Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer Research Articles

The Functional Characterization of DzCYP72A12-4 Related to Diosgenin Biosynthesis and Drought Adaptability in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial herb famous for the production of diosgenin, which is a valuable initial material for the industrial synthesis of steroid drugs. Sterol C26-hydroxylases, such as TfCYP72A616 and PpCYP72A613, play an important role in the diosgenin biosynthesis pathway. In the present study, a novel gene, DzCYP72A12-4, was identified as C26-hydroxylase and was found to be involved in diosgenin biosynthesis, for the first time in D. zingiberensis, using comprehensive methods. Then, the diosgenin heterogenous biosynthesis pathway starting from cholesterol was created in stable transgenic tobacco (Nicotiana tabacum L.) harboring DzCYP90B71(QPZ88854), DzCYP90G6(QPZ88855) and DzCYP72A12-4. Meanwhile, diosgenin was detected in the transgenic tobacco using an ultra-performance liquid chromatography system (Vanquish UPLC 689, Thermo Fisher Scientific, Bremen, Germany) tandem MS (Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, Thermo Fisher Scientific, Bremen, Germany). Further RT-qPCR analysis showed that DzCYP72A12-4 was highly expressed in both rhizomes and leaves and was upregulated under 15% polyethylene glycol (PEG) treatment, indicating that DzCYP72A12-4 may be related to drought resistance. In addition, the germination rate of the diosgenin-producing tobacco seeds was higher than that of the negative controls under 15% PEG pressure. In addition, the concentration of malonaldehyde (MDA) was lower in the diosgenin-producing tobacco seedlings than those of the control, indicating higher drought adaptability. The results of this study provide valuable information for further research on diosgenin biosynthesis in D. zingiberensis and its functions related to drought adaptability.

Identification and Characterization of the Stability of Hydrophobic Cyclolinopeptides From Flaxseed Oil.

Flaxseed (linseed) is a cultivar of the spring flowering annual plant flax (Linum usitatissimum) from the Linaceae family. Derivatives of this plant are widely used as food and as health products. In recent years, cyclic peptides isolated from flaxseed and flaxseed oil, better known as cyclolinopeptides (CLPs), have attracted the attention of the scientific community due to their roles in the inhibition of osteoclast differentiation or their antimalarial, immunosuppressive, and antitumor activities, as well as their prospects in nanotechnology and in the biomedical sector. This study describes the detection, identification, and measurement of CLPs in samples obtained from nine different flaxseed oil manufacturers. For the first time, Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer was used for CLP identification together with RP-HPLC. The routine analyses were performed using RP chromatography, measuring the absorption spectra and fluorescence detection for identifying tryptophan-containing peptides using the native fluorescence of tryptophan. In addition, existing protocols used for CLP extraction were optimized and improved in a fast and cost-efficient way. For the first time, 12 CLPs were separated using methanol/water as the eluent with RP-HPLC. Finally, the stability and degradation of individual CLPs in the respective flaxseed oil were examined over a period of 60 days at different temperatures. The higher temperature was chosen since this might reflect the cooking practices, as flaxseed oil is not used for high-temperature cooking. Using HPLC–MS, 15 CLPs were identified in total in the different flaxseed oils. The characterization of the peptides via HPLC–MS highlighted two types of CLP profiles with a substantial variation in the concentration and composition of CLPs per manufacturer, probably related to the plant cultivar. Among the observed CLPs, CLP-O, CLP-N, and CLP-B were the least stable, while CLP-C and CLP-A were the most stable peptides. However, it is important to highlight the gradual degradation of most of the examined CLPs over time, even at room temperature.

Defining Fatty Acid Changes Linked to Rumen Development, Weaning and Growth in Holstein-Friesian Heifers.

After birth, as effectively monogastric animals, calves undergo substantial physiological changes to become ruminants by 3 months of age and reach sexual maturity at approximately 15 months of age. Herein, we assess longitudinal metabolomic changes in Holstein-Friesian (HF) heifers from birth until sexual maturity during this developmental process. Sera from 20 healthy, HF heifers were sampled biweekly from 2 weeks of age until 13 months of age and then monthly until 19 months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares discriminant analysis (PLS-DA) and unsupervised hierarchical clustering analysis (HCA) of the derived metabolomes indicated changes detectable in heifers’ sera over time. Time series analyses identified 30 metabolites that could be related to rumen development and weaning at ~3 months of age. Further time series analysis identified 40 metabolites that could be correlated with growth. These findings highlight the role of acetic acid and 3-phenylpropionate (3-PP) in rumen development and growth, suggest that weaning induces elevated levels of fatty acyls in response to a post-weaning stress-induced innate immune response and demonstrate the utilization of fatty acyls in growth. The identified metabolites offer serum metabolites which could inform the nutrition and healthy development of heifers.

Metabolomic changes in polyunsaturated fatty acids and eicosanoids as diagnostic biomarkers in Mycobacterium avium ssp. paratuberculosis (MAP)-inoculated Holstein–Friesian heifers

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative organism of Johne’s disease, a chronic granulomatous enteritis of ruminants. We have previously used naturally MAP-infected heifer calves to document metabolomic changes occurring in MAP infections. Herein, we used experimentally MAP-inoculated heifer calves to identify biomarkers for MAP infections. At 2-weeks of age, 20 Holstein–Friesian (HF) calves were experimentally inoculated with MAP. These calves, along with 20 control calves, were sampled biweekly up to 13-months of age and then monthly up to 19-months of age. Sera were assessed using flow infusion electrospray high-resolution mass spectrometry (FIE-HRMS) on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer for high throughput, sensitive, non-targeted metabolite fingerprinting. Partial least squares-discriminate analysis (PLS-DA) and hierarchical cluster analysis (HCA) discriminated between MAP-inoculated and control heifer calves. Out of 34 identified metabolites, six fatty acyls were able to differentiate between experimental groups throughout the study, including 8, 11, 14-eicosatrienoic acid and cis-8, 11, 14, 17-eicosatetraenoic acid which were also detected in our previous study and so further suggested their value as biomarkers for MAP infection. Pathway analysis highlighted the role of the alpha-linoleic acid and linoleic acid metabolism. Within these pathways, two broad types of response, with a rapid increase in some saturated fatty acids and some n-3 polyunsaturated fatty acids (PUFAs) and later n-6 PUFAs, became predominant. This could indicate an initial anti-inflammatory colonisation phase, followed by an inflammatory phase. This study demonstrates the validity of the metabolomic approach in studying MAP infections. Nevertheless, further work is required to define further key events, particularly at a cell-specific level.

Metabolomic Changes in Naturally MAP-Infected Holstein-Friesian Heifers Indicate Immunologically Related Biochemical Reprogramming.

Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), causes weight loss, diarrhoea, and reduced milk yields in clinically infected cattle. Asymptomatic, subclinically infected cattle shed MAP bacteria but are frequently not detected by diagnostic tests. Herein, we compare the metabolite profiles of sera from subclinically infected Holstein–Friesian heifers and antibody binding to selected MAP antigens. The study used biobanked serum samples from 10 naturally MAP-infected and 10 control heifers, sampled monthly from ~1 to 19 months of age. Sera were assessed using flow infusion electrospray–high-resolution mass spectrometry (FIE–HRMS) on a Q Exactive hybrid quadrupole–Orbitrap mass spectrometer for high-throughput, sensitive, non-targeted metabolite fingerprinting. Partial least-squares discriminant analyses (PLS-DA) and hierarchical cluster analysis (HCA) of the data discriminated between naturally MAP-infected and control heifers. In total, 33 metabolites that differentially accumulated in naturally MAP-infected heifers compared to controls were identified. Five were significantly elevated within MAP-infected heifers throughout the study, i.e., leukotriene B4, bicyclo prostaglandin E2 (bicyclo PGE2), itaconic acid, 2-hydroxyglutaric acid and N6-acetyl-L-lysine. These findings highlight the potential of metabolomics in the identification of novel MAP diagnostic markers and particular biochemical pathways, which may provide insights into the bovine immune response to MAP.

Proteomic Evaluation of the Acute Radiation Syndrome of the Gastrointestinal Tract in a Murine Total-body Irradiation Model.

Radiation exposure to the gastrointestinal system contributes to the acute radiation syndrome in a dose- and time-dependent manner. Molecular mechanisms that lead to the gastrointestinal acute radiation syndrome remain incompletely understood. Using a murine model of total-body irradiation, C57BL/6J male mice were irradiated at 8, 10, 12, and 14 Gy and assayed at day 1, 3, and 6 after exposure and compared to nonirradiated (sham) controls. Tryptic digests of gastrointestinal tissues (upper ileum) were analyzed by liquid chromatography-tandem mass spectrometry on a Waters nanoLC coupled to a Thermo Scientific Q Exactive hybrid quadrupole-orbitrap mass spectrometer. Pathway and gene ontology analysis were performed with Qiagen Ingenuity, Panther GO, and DAVID databases. A number of trends were identified in our proteomic data including pronounced protein changes as well as protein changes that were consistently up regulated or down regulated at all time points and dose levels interrogated. Time- and dose-dependent protein changes, canonical pathways affected by irradiation, and changes in proteins that serve as upstream regulators were also identified. Additionally, proteins involved in key processes including inflammation, radiation, and retinoic acid signaling were identified. The proteomic profiling conducted here represents an untargeted systems biology approach to identify acute molecular events that will be useful for a greater understanding of animal models and may be potentially useful toward the development of medical countermeasures and/or biomarkers.

Acute Proteomic Changes in the Lung After WTLI in a Mouse Model: Identification of Potential Initiating Events for Delayed Effects of Acute Radiation Exposure

Radiation-induced lung injury is a delayed effect of acute radiation exposure resulting in pulmonary pneumonitis and fibrosis. Molecular mechanisms that lead to radiation-induced lung injury remain incompletely understood. Using a murine model of whole-thorax lung irradiation, C57BL/6J mice were irradiated at 8, 10, 12, and 14 Gy and assayed at day 1, 3, and 6 postexposure and compared to nonirradiated (sham) controls. Tryptic digests of lung tissues were analyzed by liquid chromatography-tandem mass spectrometry on a Waters nanoLC instrument coupled to a Thermo Scientific Q Exactive hybrid quadrupole-orbitrap mass spectrometer. Pathway and gene ontology analysis were performed with Qiagen Ingenuity, Panther GO, and DAVID databases. A number of trends were identified in the proteomic data, including protein changes greater than 10 fold, protein changes that were consistently up regulated or down regulated at all time points and dose levels interrogated, time and dose dependency of protein changes, canonical pathways affected by irradiation, changes in proteins that serve as upstream regulators, and proteins involved in key processes including inflammation, radiation, and retinoic acid signaling. The proteomic profiling conducted here represents an untargeted systems biology approach to identify acute molecular events that could potentially be initiating events for radiation-induced lung injury.

Simultaneously targeted and untargeted multicomponent characterization of Erzhi Pill by offline two-dimensional liquid chromatography/quadrupole-Orbitrap mass spectrometry

Large-scale targeted and untargeted metabolites characterization can be achieved by feat of different liquid chromatography/mass spectrometry (LC–MS) platforms by multiple MS experiments or using data-independent acquisition followed by precursor-product ions matching based on certain algorithms. The resulting insufficiency in efficiency and availability greatly restricts the applicability of these strategies in large-scale profiling and identification of various metabolites. A strategy simultaneously enabling both the targeted and untargeted metabolites characterization is established on a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer, by integrating precursor ions list-triggered data-dependent MS2 acquisition (PIL/dd-MS2) of the targeted components and using the “If idle-pick others” (IIPO) function to induce untargeted metabolites fragmentation. A compounds-specific mass defect filter (MDF) algorithm is proposed as a method to generate the PIL. As a proof of concept, this strategy coupled with offline two-dimensional liquid chromatography (2D-LC) was applied to identify the multicomponents of a traditional Chinese medicine formula Erzhi Pill (EZP). A rigid MDF vehicle was elaborated by orthogonal screening of the integer mass and integer mass-dependent dynamic mass defects considering a variation of 20 ppm. The Full MS/dd-MS2 method enabling PIL and IIPO exhibited better performance than Full MS/dd-MS2 and Targeted SIM/dd-MS2 (selected ion monitoring) in respect of the sensitivity in identifying the targeted components and the ability to characterize more untargeted ones. As a consequence, 270 components were separated from EZP, and 146 thereof were selectively characterized. In conclusion, it is a practical, multifaced strategy facilitating the in-depth metabolites profiling and characterization of complex herbal and biological samples.

Bioactivity-Guided Isolation of Neuritogenic Factor from the Seeds of the Gac Plant (Momordica cochinchinensis).

Nerve growth factor (NGF) is an endogenously produced protein with the capacity to induce central nervous system (CNS) neuronal differentiation and repair. NGF signaling involves its binding to tropomyosin-related kinase (Trk) receptors, internalization, and initiation of phosphorylation cascades which cause microtubule reorganization and neuronal outgrowth. Because NGF cannot cross the blood-brain barrier, its therapeutic use is limited. Synthetic peptides that can act as NGF receptor agonists (NGF mimetics) are known to attenuate neurodegenerative pathologies in experimental models of Alzheimer's disease and Parkinson's disease; however, the existence of plant-based NGF mimetics is uncertain. For this reason, we recently completed a high throughput screening of over 1100 nutraceuticals (vitamins, herbal plant parts, polyphenolics, teas, fruits, and vegetables) to identify neuritogenic factor using a PC-12 cell model. Remarkably we found only one, commonly known as the seed of Gac plant (Momordica cochinchinensis) (MCS). In the current study, we further investigated this seed for its neuritogenic effect using bioactivity-guided chemical separations. The data show no biological neuritogenic activity in any chemical solvent fraction, where activity was exclusive to the crude protein. MSC crude proteins were then separated by 1D electrophoresis, where the active neuritogenic activity was confirmed to have a molecular mass of approximately 17 kDa. Subsequently, the 17kDa band was excised, digested, and run on a UPLC-MS/MS with a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer with data evaluated diverse tools such as X! Tandem, OMS, and K-score algorithms. Proteomic evaluation of the 17kDa band confirmed evidence for 11S globulin subunit beta, napin, oleosin, Momordica trypsin inhibitors (TI) MCoTI-I /II, and many isoforms of Two Inhibitor Peptide Topologies (TIPTOPs). While all peptides identified correspond to the genus/species, Momordica cochinchinensis and Cucumis Sativus, a significant limitation of the analysis is the nonexistence of full annotation for the Momordica cochinchinensis proteome. In conclusion, these findings demonstrate that there is a stable protein within MCS having a mass of 17kDa with the capacity to induce neurite outgrowth. Future work will be required to establish the therapeutic value of the MCS for the treatment of neurodegenerative diseases.

Fast analysis of polyphenols and alkaloids in cocoa-based products by ultra-high performance liquid chromatography and Orbitrap high resolution mass spectrometry (UHPLC-Q-Orbitrap-MS/MS)

The cocoa varieties Criollo, Forastero and Trinitario, which have different organoleptic properties, quite often are mixed together in cocoa-based products. The objective of this work was to develop a rapid method for the simultaneous determination of polyphenols (n = 35) and alkaloids (n = 2) in cocoa-based products by using the high throughput advantages provided by the Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (HRMS). The proposed procedure was optimized and validated in terms of selectivity and specificity (mass accuracy <5 ppm), sensitivity (instrumental limit of detection from 15 to 30 fg), linearity (r2 > 0.990), accuracy (recovery range from 89.1 to 112.4%) and precision (relative standard deviation <10%). The method was applied to 80 cocoa-based samples. Alkaloids represented on average the 57.7% of assayed compounds. Flavanols and procyanidins were the most abundant polyphenols being quantified in a range between 261 and 668 mg/kg and from 127 to 405 mg/g, respectively. Mass equivalents but structural isomers bioflavonoids were characterized by using the HRMS-Orbitrap methodology here developed.

O1.7. PROTEOMIC ANALYSIS OF BLOOD BASED SAMPLES FROM THE OPTiMiSE (OPTIMIZATION OF TREATMENT AND MANAGEMENT OF SCHIZOPHRENIA IN EUROPE) STUDY POINT TOWARDS COMPLEMENT PATHWAY PROTEIN CHANGES

BackgroundThe OPTiMiSE (Optimization of Treatment and Management of Schizophrenia in Europe) trial may help in the identification of predictors of treatment response. Medication naïve patients with first episode schizophrenia or schizophreniform disorder were enrolled in the study and treated open-label for a four-week period with amisulpride. PANSS ratings were undertaken at baseline and following the four-week treatment. 30 non-remitters (as defined by the Andreasen criteria) with the worst change in PANSS scores and the 30 remitters with the best change in PANSS scores were selected to represent good and poor outcome groups.MethodsWe compared proteomic markers in serum collected prior to treatment in 30 patients who subsequently showed a good response to amisulpride (“responders”, and 30 patients who did not show a good response (“non-responders”). Serum samples were depleted using High Performance Liquid Chromatography (HPLC) attached to a MARS column to remove the 14 most abundant plasma proteins (albumin, IgG, antitrypsin, IgA, transferrin, haptoglobin, fibrinogen, alpha2-macroglobulin, alpha1-acid glycoprotein, IgM, apolipoprotein AI, apolipoprotein AII, complement C3, and transthyretin). The groups were matched for ethnicity, gender and age.50 µg from each sample were reduced, alkylated, tryptically digested, then zip-tipped to concentrate and purify. Samples were then run for a 90-min gradient on a Thermo Scientific Q- Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer. Raw MS data were processed by MaxQuant software and searched against the human Uniprot database, for label free quantitation of peptides and proteins. False discovery rates (FDR) were set at 1% for both peptide and protein levels in target/decoy to minimize false positives. The match between runs feature was utilized.ResultsFour hundred and sixty-four protein identifications were obtained. Samples were excluded where >30% of proteins were missing, and we used imputation, where the missingness depends upon a threshold of detection. This left 228 proteins for analysis. Of these, 21 were significantly different between responders and non-responders (p<0.05), one was FDR positive, one at trend FDR level. Pathway analysis (KEGG, David NIH) of the significant proteins determined “complement and coagulation cascades” to be the top pathway affected with six proteins from the list assigned to the pathway. These were CFI, C4A, C6, F9, VWF and SERPING1, all found to be up-regulated and with p-values ranging from 0.002 to 0.044. C6 is a constituent of the membrane attack complex (MAC) that plays a key role in the innate and adaptive immune response, while CFI belongs to the alternative pathway, C4A belongs to the classical pathway and F9 and VWF play roles in the intrinsic coagulation pathway.DiscussionThese data complement results by Sekar et al., implicating excessive complement activity in the development of schizophrenia. Our data identifies the complement proteins in treatment response and this is also consistent with our previous findings of up-regulation of the complement pathway among those at risk of future psychotic experiences.

Spectrum Effect Relationship and Component Knock-Out in Angelica Dahurica Radix by High Performance Liquid Chromatography-Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer.

Different extracts of Angelica dahuricae were available for whitening or treating vitiligo clinically. They showed inhibitory or activating effects on tyrosinase, a rate-limiting enzyme of melanogenesis. This study aimed to identify active compounds on tyrosinase in water extract of Angelica dahurica Radix. We applied spectrum-effect relationship and component knock-out methods to make it clear. HPLC was used to obtain the specific chromatograms. The effects on tyrosinase activity were examined by measuring the oxidation rate of levodopa in vitro. Partial least squares method was used to examine the spectrum-effect relationships. The knocked-out samples were prepared by HPLC method, and the identification of knocked-out compounds was conducted by the high performance liquid chromatography-four stage rod-electrostatic field orbit trap high resolution mass spectrometry. Results showed that S6, S14, S18, S21, S35, S36, S37, S40, and S41 were positively correlated to inhibitory activity of Angelica dahuricae on tyrosinase whereas S9, S11, S8, S12, S22, and S30 were negatively correlated. When the concentration of each sample was 1 g·mL−1, equal to the amount of raw medicinal herbs, oxypeucedanin hydrate, imperatorin, cnidilin, and isoimperatorin had inhibitory effects on tyrosinase activity whereas byakangelicin and bergapten had activating effects.

Determination of Protein Markers for Beef Tenderness in U.S. Select Beef

ObjectivesBeef tenderness is a primary quality feature that defines the consumer’s perception of the product quality. Therefore definition of chemical characteristics that contribute to variation in tenderness within a specific USDA grade can help refine our understanding of meat tenderness. The objective of this experiment was to identify novel protein markers for tenderness in Longissimus dorsi sarcoplasmic fractions utilizing proteomics and a valuable library of beef samples. Materials and MethodsSteaks (USDA Select grade) used in this trial were classified as low shear force (LSF, n = 12; 11.3 ± 1.6 kg) and high shear force (HSF, n = 12; 34.3 ± 4.7 kg), according to their slice shear force (SSF, kg) measurements at 14 d postmortem. Western blotting of whole-muscle samples was used to determine troponin-T degradation (indicator of overall proteolysis). Sarcoplasmic protein samples from d 2 postmortem were prepared for two-dimensional difference in gel electrophoresis (2D-DIGE). Samples from LSF and HSF were sorted according to SSF values and were matched to a pooled internal reference (representative of all samples). Protein samples were loaded (75 µg) on 24 cm strips, pH 6 to 9, and focused in immobilized pH gradient in the first dimension. For the second dimension the strips were run on 12.5% acrylamide gels (n = 12). After 2D-DIGE, protein spots found to be different in relative abundance between HSF and LSF samples, and with other proteins of interest, were identified using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (LC-MS/MS). Immunoblots were performed to confirm protein identity. ResultsThe LSF samples exhibited greater troponin-T degradation (as evidenced by abundance of a 30 kDa fragment) than the HSF samples (LSF ratio to reference was 1.8, HSF ratio to reference was < 0.01; P < 0.01). A total of 31 spots showed a significant difference (P < 0.1) in relative abundance across groups (HSF vs. LSF). Among these, 18 spots were identified as 8 different proteins (Table 1). Proteins that were more abundant in the LSF samples are metabolic enzymes involved in the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase, fructose bisphosphate aldolase A). Among the six proteins that showed greater abundance in the HSF samples, four belong to the oxidative energy metabolism pathways (malate dehydrogenase, creatine kinase M-type, myoglobin, carbonic anhydrase 3), one is involved in redox pathways (peroxiredoxin-1) and another one in protein turnover (polyubiquitin). The greater abundance of spots corresponding to oxidative metabolism enzymes suggests that aerobic metabolism may decrease potential for postmortem proteolysis. Table 1Differentially abundant proteins in high and low slice shear force groups Protein% CoverageRatio (LSF/HSF)P-valueGlyceraldehyde-3-phosphate dehydrogenase (GAPDH)26.731.980.060Fructose-bisphosphate aldolase A62.361.79< 0.050Polyubiquitin (Fragment)21.22–1.180.100Malate dehydrogenase, mitochondrial63.31–1.18< 0.050Creatine kinase M-type70.87–1.190.080Myoglobin99.35–1.290.095Carbonic anhydrase 379.12–1.56< 0.010Perociredoxin-170.85–1.240.055ConclusionThe results demonstrate that variation in tenderness within U.S. Select Top loin steaks is due in large part to variation in proteolysis. The variation in tenderness and proteolysis in these defined groups may be partially explained by a greater abundance of metabolic proteins involved in oxidative metabolism. This project was funded in part by grants from the Beef Checkoff (Denver, CO) and the Iowa Beef Industry Council (Ames, IA). The scholarship for the first author was granted by CNPq-Brazil.

Determination of Protein Markers for Beef Tenderness in U.S. Select Beef

ObjectivesBeef tenderness is a primary quality feature that defines the consumer’s perception of the product quality. Therefore definition of chemical characteristics that contribute to variation in tenderness within a specific USDA grade can help refine our understanding of meat tenderness. The objective of this experiment was to identify novel protein markers for tenderness in Longissimus dorsi sarcoplasmic fractions utilizing proteomics and a valuable library of beef samples. Materials and MethodsSteaks (USDA Select grade) used in this trial were classified as low shear force (LSF, n = 12; 11.3 ± 1.6 kg) and high shear force (HSF, n = 12; 34.3 ± 4.7 kg), according to their slice shear force (SSF, kg) measurements at 14 d postmortem. Western blotting of whole-muscle samples was used to determine troponin-T degradation (indicator of overall proteolysis). Sarcoplasmic protein samples from d 2 postmortem were prepared for two-dimensional difference in gel electrophoresis (2D-DIGE). Samples from LSF and HSF were sorted according to SSF values and were matched to a pooled internal reference (representative of all samples). Protein samples were loaded (75 µg) on 24 cm strips, pH 6 to 9, and focused in immobilized pH gradient in the first dimension. For the second dimension the strips were run on 12.5% acrylamide gels (n = 12). After 2D-DIGE, protein spots found to be different in relative abundance between HSF and LSF samples, and with other proteins of interest, were identified using Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (LC-MS/MS). Immunoblots were performed to confirm protein identity. ResultsThe LSF samples exhibited greater troponin-T degradation (as evidenced by abundance of a 30 kDa fragment) than the HSF samples (LSF ratio to reference was 1.8, HSF ratio to reference was < 0.01; P < 0.01). A total of 31 spots showed a significant difference (P < 0.1) in relative abundance across groups (HSF vs. LSF). Among these, 18 spots were identified as 8 different proteins (Table 1). Proteins that were more abundant in the LSF samples are metabolic enzymes involved in the glycolytic pathway (glyceraldehyde-3-phosphate dehydrogenase, fructose bisphosphate aldolase A). Among the six proteins that showed greater abundance in the HSF samples, four belong to the oxidative energy metabolism pathways (malate dehydrogenase, creatine kinase M-type, myoglobin, carbonic anhydrase 3), one is involved in redox pathways (peroxiredoxin-1) and another one in protein turnover (polyubiquitin). The greater abundance of spots corresponding to oxidative metabolism enzymes suggests that aerobic metabolism may decrease potential for postmortem proteolysis. Table 1Differentially abundant proteins in high and low slice shear force groups Protein% CoverageRatio (LSF/HSF)P-valueGlyceraldehyde-3-phosphate dehydrogenase (GAPDH)26.731.980.060Fructose-bisphosphate aldolase A62.361.79< 0.050Polyubiquitin (Fragment)21.22–1.180.100Malate dehydrogenase, mitochondrial63.31–1.18< 0.050Creatine kinase M-type70.87–1.190.080Myoglobin99.35–1.290.095Carbonic anhydrase 379.12–1.56< 0.010Perociredoxin-170.85–1.240.055ConclusionThe results demonstrate that variation in tenderness within U.S. Select Top loin steaks is due in large part to variation in proteolysis. The variation in tenderness and proteolysis in these defined groups may be partially explained by a greater abundance of metabolic proteins involved in oxidative metabolism. This project was funded in part by grants from the Beef Checkoff (Denver, CO) and the Iowa Beef Industry Council (Ames, IA). The scholarship for the first author was granted by CNPq-Brazil.

Metabolomic profiling predicts outcome of rituximab therapy in rheumatoid arthritis

ObjectiveTo determine whether characterisation of patients' metabolic profiles, utilising nuclear magnetic resonance (NMR) and mass spectrometry (MS), could predict response to rituximab therapy. 23 patients with active, seropositive rheumatoid arthritis...

Proteomic analysis of the palmitoyl protein thioesterase 1 interactome in SH-SY5Y human neuroblastoma cells

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited progressive childhood disorders, characterized by early accumulation of autofluorescent storage material in lysosomes of neurons or other cells. Clinical symptoms of NCL include: progressive loss of vision, mental and motor deterioration, epileptic seizures and premature death. CLN1 disease (MIM#256730) is caused by mutations in the CLN1 gene, which encodes palmitoyl protein thioesterase 1 (PPT1). In this study, we utilised single step affinity purification coupled to mass spectrometry (AP-MS) to unravel the in vivo substrates of human PPT1 in the brain neuronal cells. Protein complexes were isolated from human PPT1 expressing SH-SY5Y stable cells, subjected to filter-aided sample preparation (FASP) and analysed on a Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer. A total of 23 PPT1 interacting partners (IP) were identified from label free quantitation of the MS data by SAINT platform. Three of the identified PPT1 IP, namely CRMP1, DBH, and MAP1B are predicted to be palmitoylated. Our proteomic analysis confirmed previously suggested roles of PPT1 in axon guidance and lipid metabolism, yet implicates the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway. Biological significanceThe significance of this work lies in the unravelling of putative in vivo substrates of human CLN1 or PPT1 in brain neuronal cells. Moreover, the PPT1 IP implicate the enzyme in novel roles including: involvement in neuronal migration and dopamine receptor mediated signalling pathway.

Determination of ethyl carbamate in fermented liquids by ultra high performance liquid chromatography coupled with a Q Exactive hybrid quadrupole-orbitrap mass spectrometer

In this study, the determination of ethyl carbamate (EC) in fermented liquids (red wines, Chinese liquors and yellow wines) was carried by using ultra high performance liquid chromatography coupled with a Q Exactive hybrid quadrupole-orbitrap mass spectrometer (UHPLC–MS/MS). The parameters usually examined in the method validation were firstly evaluated. Good linearity was obtained with a correlation coefficient of 0.9999. The limits of detection (LOD) and quantitation (LOQ) of EC in sample were 1.8 and 4.0μg/L, respectively. Recoveries ranged between 107.19% and 110.98%. The precision (relative standard deviation, RSD) of the method was <5%. Furthermore, red wines, Chinese liquors and yellow wines for sale were detected using this method. The results showed that the red wines and Chinese liquors had the normal EC level that was within the limits of the Canadian legislation. The proposed method was simple in sample preparation without using organic solvents in pre-treatment and could be used for the determination of the content of EC in fermented liquids.

Overexpression of Pro-Osteoclastogenic Cytokine Receptors and Chemokines By Bone Marrow CD14+ Monocytes of Multiple Myeloma (MM) Patients As Compared to Smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS): Role of Interleukin(IL)-21 Receptor/IL-21 Axis in MM-Induced Osteoclastogenesis

Multiple myeloma (MM) is characterized by the uncoupled increase in bone marrow (BM) of osteoclast formation and activation, which lead to bone destruction, as compared to patients with smoldering MM (SMM) and monoclonal gammopathy of uncertain significance (MGUS). Although the molecular analysis of clonal plasma cells (PCs) identified several genes whose overexpression is associated with the occurrence of bone lesions, a clear transcriptional fingerprint able to distinguish the different PC dyscrasias is lacking. As the close relationship between PCs and BM microenvironment plays a pivotal role in MM pathogenesis, ongoing studies are focusing on the presence of potential molecular alterations in the microenvironment. Among the different cell type of BM microenvironment, monocytes are known to be primarily involved in osteoclastogenesis, angiogenesis and immune function. The aim of this study was to analyze the transcriptional and proteomic profiles of the BM CD14+ cells across the different types of monoclonal gammopathies and to identify alterations potentially involved in the pathogenesis of the increased osteoclastogenesis.The expression profiles of CD14+ samples were evaluated by GeneChip HG-U133Plus 2.0 arrays (Affymetrix®) in 25 MM, 11 SMM and 8 MGUS patients. The proteomic analysis of CD14+ cells of 5 MM, 5 SMM and 5 MGUS was run on Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific®) and the data analyzed with Proteome Discoverer 1.4 software.A multiclass analysis identified 18 differentially expressed genes in MGUS, SMM and MM. The comparison of MM with both SMM and MGUS samples identified 61 genes differentially expressed in CD14+ cells (37 up-regulated and 24 down-regulated). Interestingly, we found specific cytokine receptors (IL21R and IL-15R) and pro-osteoclastogenic chemokines (CXCL10 and CXCL11) that were over-expressed in CD14+ of MM patients, as compared to SMM and MGUS. Similarly, the proteomic analysis reinforced that different CD14+ monocyte protein profiles were found comparing MM patients with MGUS and SMM ones. Interestingly, MM monocytes over-expressed proteins involved in cell adhesion and inflammation and down-regulated molecules implicated in antimicrobial functions.Because recent data indicate that IL-21 is a growth factor for MM cells and may promote osteoclastogenesis in some pathological conditions such as rheumatoid arthritis, we further investigate the potential role of IL-21R over-expression by MM CD14+ cells in osteoclastogenesis. Firstly, we confirmed the IL-21R up-regulation by CD14+ of MM patients at both mRNA and protein level as compared to both SMM and MGUS. Consistently we found that the conditioned media of human MM cell lines up-regulated IL21R mRNA expression by CD14+ cells. On the other hand any significant difference was not observed in the BM plasma IL-21 levels between MM, SMM and MGUS in a larger cohort of 160 patients. The treatment with rhIL-21, at the concentration observed in the BM plasma increased the osteoclast differentiation of the monocytic cell lines THP-1 and stimulated CD14+-derived in vitro osteoclastogenesis increasing the number and the size of osteoclasts, in MM patients but not in SMM and MGUS; this suggested higher sensitivity to the IL-21-dependent pro-osteoclastogenic differentiation effect in MM patients. Finally the overexpression or the knockout of IL-21R by lentivirus vectors, as well as the use of an IL-21R signaling inhibitor, was analyzed in the monocytic cell lines THP-1 to delineate a new anti-osteoclastogenic in vitro strategy. In conclusion our results indicate that different expression fingerprints characterize BM CD14+ monocytes of patients with MM as compared to those with SMM and MGUS, including over-expression of IL-21R, putatively involved in MM-induced osteoclast formation and activation through an increased sensitivity to IL-21. DisclosuresGiuliani:Celgene Italy: Research Funding.

Human Platelet Exosome Proteomics Leads to the Identification of WNT Positive Exosomes Which Impact Canonical WNT Signalling in Target Cells

Upon activation platelets release a myriad of soluble proteins as well as two distinct membrane vesicle populations- exosomes and microparticles (MPs) into the external milieu [1]. Although there have been many studies characterising platelet MPs and their increased circulating numbers in disease [2, 3], platelet exosomal vesicles have been characterised to date only by transmission electron microscopy (TEM) [4]. Here, we characterize the proteome of human platelet exosomes and reveal that a population of these vesicles carry active WNT glycoproteins on their surface that can modulate WNT signalling activity in both endothelial and monocytic cells.To establish the exosomal proteome of human platelets we compared platelet exosomes isolated from three healthy volunteers by (i) differential ultracentrifugation (DC) and (ii) ExoQuickTM (ExoQ) precipitation. Assessment of both exosomal populations was performed using nanoparticle tracking analysis (NTA), TEM, western blot and proteomics analysis (in triplicate) using a high performance Q Exactive Hybrid Quadrupole-Orbitrap mass spectrometer coupled with quantitative computational analysis using MaxQuant software. We concluded that DC was the superior isolation method for platelet exosomes, for example, our NTA revealed a 2-fold greater enrichment (6x108 /ml) of platelet exosomes following isolation by DC in comparison to ExoQ. Proteomic analysis of platelet exosomes identified 704 proteins across 18 MS runs, 119 of which were present in all 18 runs and represented the core human platelet exosomal proteome. Using western blotting and immunogold TEM, we confirmed the presence of numerous well-established exosomal membrane markers (CD63, CD9, and HSP70) in our proteome, as well as several platelet-specific proteins including GPIb, as previously described [4], and GPV.We also detected several of the 19 human WNT glycoproteins to be specifically secreted in the exosomal fraction upon platelet activation and verified their surface expression on platelet-derived exosomes using immunogold TEM. As we have previously demonstrated, platelets themselves can be modulated directly by WNT glycoproteins [5], hence here, we assessed the paracrine impact of these WNT-positive platelet exosomes on intracellular signalling events in monocytes and endothelial cells, both cell types closely linked to the pathogenesis of atherosclerosis. Using fluorescence microscopy, we found that these platelet exosomes were readily endocytosed by both endothelial (EAHY296) and monocytic (THP-1) cell lines where they specifically modulated beta-catenin nuclear expression and canonical WNT signalling.In conclusion our proteomic analysis has revealed that platelet exosomes are new players in the regulation of WNT signalling in both endothelial and monocytic cells.1. Coppinger, J.A., et al., Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood, 2004. 103(6): p. 2096-104.2. Knijff-Dutmer, E.A., et al., Elevated levels of platelet microparticles are associated with disease activity in rheumatoid arthritis. Arthritis Rheum, 2002. 46(6): p. 1498-503.3. van der Zee, P.M., et al., P-selectin- and CD63-exposing platelet microparticles reflect platelet activation in peripheral arterial disease and myocardial infarction. Clin Chem, 2006. 52(4): p. 657-64.4. Heijnen, H.F., et al., Activated platelets release two types of membrane vesicles: microvesicles by surface shedding and exosomes derived from exocytosis of multivesicular bodies and alpha-granules. Blood, 1999. 94(11): p. 3791-9.5. Steele, B.M., et al., Canonical Wnt signaling negatively regulates platelet function. Proc Natl Acad Sci U S A, 2009. 106(47): p. 19836-41. DisclosuresNo relevant conflicts of interest to declare.

Proteome analysis of stratum corneum from atopic dermatitis patients by hybrid quadrupole-orbitrap mass spectrometer

Proteome analysis of stratum corneum from atopic dermatitis patients by hybrid quadrupole-orbitrap mass spectrometer