Orbitrap Velos Research Articles

Hypothalamic protein profiling from mice subjected to social defeat stress.

The Hypothalmic-Pituitary-Adrenal axis also known as the HPA axis is central to stress response. It also acts as the relay center between the body and the brain. We analysed hypothalamic proteome from mice subjected to chronic social defeat paradigm using iTRAQ based quantitative proteomics to identify changes associated with stress response. We identified greater than 2000 proteins after processing our samples analysed through Q-Exactive (Thermo) and Orbitrap Velos (Thermo) at 5% FDR. Analysis of data procured from the runs showed that the proteins whose levels were affected belonged primarily to mitochondrial and metabolic processes, translation, complement pathway among others. We also found increased levels of fibrinogen, myelin basic protein (MBP) and neurofilaments (NEFL, NEFM, NEFH) in the hypothalamus from socially defeated mice. Interestingly, research indicates that these proteins are upregulated in blood and CSF of subjects exposed to trauma and stress. Since hypothalamus secreted proteins can be found in blood and CSF, their utility as biomarkers in depression holds an impressive probability and should be validated in clinical samples.

Metagenomic and metaproteomic analyses of microbial amino acid metabolism during Cantonese soy sauce fermentation.

Cantonese soy sauce is an important type of traditional Chinese brewed soy sauce that was developed in southern China, mainly in Guangdong. Due to the long fermentation period and complex microbiota in Cantonese soy sauce, there are few reports on the microbial metaproteomics of Cantonese soy sauce. In this study, integrative metagenomic and metaproteomic analyzes were used to identify the changes in the dominant microbiota and amino acid synthesis-related enzymes and metabolism during Cantonese soy sauce fermentation. Metagenomic analysis revealed that Tetragenococcus halophilus, Weissella confusa, Weissella paramesenteroides, Enterobacter hormaechei, and Aspergillus oryzae were the dominant microbiota. Using the Top 15 dominant microbiota identified by metagenomics as the database, LTQ Orbitrap Velos Pro ETD mass spectrometry was used to obtain metaproteomic information about the microbes in the soy sauce, and the results indicated that the active enzymes involved in the metabolism of amino acids were secreted by microorganisms such as A. oryzae, T. halophilus, and Zygosaccharomyces rouxii. During the Cantonese soy sauce fermentation process. Among them, early fermentation (0-15d) was dominated by A. oryzae and T. halophilus, mid-term fermentation (60-90d) was dominated by Z. rouxii, A. oryzae, and T. halophilus, and late fermentation (90-120d) was dominated by A. oryzae, Z. rouxii, and T. halophilus. Kyoto Encyclopedia of Genes and Genomes analysis revealed that the main enzymes involved in the metabolism of umami amino acids were aspartate aminotransferase, citrate synthase, aconitase, and isocitrate dehydrogenase, which were produced by Z. rouxii and A. oryzae during early fermentation (0-15 d) and the middle fermentation stage (60-90 d). This study constructed a regulatory network of enzymes potentially involved in the metabolism of flavor amino acids, which provided a theoretical basis for studying the amino acid metabolism of Cantonese soy sauce.

Unveiling the Protein Components of the Secretory-Venom Gland and Venom of the Scorpion Centruroides possanii (Buthidae) through Omic Technologies.

Centruroides possanii is a recently discovered species of "striped scorpion" found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD50 to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.

Pesticide Residue Fast Screening Using Thermal Desorption Multi-Scheme Chemical Ionization Mass Spectrometry (TD-MION MS) with Selective Chemical Ionization.

In this work, the detection characteristics of a large group of common pesticides were investigated using a multi-scheme chemical ionization inlet (MION) with a thermal desorption unit (Karsa Ltd.) connected to an Orbitrap (Velos Pro, Thermo Fisher Scientific) mass spectrometer. Standard pesticide mixtures, fruit extracts, untreated fruit juice, and whole fruit samples were inspected. The pesticide mixtures contained 1 ng of each individual target. Altogether, 115 pesticides were detected, with a set of different reagents (i.e., dibromomethane, acetonylacetone, and water) in different polarity modes. The measurement methodology presented was developed to minimize the common bottlenecks originating from sample pretreatments and nonetheless was able to retrieve 92% of the most common pesticides regularly analyzed with standardized UHPLC-MSMS (ultra-high-performance liquid chromatography with tandem mass spectrometry) procedures. The fraction of detected targets of two standard pesticide mixtures generally quantified by GC-MSMS (gas chromatography with tandem mass spectrometry) methodology was much less, equaling 45 and 34%. The pineapple swabbing experiment led to the detection of fludioxonil and diazinon below their respective maximum residue levels (MRLs), whereas measurements of untreated pineapple juice and other fruit extracts led to retrieval of dimethomorph, dinotefuran, imazalil, azoxystrobin, thiabendazole, fludioxonil, and diazinon, also below their MRL. The potential for mutual detection was investigated by mixing two standard solutions and by spiking an extract of fruit with a pesticide's solution, and subsequently, individual compounds were simultaneously detected. For a selected subgroup of compounds, the bromide (Br-) chemical ionization characteristics were further inspected using quantum chemical computations to illustrate the structural features leading to their sensitive detection. Importantly, pesticides could be detected in actual extract and fruit samples, which demonstrates the potential of our fast screening method.

Diabetes Retinopathy: New Ways to Detect and Treat.

Recent clinical trials demonstrated strong association between lipid abnormalities and progression of diabetic retinopathy (DR); however, whether circulating lipid levels or retinal lipid metabolism, or both, contributes to the pathogenesis of DR is not well understood. Limited amounts of retinal tissue available from animal models, such as mouse models of DR, have proved. Limited amount of retinal tissue was especially challenging for cholesterol and oxysterol detection as it precluded identification of individual isomers of each nonesterified sterol class. To measure cholesterol and oxysterols from limited retinal tissue samples, we developed extremely sensitive electrospray ionization liquid chromatography high-resolution/accurate mass measurements on an LTQ Orbitrap Velos mass spectrometer that are able to resolve sterols and oxysterols separated by reverse-phase HPLC using a gradient of 85-100% methanol containing 0.1% formic acid, with subsequent detection in positive ionization mode. This methodology will aid in our understanding of diabetes-induced changes in retinal cholesterol and oxysterol metabolism.

Diagnosis of renal amyloidosis by liquid chromatography-tandem mass spectrometry: experience from a single-center cohort study in China.

Diagnosis of renal amyloidosis by liquid chromatography-tandem mass spectrometry: experience from a single-center cohort study in China.

Probing the influence of hydrogen cyanide on PAH chemistry

Hydrogen cyanide (HCN) is an important and highly toxic intermediate in coal, nitrogen-rich biomass, and ammonia combustion. It may play an important role in polycyclic aromatic hydrocarbons (PAH) and soot reduction, but the influence of HCN on PAH chemistry is unclear due to the lack of experimental data. With this motivation, the experimental and numerical investigations were performed in this work. Key intermediates were detected, identified, and quantified by the combination of Linear Trap Quadropole (LTQ) Velos Orbitrap mass spectrometer and gas chromatography-mass spectrometer in a jet-stirred reactor fueled by C2H2/HCN/N2 in the temperature range of 800-1200 K. The results reveal that the formation of benzene increases with the addition of HCN, but the PAH formation decreases. PAH reduction can be attributed to the formation of N-containing PAH (NPAH) via HCN-PAH interaction reactions, which were investigated by quantum chemistry and the Rice–Ramsperger–Kassel–Marcus theory with solving the master equation (RRKM-ME). Reaction rate comparison suggests that the HCN addition pathways to NPAH compete with the C2H2 addition pathways to PAH. The product yields in the systems of 1-naphthyl radical + C2H2 and 1-naphthyl radical + HCN were evaluated (T=800-2500 K and p=0.1-100 atm). The results indicate that the increasement of a new aromatic ring via cyclization is difficult in the 1-naphthyl radical + HCN system due to the high energy barrier, and the growth of larger NPAH is limited since the saturated N atoms in the heterocycle rings and CN functional group inhibit further carbon addition.

DDA-imaging with structural identification of lipid molecules on an Orbitrap Velos Pro mass spectrometer.

Matrix‐assisted laser desorption/ionization‐mass spectrometry imaging (MALDI‐MSI) is a useful technique for visualizing the spatial distribution of lipid molecules in tissues. Nevertheless, the use of MSI to investigate local lipid metabolic hallmarks has until recently been hampered by a lack of adequate technology that supports confident lipid identification. This limitation was recently mitigated by the development of DDA‐imaging technology where high‐resolution MSI is combined with parallel acquisition of lipid tandem MS2 spectra on a hybrid ion trap‐Orbitrap Elite mass spectrometer featuring a resolving power of 240,000 and a scan time of 1 s. Here, we report the key tenets related to successful transfer of the DDA‐imaging technology onto an Orbitrap Velos Pro instrument featuring a resolving power of 120,000 and a scan time of 2 s. Through meticulous performance assessments and method optimization, we tuned the DDA‐imaging method to be able to confidently identify 73 molecular lipid species in mouse brain sections and demonstrate that the performance of the technology is comparable with DDA‐imaging on the Orbitrap Elite. Altogether, our work shows that DDA‐imaging on the Orbitrap Velos Pro instrument can serve as a robust workhorse for lipid imaging in routine applications.

Identification of a Novel ACE-inhibitory Peptide from Sheep Casein and Evaluation of the Molecular Binding Mechanism

The purpose of this study was to prepare a novel Angiotensin-I-Converting Enzyme (ACE) inhibitory peptide by hydrolyzing sheep casein with protease and analyze its molecular inhibitory mechanism, so as to provide technical support for the development of functional sheep milk polypeptide dairy products. In this experiment, sheep casein was used as raw material, with hydrolysis degree, ACE inhibition rate and molecular weight distribution as indexes, the most suitable protease was selected from four proteases, and the components with less than <3 kDa was identified by LTQ Orbitrap Velos mass spectrometer. the potential ACE inhibitory peptides were selected for artificial synthesis and it half inhibitory concentration (IC50) was measured. The inhibition kinetics of enzyme was determined by Linewaver-Burk mapping, and the inhibition mechanism of peptide segment was further explained by molecular docking. The results showed that alkaline protease was the best hydrolytic protease. A new ACE inhibitory peptide KYIPIQY was screened from κ -casein and its IC50 was 5.73 μmol/L. Linewaver-Burk diagram showed that the peptide showed a mixed inhibition mode on ACE. Molecular docking showed that KYIPIQY can form hydrogen bonds with S1 and S2 active pockets of ACE, and closely combine with hydrophobic force. KYIPIQY can distort the Zn2+ tetrahedron of ACE, and inhibit the deactivation of ACE catalytic activity, thus exerting high-efficiency ACE inhibitory activity.

Ancient enamel peptides recovered from the South American Pleistocene species Notiomastodon platensis and Myocastor cf. coypus

We used two fossil teeth from South American Pleistocene mammals to obtain subsuperficial acid etching samples. We employed samples from the species Notiomastodon platensis and Myocastor cf. coypus for the enamel etchings. The controls included an extant rodent (rat). After the first etching was discarded, a second 20-s etching (i.e., subsuperficial) was directly collected with a ZipTip and injected into an LTQ Orbitrap Velos for MS analysis. The peptides were identified with different software programs that used Peptide Spectrum Match (PSM) and de novo sequencing including similarity search strategies. Most of the peptides that were recovered from the enamel of the fossils belonged to enamel-specific proteins. To our knowledge, this is the first study that has described the recovery of enamel peptide molecules from extinct South American taxa, indicating that enamel peptide data from late Pleistocene fossils can be employed as an additional parameter for phylogenetic analysis, and that the sample can be obtained by a very conservative acid etching, with almost no damage to the fossils. SignificanceThis study shows that it is possible to obtain information based on plenty of ancient peptides recovered from subsuperficial enamel of fossil teeth from South American Pleistocene. The quality of the data suggests that peptides are likely the best preserved biomolecules under certain harsh environmental conditions. The recovery procedure only lasted 20 s and was minimally destructive to the fossils. This opens a myriad of new possibilities for the study of the past.

Proteomic profile of bone "collagen" extracted for stable isotopes: Implications for bulk and single amino acid analyses.

Protein studies in archaeology and paleontology have been dominated by stable isotope studies to understand diet and trophic levels, but recent applications of proteomic techniques have resulted in a more complete understanding of protein diagenesis than stable isotopes alone. In stable isotope analyses, samples are retained or discarded based on their properties. Proteomics can directly determine what proteins are present within the sample and may be able to allow previously discarded samples to be analyzed. Protein samples that had been previously analyzed for stable isotopes, including those with marginal and poor sample quality, were characterized by liquid chromatography/mass spectrometry using an LTQ Orbitrap Velos mass spectrometer after separation on a Dionex Ultimate 3000 LC system. Data were analyzed using MetaMorpheus and custom R scripts. We found a variety of proteins in addition to collagen, although collagen I was found in the majority of the samples (most samples >80%). We also found a positive correlation between total deamidation and wt% N, suggesting that deamidation may impact the overall nitrogen signal in bulk analyses. The amino acid profiles of samples, including those of marginal or poor stable isotope quality, reflect the expected collagen I percentages, allowing their use in single amino acid stable isotope analyses. All the samples regardless of quality were found to have high concentrations of collagen I, making interpretations of dietary routing based on collagen I reasonably valid. The amino acid profiles on the marginal and poor samples reflect an expected collagen I profile and allow these samples to be recovered for single amino acid analyses.

Pan-Proteomic Analysis and Elucidation of Protein Abundance among the Closely Related Brucella Species, Brucella abortus and Brucella melitensis.

Brucellosis is a zoonotic infection caused by bacteria of the genus Brucella. The species, B. abortus and B. melitensis, major causative agents of human brucellosis, share remarkably similar genomes, but they differ in their natural hosts, phenotype, antigenic, immunogenic, proteomic and metabolomic properties. In the present study, label-free quantitative proteomic analysis was applied to investigate protein expression level differences. Type strains and field strains were each cultured six times, cells were harvested at a midlogarithmic growth phase and proteins were extracted. Following trypsin digestion, the peptides were desalted, separated by reverse-phase nanoLC, ionized using electrospray ionization and transferred into an linear trap quadrapole (LTQ) Orbitrap Velos mass spectrometer to record full scan MS spectra (m/z 300–1700) and tandem mass spectrometry (MS/MS) spectra of the 20 most intense ions. Database matching with the reference proteomes resulted in the identification of 826 proteins. The Cluster of Gene Ontologies of the identified proteins revealed differences in bimolecular transport and protein synthesis mechanisms between these two strains. Among several other proteins, antifreeze proteins, Omp10, superoxide dismutase and 30S ribosomal protein S14 were predicted as potential virulence factors among the proteins differentially expressed. All mass spectrometry data are available via ProteomeXchange with identifier PXD006348.

Evaluating Combined Precursor Isotopic Labeling and Isobaric Tagging Performance on Orbitraps To Study the Peripheral Proteome of Alzheimer's Disease.

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced multiplexing strategy currently capable of analyzing up to 24 samples simultaneously. This capability is especially helpful when studying multiple tissues and biological replicates in models of disease, such as Alzheimer's disease (AD). Here, cPILOT was used to study proteomes from heart, liver, and brain tissues in a late-stage amyloid precursor protein/presenilin-1 (APP/PS-1) human transgenic double-knock-in mouse model of AD. The original global cPILOT assay developed on an Orbitrap Velos instrument was transitioned to an Orbitrap Fusion Lumos instrument. The advantages of faster scan rates, lower limits of detection, and synchronous precursor selection on the Fusion Lumos afford greater numbers of isobarically tagged peptides to be quantified in comparison to the Orbitrap Velos. Parameters such as LC gradient, m/z isolation window, dynamic exclusion, targeted mass analyses, and synchronous precursor scan were optimized leading to >600 000 PSMs, corresponding to 6074 proteins. Overall, these studies inform of system-wide changes in brain, heart, and liver proteins from a mouse model of AD.

Simultaneously quantitative analysis of peptides and chemical components in Cervus and Cucumis polypeptide injection (Songmeile®) using reversed phase liquid chromatography-hydrophilic interaction liquid chromatography–tandem mass spectrometry

Co-occurrences of peptides and chemical components are usually observed in complicated matrices. Notably, those traditional Chinese medicine prescriptions (TCMPs) contain both plant and animal ingredients. It is still challenging to simultaneously monitor peptides and chemical components attributing to their different liquid chromatographic (LC) and mass spectrometric (MS) behaviors. Herein, efforts were made to pursue an eligible approach enabling simultaneous determination of peptides and chemical components in a TCMP namely Cervus and Cucumis polypeptide injection (CCPI, Songmeile®) that is prepared from the acid hydrolytic peptide-enriched extract of Sika deer (Cervus nippon Temminck) bone and the aqueous extract of muskmelon (Cucumis melo L.) seeds. Reversed phase liquid chromatography and hydrophilic interaction liquid chromatography were serially connected (RPLC-HILIC) to achieve comprehensive retention and separation. Sensitive detection was accomplished with selected reaction monitoring (SRM) mode, and multiply charged and singly charged ion transitions were defined for peptides and chemical components, respectively. Inter-batch variations of CCPI were evaluated in an authentic compound-independent manner. In particular, online energy-resolved MS was proposed to gain optimal parameters for five targeted peptides after that CCPI peptidome was profiled using nanoLC-LTQ Orbitrap Velos Pro MS. A so-called universal metabolome standard (UMS) sample was built for calibration curve construction and subsequently applied to acquire the quasi-contents of all 31 analytes, including five peptides and 26 chemical components, in ten batches of CCPI (CCPI1–CCPI10). The quantitative dataset revealed mild fluctuation for the quasi-content profiles of analytes-of-interest within different batches. More importantly, RPLC-HILIC–SRM is a promising method to fully address the demands of simultaneous measurement of peptides and chemical components in complicated matrices, and it might be a robust analytical tool for in-depth quality evaluation of CCPI as well as other TCMPs.

Salivary proteins as markers of radiation-related oral mucositis

Objectives The aim of this study was to characterize the salivary proteomic profile of patients treated for head and neck cancers (HNCs) and correlate it with the risk of developing severe radiation-related oral mucositis (OM). Study Design Forty-one patients with oral squamous cell carcinoma (OSCC) who underwent adjuvant radiotherapy (RT) or chemoradiotherapy (CRT) were included. All patients were submitted to dental conditioning protocols prior to RT. OM and OM-related pain were evaluated daily during RT and graded according to the Common Toxicity Criteria for Adverse Events (National Cancer Institute, version 4.0, 2010) and the visual analogue scale (VAS). For the molecular analysis, whole saliva was collected immediately before RT and subjected to proteomic analysis by means of liquid chromatography–mass spectrometry (LTQ Orbitrap Velos MS; Thermo Fisher Scientific, Bremen, Germany) and label-free protein quantification. The results obtained from the targeted proteomic analysis were compared with OM clinical outcomes. Statistical analysis was performed by using Wilcoxon's test. Results Of the study patients, 73% presented with stage III/IV OSCC; 58% were submitted to CRT protocols, with a mean RT dose of 66 Gy. Most of the patients (66%) had visible tumor areas at the time of saliva collection; 43.9% had grade 2 and 31.7% had grade 3 as the highest OM grade during RT, with a mean highest reported VAS score of 3.53. For the target proteomics analysis, a total of 65 proteins were observed to be mostly related to biologic processes, such as immune responses, peptidase inhibitor activity, and the inflammatory system. The macrophage migration inhibitory factor (MIF HUMAN) was statistically significant when correlated with OM grade. MIF was observed in patients with grades 1 to 4 OM and showed higher abundance for OM grades 3 and 4 compared with grades 1 and 2 (P = .04). Conclusions The correlation of MIF with the severity of OM is compatible with the role of MIF as a proinflammatory protein. This seems to be the first study to describe this association; therefore, future prospective studies would be ideal to observe pre- and post-RT alterations in salivary MIF levels to validate this result and better understand the role of MIF in the pathogenesis of OM.

Proteomic analysis of whole saliva in chronic periodontitis

Periodontitis is a chronic inflammatory disease resulting from a dysbiosis of the dental biofilm and a dysregulated host response in susceptible individuals. It is characterized by periodontal attachment destruction, bone resorption and eventual tooth loss. Salivary biomarkers have been sought to predict and prevent periodontitis. This comparative study analyzed the salivary proteome of individuals with chronic periodontitis (CP) and periodontal health (PH) and correlated specific proteins with clinical parameters of disease by using mass spectrometry. Stimulated whole saliva was obtained 10 PH and 30 CP patients and pooled into 5 healthy control samples and 15 CP samples. After precipitation with TCA, samples were digested enzymatically with trypsin and analyzed by a LTQ Orbitrap Velos equipped with a nanoelectrospray ion source. A wide range of salivary proteins of various functions was significantly reduced in CP individuals, whereas salivary acidic proline-rich phosphoprotein, submaxillary gland androgen-regulated protein, histatin-1, fatty acid binding protein, thioredoxin and cystatin-SA were predominant in diseased patients and correlated significantly with signs of periodontal attachment loss and inflammation. In conclusion, few specific salivary proteins were associated with CP. These findings may contribute to the identification of disease indicators or signatures for the improvement of periodontal diagnosis. SignificancePeriodontitis is a chronic inflammatory disease that results in periodontal attachment destruction, bone resorption and eventual tooth loss. Salivary biomarkers have been sought to predict periodontitis. The analysis of the salivary proteome of individuals with chronic periodontitis indicated that several proteins of various functions were significantly reduced in these individuals, except for salivary acidic proline-rich phosphoprotein, submaxillary gland androgen-regulated protein, histatin, fatty acid binding protein, thioredoxin and cystatin. Differences in salivary proteome profiles between periodontal health and periodontitis may contribute to the identification of disease indicators and to the improvement of periodontal diagnosis and treatment.

122 Proteome of extracellular vesicles from follicular fluid of bovine 3- to 6-mm follicles: Similarity and specificity compared with granulosa cells

Extracellular vesicles (EVs) are released from cells and are present in most bodily fluids. Small EVs, 30-150nm in diameter, known as exosomes, transport regulatory noncoding RNA, lipids, and proteins, and thus participate in cell-to-cell communications and signalling. Exosomes from bovine ovarian follicular fluid (FF) can stimulate proliferation of granulosa cells (GCs), cellular uptake, and cumulus expansion, and therefore might be useful in reproductive technologies. The aim of our study was to characterise the protein content of bovine FF exosomes and compare it with the GC proteome in order to better understand the origin of these EVs and their possible functions. We extracted EVs from FF of bovine 3- to 6-mm ovarian follicles (enclosed oocytes routinely used for invitro embryo production (IVEP)) via a series of centrifugations at 2,000g to 100 000g. Transmission electron microscopy analysis revealed that 87.9% of extracted EVs had a larger size of exosomes, and 11.8% had smaller size (&amp;lt;30nm); only 0.3% of EVs ranged from 150 to 250nm. For proteome analysis, 80µg of total proteins extracted from EVs and GCs originating from the same follicles were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After trypsin proteolytic digestion, peptide mixtures were analysed via nanoflow liquid chromatography coupled to high-resolution tandem mass spectrometry (MS/MS) using LTQ Orbitrap Velos (Thermo Fisher Scientific). We used Mascot engine (Matrix Science) against the NCBIprot mammalian database to perform MS/MS ion searches; proteins identified with at least two peptides were clustered with Scaffold 4.0 software. We identified 542 protein isoforms that represented 221 and 299 clusters of unique proteins in EVs and GCs, respectively. Of these, 158 are common among EVs and GCs and demonstrate significant enrichment (P&amp;lt;0.01) in Gene Ontology (GO) terms related to protein folding in endoplasmic reticulum, receptor-mediated endocytosis, cell-cell adhesion, translation processes, binding of proteins, RNA or cholesterol, and antioxidant activity. Of common proteins, 69.6% are mapped to the GO “extracellular exosome.” Of the 63 proteins over-represented in EVs, 37 are ribosomal proteins involved in translation, rRNA processing, and mRNA catabolic processes. Of the proteins over-represented in GCs, 141 are mainly involved in carbon, fatty acid, and tricarboxylic acid (TCA) metabolism, oxidation-reduction processes, NAD and poly(A) RNA binding, and cell cycle regulation. These proteins are enriched in GO terms related to membrane, mitochondrion, and extracellular exosomes. Our findings indicate that proliferating GCs of antral follicles actively synthesise and secrete exosomes, which contain significant quantity of different ribosomal proteins. These RNA-binding proteins may serve to compact the cargo of different types of RNAs-both regulatory and designated for degradation, which are transported by FF exosomes. Funds were provided by INRA PHASE France and by the Russian Science Foundation (project 19-16-00115).

Tandem Mass Tag Labeling to Identify Proteome Changes in Beef Longissimus Lumborum and Psoas Major Muscles During Early Postmortem Period

ObjectivesLongissimus lumborum (LL) and psoas major (PM) are important muscles in beef hindquarters that exhibit variation in meat quality attributes. Postmortem metabolism (muscle-to-meat conversion) affects biochemical properties of muscles and in turn influence the meat quality. Although previous research has indicated that variation in the proteome profile of LL and PM post-rigor influences meat quality attributes such as tenderness and color stability during retail display, limited research has examined the influence of early postmortem metabolism on meat quality. Tandem mass tag (TMT) labeling is a chemical labeling approach used for accurate mass spectrometry-based quantification and identification of biological macromolecules. Therefore, the objective of this study was to use TMT labeling to examine proteome profile variation between beef LL and PM during the early postmortem period.Materials and MethodsMuscle biopsy samples were collected from carcasses (n = 4) at 45 min, 12 h, and 36 h postmortem from a commercial beef processing facility. Samples were frozen immediately in liquid nitrogen and stored at –80°C until proteomic analysis. Proteome was analyzed using TMT label containing ten different isobaric compounds with the same mass and chemical structure composed of an amine-reactive NHS-ester group, a spacer arm, and a mass reporter. After labeling and peptide fractionation, all the samples were multiplexed and ran through the Orbitrap Velos mass spectrometer equipped with a Nanospray Flex ion source to identify differentially abundant proteins. The proteins exhibiting 1.5-fold or more intensity difference and a statistical difference (P &lt; 0.05) between LL and PM or within the muscles during the postmortem were reported as differentially abundant.ResultsSeventy differentially abundant proteins (P &lt; 0.05) were identified from three comparisons between the muscles (31 proteins in PM 45 min vs. LL 45 min, 41 proteins in PM 12 h vs. LL 12 h, 49 proteins in PM 36 h vs. LL 36 h). However, no difference (P &gt; 0.05) in protein expression within a muscle was observed during these time points. The differentially abundant proteins were mainly involved in oxidative phosphorylation and ATP-related transport, tricarboxylic acid cycle, NADPH regeneration, fatty acid degradation, muscle contraction, calcium signaling, chaperone activity, oxygen transport, as well as degradation of the extracellular matrix. At early postmortem, overabundant anti-apoptotic proteins in LL could cause high metabolic stability, enhanced autophagy, and delayed apoptosis, while overabundant metabolic enzymes and pro-apoptotic proteins in PM could accelerate the reactive oxygen species generation and programmed cell death.ConclusionDifferentially abundant proteins between LL and PM during the early postmortem were primarily associated with cellular metabolism and programmed cell death. The greater oxidative and color stability in LL compared to PM could be related to the increased expression of anti-apoptotic proteins and the decreased expression of metabolic enzymes and proapoptotic proteins in LL.

Integration of High-Resolution Mass Spectrometry with Cryogenic Ion Vibrational Spectroscopy.

We describe an instrumental configuration for the structural characterization of fragment ions generated by collisional dissociation of peptide ions in the typical MS2 scheme widely used for peptide sequencing. Structures are determined by comparing the vibrational band patterns displayed by cryogenically cooled ions with calculated spectra for candidate structural isomers. These spectra were obtained in a linear action mode by photodissociation of weakly bound D2 molecules. This is accomplished by interfacing a Thermo Fisher Scientific Orbitrap Velos Pro to a cryogenic, triple focusing time-of-flight photofragmentation mass spectrometer (the Yale TOF spectrometer). The interface involves replacement of the Orbitrap's higher-energy collisional dissociation cell with a voltage-gated aperture that maintains the commercial instrument's standard capabilities while enabling bidirectional transfer of ions between the high-resolution FT analyzer and external ion sources. The performance of this hybrid instrument is demonstrated by its application to the a1, y1 and z1 fragment ions generated by CID of a prototypical dipeptide precursor, protonated L-phenylalanyl-L-tyrosine (H+-Phe-Tyr-OH or FY-H+). The structure of the unusual z1 ion, nominally formed after NH3 is ejected from the protonated tyrosine (y1) product, is identified as the cyclopropane-based product is tentatively identified as a cyclopropane-based product.

SUN-229 The Role of Testosterone in Spermatogenesis: Lessons from Proteome Profiling of Human Spermatozoa in Testosterone Deficiency

Testosterone is required during spermatogenesis to mantain the blood-testis barrier and for meiosis, Sertoli-spermatid adhesion and sperm release. Aim of the study was to better understand the effect of hypothalamic-pituitary-testicular axis on sperm protein composition and function.12 male patients with low testosterone and LH levels were consecutively enrolled for this study. Five normogonadic men were enrolled as control group in the protocol. If spermatozoa were detectable in seminal samples, the the ejaculates were washed and the sperm cells were selected using 50% PercollTM. The recovered sperm cells were then suspended in PBS and subjected to a residual leukocyte depletion using CD45 Dynabeads® magnetic cell sorting. The absence of leukocytes was further confirmed both by microscopic observation after staining and by performing a RT-PCR for CD45. Protein solubilization was independently performed on each sperm sample. A comparative proteomic analysis was performed comparing two pools: controls vs hypogonadic patients. The TMTduplexTM Isobaric Mass Tagging Kit was used for the peptide labeling. MS/MS analysis was performed using a LTQ Orbitrap Velos. Data were processed using Proteome Discoverer 1.4.1.14 software, using very stringent filtering criteria. Proteins identified by SEQUEST were analyzed using the PANTHER classification system. Proteomic results have been confirmed by Western Blot. The following primary antibody were used: anti-Lactoferrin polyclonal antibody and anti-PIP polyclonal antibodyWe included in our study 5 oligozoospermic hypogonadic patients with a sperm count >10 x 106/ml and without leukocyte contamination. 42 proteins were under-expressed in the pool of hypogonadic patients; 12 of these 42 increased proteins have been previously reported to be related with spermatogenesis, sperm motility, sperm-oocyte interaction and acrosomal reaction. The distribution of proteins for molecular function revealed that the protein pattern impaired by low blood testosterone is involved in protein binding and hydrolase. Twelve proteins have been over-expressed in the pool of hypogonadic patients, including markers of defective spermatogenesis. Western-blot analysis confirmed proteomic data. This is the first application of high resolution mass spectrometry -based proteomics aimed to reveal an array of proteins in spermatozoa reflecting an impairment of spermatogenesis and sperm function in male secundary hypogonadism.