was filtered based on an ANOVA p-value of ≤0.05, fold change >1.5 between experimental groups, and proteins with ≥1 unique peptides contributing to the identification. Proteins with less than 70% valid values were removed from the analysis. G:profiler and STRING were utilized for functional enrichment and the characterisation of protein interaction patterns.Results: Our quantitative MS-based proteomic analysis identified a total of 492 proteins with significantly altered abundances between EMM and MM bone marrow MNC. Of these significant proteins, 275 were found to be increased in EMM compared to medullary MM and 217 were found to be decreased in EMM compared to medullary MM. Hierarchical clustering was performed to highlight the proteomic profile associated with extramedullary disease (Figure 1A). KEGG pathway analysis and gene ontology (GO) analysis of proteins found to be increased in EMM indicated an increase in proteins associated with cell adhesion, invasion, and migration pathways (Figure 1B). Interestingly, several proteins involved in leukocyte transendothelial migration were significantly increased in EMM indicating their potential involvement in the dissemination of MM cells from the bone marrow microenvironment to distal tissues (Figure 1C). Among the proteins found to be involved in this biological pathway was junctional adhesion molecule-A (F11R), a protein previously reported to play a role in EMD pathophysiology [1]. Other proteins involved in MM invasion and migration including Rho-associated protein kinase 2 (ROCK2), Ras-related C3 botulinum toxin substrate 1 (Rac1) and platelet endothelial cell adhesion molecule (PECAM-1) were significantly increased in EMM.Conclusion: Using high-resolution mass spectrometry to characterise the tumour proteome of MM patients with extramedullary disease, we have identified a significant increase in the abundance of proteins associated with leukocyte transendothelial invasion in primary EMM samples. Our study provides further insight into the molecular mechanisms within EMM and thus holds potential to enhance current efforts to provide a more personalised therapeutic approach for EMM patients.

Suspect screening and targeted analysis of acyl coenzyme A thioesters in bacterial cultures using a high-resolution tribrid mass spectrometer

Analysis of acyl coenzyme A thioesters (acyl-CoAs) is crucial in the investigation of a wide range of biochemical reactions and paves the way to fully understand the concerned metabolic pathways and their superimposed networks. We developed two methods for suspect screening of acyl-CoAs in bacterial cultures using a high-resolution Orbitrap Fusion tribrid mass spectrometer. The methods rely on specific fragmentation patterns of the target compounds, which originate from the coenzyme A moiety. They make use of the formation of the adenosine 3′,5′-diphosphate key fragment (m/z 428.0365) and the neutral loss of the adenosine 3′-phosphate-5′-diphosphate moiety (506.9952) as preselection criteria for the detection of acyl-CoAs. These characteristic ions are generated either by an optimised in-source fragmentation in a full scan Orbitrap measurement or by optimised HCD fragmentation. Additionally, five different filters are included in the design of method. Finally, data-dependent MS/MS experiments on specifically preselected precursor ions are performed. The utility of the methods is demonstrated by analysing cultures of the denitrifying betaproteobacterium “Aromatoleum” sp. strain HxN1 anaerobically grown with hexanoate. We detected 35 acyl-CoAs in total and identified 24 of them by comparison with reference standards, including all 9 acyl-CoA intermediates expected to occur in the degradation pathway of hexanoate. The identification of additional acyl-CoAs provides insight into further metabolic processes occurring in this bacterium. The sensitivity of the method described allows detecting acyl-CoAs present in biological samples in highly variable abundances.Graphical abstract

Multiomic Profiling of Iron Deficient Infant Monkeys Detects Biochemical Signatures of Inflammatory and Neural Dysfunction in Blood and CSF Prior to Anemia

Background: Pre-anemic iron deficiency (ID) and ID anemia in infancy is associated with long-term effects on brain development. Iron is an essential micronutrient needed for multiple neurodevelopmental processes, including energy production, myelination and neurotransmission. Serum and cerebrospinal fluid (CSF) metabolomic and proteomic analyses can potentially characterize the metabolic and protein signatures of aberrant iron-dependent brain development pathways. A better understanding of the timing and course of the biochemical signatures of ID-induced dysfunction in the heme and intrathecal compartments may help with early diagnosis and treatment of iron deficiency prior to onset of anemia. Objective: To determine proteomic and metabolomic signatures of ID-induced dysfunction in serum and CSF of infant monkeys prior to the onset of iron deficiency anemia (IDA). Methods: Hematological parameters were determined in rhesus monkey infants (Macaca mulatta) at 4 and 6 months of age as they progressed from preanemic ID to IDA (ID group; n=6) or remained iron-sufficient(IS group; n=12). Blood and CSF were collected at 4 and 6 months and metabolically profiled using HPLC/MS with isobaric standards for identification and quantification. Proteomic quantification utilized a custom TMT-labeled workflow with an Orbitrap Fusion Tribrid mass spectrometer. Serum and CSF metabolomic and proteomic profiles of the ID and IS groups were compared, along with a a third group of infants that became IDA earlier at 4 months of age (IDA group; n=3). Results: Evidence of pre-anemic ID was apparent in some infant at 4 months of age with clinical IDA evident at 6 months A (Hgb &amp;lt;10 g/dL and MCV &amp;lt;60 fL). A total of 1,128 CSF proteins and 251 serum proteins were identified and quantified. Over 4,000 metabolite features were observed in serum and CSF across three modalities including reverse phase positive, reverse phase negative and HILIC mode. Pathway analyses provided evidence of increased inflammatory activity, impacts on neural pathways, and bioindicators of acute phase responses in ID and IDA groups. Signs of these metabolic changes were already present in the preanemic ID? period (4 months). Conclusions: Biomarkers indicating indicative of increased inflammatory activity with downstream effects on pathways involved in normal neural function were detectable in both the CSF and serum of ID monkeys prior to the onset of clinical anemia. Brain-specific proteomic and metabolomic profiling of novel analytes in circulation can facilitate earlier diagnosis and treatment of infantile ID to reduce neurodevelopmental risk. Funded by NIH/NICHD Grant HD089989, this abstract is sponsored by ASH member Zohar Sachs MD, PhD Disclosures No relevant conflicts of interest to declare.

Quantitative proteomic analysis of cerebrospinal fluid of women newly diagnosed with multiple sclerosis

Purpose The lack of reliable diagnostic and/or prognostic biomarkers for multiple sclerosis (MS) is the major obstacle to timely and accurate patient diagnosis in MS patients. To identify new proteins associated with MS we performed a detailed proteomic analysis of cerebrospinal fluid (CSF) of patients newly diagnosed with relapsing-remitting MS (RRMS) and healthy controls. Material Reflecting significantly higher prevalence of MS in women we included only women patients and controls in the study. To eliminate a potential effect of therapy on the CSF composition, only the therapy-naïve patients were included. Methods Pooled CSF samples were processed in a technical duplicate, and labeled with stable-isotope coded TMT tags. To maximize the proteome coverage, peptide fractionation using 2D-LC preceded mass analysis using Orbitrap Fusion Tribrid Mass Spectrometer. Differential concentration of selected identified proteins between patients and controls was verified using specific antibodies. Results Of the identified 900 CSF proteins, we found 69 proteins to be differentially abundant between patients and controls. In addition to several proteins identified as differentially abundant in MS patients previously, we observed several linked to MS for the first time, namely eosinophil-derived neurotoxin and Nogo receptor. Conclusions Our data confirm differential abundance of several previously proposed protein markers, and provide indirect support for involvement of copper–iron disbalance in MS. Most importantly, we identified two new differentially abundant CSF proteins that seem to be directly connected with myelin loss and axonal damage via TLR2 signaling and Nogo-receptor pathway in women newly diagnosed with RRMS.

Metabolomic analysis of primary human skeletal muscle cells during myogenic progression

Skeletal muscle constitutes more than 30% of total body mass using substrates such as glycogen, glucose, free fatty acids, and creatinine phosphate to generate energy. Consequently, multinucleated myofibers and resident mononucleated stem cells (satellite cells) generate several metabolites, which enter into circulation affecting the function of other organs, especially during exercise and atrophy. The present study was aimed at building a comprehensive profile of metabolites in primary human skeletal muscle cells during myogenic progression in an untargeted metabolomics approach using a high resolution Orbitrap Fusion Tribrid Mass Spectrometer. Identification of metabolites with multivariate statistical analyses showed a global shift in metabolomic profiles between myoblasts undergoing proliferation and differentiation along with distinctly separable profiles between early and late differentiating cultures. Pathway analyses of 71 unique metabolites revealed that Pantothenate metabolism and Coenzyme A biosynthesis and Arginine Proline metabolism play dominant roles in proliferating myoblasts, while metabolites involved in vitamin B6, Glyoxylate and Dicarboxylate, Nitrogen, Glutathione, and Tryptophan metabolism were upregulated during differentiation. We found that early and late differentiating cultures displayed differences in Phenylalanine, Tyrosine, Glycine, Serine and Threonine metabolism. Our results identify metabolites during maturation of muscle from progenitor myoblasts that have implications in muscle regeneration and pathophysiology.

Occurrence of short- and medium-chain chlorinated paraffins in soils and sediments from Dongguan City, South China

As a group of emerging organic pollutants, chlorinated paraffins (CPs) have attracted rising global attention due to their persistence and toxicity. In this study, we have investigated the concentration levels and profiles of short-chain chlorinated paraffins (SCCPs) and medium-chain chlorinated paraffins (MCCPs) in soils and sediments from Dongguan City, an industrial area in South China, and have also screened very short-chain chlorinated paraffins (vSCCPs) by means of ultra-high resolution liquid chromatograph coupled with an Orbitrap Fusion Tribrid mass spectrometer. The results indicated that total SCCP concentrations ranged from 6.75 to 993 ng/g (mean 172 ng/g) in soils and from 4.00 to 613 ng/g (mean 153 ng/g) in sediments, respectively. Higher MCCP levels were observed with a range of 23.9–2427 ng/g (mean 369 ng/g) in soils and 14.0–1581 ng/g (mean 493 ng/g) in sediments, respectively. The results indicated that MCCPs dominated over SCCPs in the studied region. The dominant homologues in soils and sediments were C13Cl6–7 and C14Cl7–8, C13Cl7, and C14Cl7–8, respectively. Furthermore, six vSCCP homologues (C8Cl7–8 and C9Cl5–8) in soils and four vSCCPs (C9Cl5–8) in sediments have been identified. Because of their higher detection frequencies, further studies should focus on the transformation mechanisms and toxicities of these vSCCPs in environmental media and biota.

Quantitative Proteomics in Diffuse Large B-Cell Lymphoma Patients Reveal Novel Overexpressed Proteins and Potentially Druggable Targets in the ABC Subtype

Background: The cell-of-origin (COO) concept, based on gene expression profiling (GEP), dividing diffuse large B-cell lymphoma (DLBCL) patients into germinal center B cell (GCB) or activated B cell (ABC) subtypes, is a well-established subclassification where ABC patients have an inferior survival. The hallmark the ABC-type is constitutive activation of nuclear factor kappa B (NF-κB), often due to mutations in the B-cell receptor (BCR) signaling pathway. This has been the underlying rationale for adding newer drugs, such as bortezomib, ibrutinib or lenalidomide, to R-CHOP for ABC patients. However, none of these combinations studied in phase III trials have shown any clinical benefit. So, the complexity of ABC DLBCL is probably not only explained by genetic alterations and transcriptional changes as gene expression not necessarily correlate with protein expression, and protein action and dynamics are not caught by genomics-based techniques. Instead, using methods to measure global protein expression and interactions could offer new insights into the ABC subtype and possibly aid in the identification of novel drug targets. Aim: To study possible differences in global protein expression between ABC and GCB DLBCL subtypes using quantitative proteomics. Patients: A total of 213 adult DLBCL patients in western Sweden diagnosed between 1/1 2004 and 31/12 2016, were included. All patients received immunochemotherapy (R-CHOP). Primary mediastinal large B-cell lymphoma, primary CNS lymphoma, HIV-related lymphoma and transformed lymphoma were excluded. From archived formalin-fixed, paraffin-embedded (FFPE) tissue sections, from the time of diagnosis, a core biopsy (1 mm diameter) were obtained from each patient sample. Methods: COO was determined using the Hans immunohistochemistry algorithm. For 92 of the 213 patients, COO was also determined using the gene expression Lymph2cx chip: 14% changed subtype group from either non-GCB to GCB (n=8), GCB to ABC (n=4) or GCB to unclassified (n=1). From the FFPE samples a proteomic analysis was performed. In short, peptides were labelled using tandem mass tag (TMT) according to the manufacturer instructions and samples were analysed on an Orbitrap Fusion Tribrid mass spectrometer. The data files were merged for identification and relative quantification using Proteome Discoverer version 1.4.The search used the Human Swissprot Database version August 2016 using Mascot 2.3 as a search engine. The differentially expressed proteins were analysed using STRING version 10.0, for pathway analysis we used the Reactome database resource, and for potentially druggable proteins we used the Human Protein Atlas website which holds protein information of the current FDA approved drugs directed to 672 separate human proteins. Results: In all, 3078 proteins could be identified in all patients and 793 proteins were differentially expressed (p&lt;0.05 adjusted for mass significance according to Benjamin-Hochberg) between ABC and GCB patients. Of these, 410 proteins were overexpressed in the ABC group. Among the most expressed proteins were several well-known ABC-associated proteins (such as IRF4/MUM1, HSP90B1, CCDC50 and STAT3) in addition to a large number of proteins previously not described in ABC DLBCL, e.g. neudesin, BLNK, MPST, BPGM, SUB1, SP140, PCK2, PARP4, SRP54, SRP68, SRP72, TRPV2, IGF2R and FGD2. A majority of the 410 proteins were closely linked with an enrichment p-value &lt; 1x10-16(Fig. 1) and the most enriched pathways were immune system (FDR rate 3.3 x 10-27), interferon signaling (2.9 x 10-7), antigen processing (8.8 x 10-7) and down-modulation of cell surface receptors (4.7 x 10-5). Most interestingly, we also found that 16 proteins overexpressed in the ABC group could be potential drug targets for an FDA approved drug, e.g. high affinity immunoglobulin gamma Fc receptor I, CD47, HDAC2, ELANE and carbonic anhydrase 1. Conclusions: In this large proteomic study we found a number of overexpressed proteins in the ABC subtype, previously not described in DLBCL. Even though functional studies aimed at individual proteins and protein interactions to evaluate potential clinical effect are needed, our findings reveal novel proteins that could be potential druggable targets in ABC DLBCL patients. Figure 1 Disclosures Enblad: Kite/Gilead: Membership on an entity's Board of Directors or advisory committees. Mellqvist:Amgen, Janssen, Oncopeptides, Sanofi, Sandoz, Takeda: Honoraria. Andersson:Abbvie and Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead, Janssen and Roche: Consultancy; Gilead: Research Funding.

Abstract 603: Signatures of chemoprotection from ambient exposure to outdoor air pollution in the human serum albumin Cys34 adductome

Abstract Many components of outdoor air pollution, such as benzene, are known to be carcinogenic to humans. However, due to spatial and temporal variation in air pollution, ambient measures of single pollutant exposures may be limited for generalizing across diverse settings. Covalent adducts of air pollutants with the free thiol at the Cys34 residue of human serum albumin represent biomarkers of recent air pollution exposure (&amp;lt;3 months), while adductomic interrogation enables characterization of exposures to complex air pollution mixtures. We have used Cys34 adductomics to further characterize the protective effects of a successful intervention trial in the Yangtze River delta - a region characterized by consistent and substantial levels of airborne pollution, as well as a rising incidence of lung cancer. Residents of Qidong, China were recruited to participate in a 12-week, randomized, placebo-controlled clinical trial. Participants consumed either a placebo (P) or broccoli sprout beverage (B) daily, and urine and serum samples were collected before and throughout the intervention. Previously published results revealed that, relative to P treatment, B significantly increased detoxication and urinary excretion of benzene (+61%, as S-phenylmercapturic acid [SPMA]). For this pilot study, the serum albumin Cys34adductome was interrogated in n=8 non-smoking women at study day 56 from each of the P and B treatment groups, selecting subjects whose day 56 SPMA excretion was closest to their respective group’s geometric mean. Albumin was precipitated from serum, digested with lysyl endopeptidase, separated by nanoflow LC, and Cys34adducts were identified and quantified on a Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer. We detected four serum albumin Cys34 adducts arising from three metabolites of benzene: benzoquinone (BQ; BQ1 and BQ2), benzene oxide (BO), and benzene diolepoxide (BDE). In accordance with elevated urinary SPMA excretion, BO levels trended lower with B treatment (-39%, p=0.06), while BQ2 was strongly and significantly diminished (-51%, p=0.005); no effect was observed in BQ1 or BDE adduct levels. Incubation of 1,4-BQ with human serum in vitro suggested that the BQ adducts observed in vivo reflect keto-enol tautomerism of a 1,4-BQ-derived adduct, rather than 1,4-BQ and its structural isomer, 1,2-BQ. Along with benzene-derived adducts, we identified several other putative adducts (cyano, ethylene oxide, aldehydes, oxidation, mixed disulfides), revealing a multidimensional signature. To our knowledge, this is the first report to detect benzene-derived adducts, using an adductomics approach, in humans with only ambient exposures. Work is underway to expand upon these results. Cys34 adductomics represents a promising approach for the discovery and quantification of air pollution exposure biomarkers. Citation Format: Joshua W. Smith, Robert N. O'Meally, Derek Ng, Thomas W. Kensler, Robert N. Cole, John D. Groopman. Signatures of chemoprotection from ambient exposure to outdoor air pollution in the human serum albumin Cys34 adductome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 603.

Varroa destructor parasitism has a greater effect on proteome changes than the deformed wing virus and activates TGF-\u03b2 signaling pathways

Honeybee workers undergo metamorphosis in capped cells for approximately 13 days before adult emergence. During the same period, Varroa mites prick the defenseless host many times. We sought to identify proteome differences between emerging Varroa-parasitized and parasite-free honeybees showing the presence or absence of clinical signs of deformed wing virus (DWV) in the capped cells. A label-free proteomic analysis utilizing nanoLC coupled with an Orbitrap Fusion Tribrid mass spectrometer provided a quantitative comparison of 2316 protein hits. Redundancy analysis (RDA) showed that the combination of Varroa parasitism and DWV clinical signs caused proteome changes that occurred in the same direction as those of Varroa alone and were approximately two-fold higher. Furthermore, proteome changes associated with DWV signs alone were positioned above Varroa in the RDA. Multiple markers indicate that Varroa activates TGF-β-induced pathways to suppress wound healing and the immune response and that the collective action of stressors intensifies these effects. Furthermore, we indicate JAK/STAT hyperactivation, p53-BCL-6 feedback loop disruption, Wnt pathway activation, Wnt/Hippo crosstalk disruption, and NF-κB and JAK/STAT signaling conflict in the Varroa–honeybee–DWV interaction. These results illustrate the higher effect of Varroa than of DWV at the time of emergence. Markers for future research are provided.