ABSTRACT: Cerebrospinal fluid is a diagnostic biofluid that is reflective of the overall health of the patient. Proteomic profiling of human CSF has been performed on a variety of disease states, but a comprehensive proteomic profile of equine CSF has not been completed until now. A total of 320 proteins were confidently identified in a pooled sample representative of 6 healthy horses. Gene Ontology terms were mapped in Uniprot, and normalized spectral abundance factors were calculated as a measure of relative abundance. Briefly, CSF was collected from healthy horses via subarachnoid catheter or manual draw, protease inhibitors were added (Pierce, Rockford, IL), and samples were frozen at -80oC (Figure 1). Protein concentrations were determined via Bradford Assay (Thermo Scientific, Rockford, IL) and 30 ug of each sample underwent in-solution digestion using ProteaseMAX (Promega, Madison, WI) and urea. Samples were solubilized in 8 M urea, 0.2% protease max, then reduced, alkylated, and digested with 1% protease max and trypsin at 37oC for 3 hours. Samples were dried in a Speed Vac® vacuum centrifuge, desalted using Pierce PepClean C18 spin columns (Pierce, Rockford, IL), dried and resuspended in 30 ul 3% ACN, 0.1% formic acid. All solvents, water, and acid were LC-MS/MS grade from Sigma (St. Louis, MO). Online 2-dimensional LC-MS/MS with SCX (strong cation exchange) and subsequent reverse phase chromatography was performed as follows. 10 ug of digested peptides from a sample were loaded onto a Zorbax BIO-SCX II 3.5 umm, 50 x 0.8 mm column (Agilent Technologies, Santa Clara, CA). Peptides were eluted off of the SCX column step-wise using increasing concentrations of NaCl in 0.3% ACN, 0.1% FA (20 ul NaCl salt injections: 15, 30, 45, 60, 75, 90, 120, 150, 300, 500 mM). Peptides from each individual salt injection were then purified and concentrated using an on-line enrichment column (Agilent Zorbax C18, 5 um, 5 x 0.3mm). Subsequent chromatographic separation was performed on a reverse phase nanospray column (Agilent 1100 nanoHPLC, Zorbax C18, 5um, 75 um ID x 150mm column) using a 60 minute linear gradient from 25%-55% buffer B (90% ACN, 0.1% formic acid) at a flow rate of 300 nanoliters/min. Peptides were eluted directly into the mass spectrometer (Thermo Scientific LTQ linear ion trap) and spectra were collected over a range of 200-2000 m/z using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). Compound lists of the resulting spectra were generated using Bioworks 3.0 software (Thermo Scientific) with an intensity threshold of 5,000 and 1 scan/group. This workflow generates 10 raw data files per sample. MS/MS spectra were searched against the NCBInr equine database concatenated to a reverse database (14,473 entries) using the Mascot database search engine (Matrix Science, version 2.3.02) and SEQUEST (version v.27, rev. 11, Sorcerer, Sage-N Research). The following search parameters were used: average mass, peptide mass tolerance of 2.5 Da, fragment ion mass tolerance of 1.0 Da, complete tryptic digestion allowing one missed cleavage, variable modification of methionine oxidation, and a fixed modification of cysteine carbamidomethylation. Peptide identifications from both of the search engines were combined using protein identification algorithms in Scaffold 3 (Version 3.6.4, Proteome Software, Portland, OR). All data files were then combined using the MudPIT option in Scaffold 3 generating a composite listing for all proteins identified across all runs. Thresholds were set to 99% and 95% protein and peptide probability respectively, and a 2 unique peptide minimum was required. The peptide false discovery rate (FDR) was less than 0.2% after manual validation of all proteins identified by 2 unique peptides. Criteria for manual validation included the following: 1) a minimum of at least 3 theoretical y or b ions in consecutive order that are peaks greater than 5% of the maximum intensity; 2) an absence of prominent unassigned peaks greater than 5% of the maximum intensity; and 3) indicative residue specific fragmentation, such as intense ions N-terminal to proline and immediately C-terminal to aspartate and glutamate.
Project description:Herein, proteomic profiling was conducted on susceptible sugarbeet infected with BNYVV to clarify the types of proteins prevalent during compatible virus-host plant interactions. Total protein was extracted from sugarbeet leaf tissue infected with BNYVV (beet necrotic yellow vein virus), quantified, and analyzed by mass spectrometry. A proprietary sugarbeet variety, KWS-rzNI, susceptible to all forms of BNYVV, was provided by KWS (Einbeck, Germany). To enable detection of a broad range of proteins associated with compatible interactions, two sources of BNYVV were used; standard BNYVV pathotype A, originally collected from Spence Field at the USDA-ARS in Salinas, CA, and resistance-breaking BNYVV-IV originating from the field, Rockwood 158, Imperial County, CA. Pooled leaf tissue from each cup was ground in liquid nitrogen and lyophilized. Total protein was extracted from 100 mg (dry weight) of lyophilized leaf tissue of each sample using the Plant Total Protein Extraction Kit (Sigma, St. Louis, MO) according to manufacturer’s recommendations. Protease inhibitors were added to liquid protein extracts (Pierce, Rockford, IL), and samples were stored at -80oC prior to analysis. Protein concentrations were determined via Bradford Assay  (Thermo Scientific, Rockford, IL) and 30 g of each sample underwent in-solution proteolytic digestion as previously described . Briefly, samples were solubilized in 8 M urea, 0.2% Protease Max (Promega, Madision, WI), then reduced with dithiothreitol, alkylated with iodoacetamide, and digested with 1% Protease Max and trypsin at 37oC for 3 hours. Samples were dried in a Speed Vac® vacuum centrifuge, desalted using Pierce PepClean C18 spin columns (Pierce, Rockford, IL), dried and resuspended in 30 ull 3% ACN, 0.1% formic acid. All solvents, water, and acid were LC-MS/MS grade from Sigma (St. Louis, MO). 1 l of each sample (three replicates from each soil type) was analyzed in duplicate injections via LC-MS/MS . Peptides were purified and concentrated using an on-line enrichment column (Thermo Scientific 5m, 100 m ID x 2 cm C18 columns). Subsequent chromatographic separation was performed on a reverse phase nanospray column (Thermo Scientific EASYnano-LC, 3m, 75 m ID x 100 mm C18 column) using a 90 minute linear gradient from 10%-30% buffer B (100% ACN, 0.1% formic acid) at a flow rate of 400 nanoliters/min. Peptides were eluted directly into the mass spectrometer (Thermo Scientific Orbitrap Velos Pro) and spectra collected over a m/z range of 400-2000 Da using a dynamic exclusion limit of 2 MS/MS spectra of a given peptide mass for 30 s (exclusion duration of 90 s). Compound lists of the resulting spectra were generated using Xcalibur 2.2 software (Thermo Scientific) with a S/N threshold of 1.5 and 1 scan/group. MS/MS spectra were searched against a Uniprot Amaranthacae database (3,645 entries, version 03/06/13) concatenated to a reverse database and the Uniprot Mus musculus database (50,665 entries, version 03/06/13) using the Mascot database search engine (Matrix Science, version 2.3.02) and SEQUEST (version v.27, rev. 11, Sorcerer, Sage-N Research). Due to the limited size of the Amaranthaceae database, the Mus musculus sequences were added to ensure adequate database size for statistical scoring and calculation of false discovery rates. The following search parameters were used in Mascot: monoisotopic mass, parent mass tolerance of 20 ppm, fragment ion mass tolerance of 0.8 Da , complete tryptic digestion allowing two missed cleavages, variable modification of methionine oxidation, and a fixed modification of cysteine carbamidomethylation. SEQUEST search parameters were the same except for a fragment ion mass tolerance of 1.0 Da and a parent ion tolerance of 0.0120 Da. Peptide identifications from both of the search engines were combined using probabilistic protein identification algorithms in Scaffold 4 [13, 14] (Version 4.0.3, Proteome Software, Portland, OR) with protein clustering enabled. All data files were then combined using the “mudpit” option in Scaffold 4 generating a composite listing for all proteins identified. Thresholds were set to 99% protein probability, 95% peptide probability, and a 2 unique peptide minimum was required. The peptide FDR was 0% after manual validation of all proteins identified by 2 unique peptides . Criteria for manual validation included the following: 1) a minimum of at least 5 theoretical y or b ions in consecutive order that are peaks greater than 5% of the maximum intensity; 2) an absence of prominent unassigned peaks greater than 5% of the maximum intensity; and 3) indicative residue specific fragmentation, such as intense ions N-terminal to proline and immediately C-terminal to aspartate and glutamate.
Project description:Female Sprague-Dawley rats were randomly separated into two groups: i) trained (Ex group) and ii) sedentary (Cont group). Animals from the Ex group were adapted to treadmill exercise for two consecutive weeks, involving a gradual increase in the running time till 60 min/day at 20 m/min, 0% grade, 5 days/week. This exercise program remained constant until the end of the 54 weeks. Mitochondria isolation from 10 animals (5 Ex group and 5 Cont group) was performed using the conventional methods of differential centrifugation. Mitochondrial protein extracts were reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin using the FASP procedure (18). Briefly, each sample was solubilized in 4 % SDS, 0.1 M DTT, 0.1 M HEPES and incubated for 30 min at 60 C. Then, samples were mixed with 0.2 mL of a solution of 8 M Urea, 0.1 M HEPES, pH 8.5 (UH solution), loaded into the filtration devices (3k Microcon, Millipore), centrifuged at 14,000 g for 20 min, and washed twice with UH solution. After centrifugation, the concentrates were alkylated with 50 mM iodoacetamide in UH solution (dark, 25 C, 20 min), and washed twice with UH solution. The concentrate was diluted with 0.1 mL of 50 mM ammonium bicarbonate (ABC) and digested overnight (37 C) with the addition of 2 ug of trypsin diluted in 30 uL of 50 mM ABC. The resulting tryptic peptides were collected by centrifugation and the filter were washed twice with 50 uL of 50 mM ABC. Eluted peptides were acidified with 10 uL of formic acid and cleaned up on a homemade Empore C18 column (3M, St. Paul, MN, USA) (ref) for subsequent phospho-enrichment and/or analysis by LC-MS/MS. The dried protein digest was dissolved with 10 uL (3 % ACN, 0.1 % TFA), diluted 1:5 with loading buffer (1 M glycolic acid, 5 % TFA, 80 % ACN) and phosphopeptides were enriched on TiO2-beads as previously described (19). Briefly 5 mg of "Titansphere TiO2 5 um" were suspended in 100 uL of 100 % ACN, immobilized in a pipette-column and washed using 5 uL of loading buffer. Each sample was loaded in each pipette-column and then washed with 30 uL of washing buffer (1 % TFA, 80 % ACN). Phosphopeptides were eluted from the beads with 25 uL of 25 % ACN (v/v) containing 25 % NH4OH (m/v), acidified with 10 uL of 10 % TFA and vacuum- concentrated to approximately 4 uL. Samples were finally diluted to 10 uL with H2O + 0.1% formic acid prior to injection. The peptides mixtures were analyzed by online nanoflow liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) on an EASY-nLC system (Proxeon Biosystems, Odense, Denmark) connected to the LTQ Orbitrap Velos instrument (Thermo Fisher Scientific, Bremen, Germany) through a nanoelectrospray ion source. Six microliters of the peptide mixture were auto-sampled directly onto the analytical HPLC column (120 mm x75 um I.D., 3 um particle size (Nikkyo Technos Co., Ltd.) with a 120-min gradient from 5 % to 50 % ACN in 0.1 % FA. The effluent from the HPLC column was directly electrosprayed into the mass spectrometer. The LTQ Orbitrap Velos instrument was operated in data-dependent acquisition mode to automatically switch between full scan MS and MS/MS acquisition. The MS survey scan was performed in the FT cell recording a window between 350 and 2000 m/z. The resolution was set to 60 000, and the automatic gain control was set to 1,000,000 ions with a maximal acquisition time of 400 ms. The 20 most intense peptide ions with charge states great than or equal to 2 were sequentially isolated to a target value of 5,000 and fragmented in the high-pressure linear ion trap by low-energy CID with normalized collision energy of 35% Q value of 0.25, and an activation time of 10 ms. Raw files were processed using Proteome Discoverer version 1.3 (Thermo Fisher Scientific, Bremen). Peak lists were searched using Mascot software version 2.3 (Matrix Science, UK) against a SwissProt database containing entries corresponding to Rattus norvegicus (version of July 2012), a list of common contaminants, and all the corresponding decoy entries. Trypsin was chosen as enzyme and a maximum of two miscleavages were allowed. Carbamidomethylation (C) was set as a fixed modification, whereas oxidation (M) and phosphorylation (STY) were used as variable modifications. Searches were performed using a peptide tolerance of 7 ppm, a product ion tolerance of 0.5 Da. Resulting data files were filtered for FDR less than 1 % at peptide level.
Project description:The experiment consists of M.hyp 232 cultures digests analyzed on two mass specs, LTQ Velos Pro and LTQ FT Ultra LTQ Velos Pro: Six cell culture replicates of M.hyo 232 were hydrophobically separated using tree detergents: Digitonin, Tween, and SDS. Each francion, including the insoluble pellet, was trypsin digested and then cleaned using an SCX trap follwed by a RP trap to remove detergents. Each fraction was sepated using a Dionex U3000 splitless nanoflow system operating at 333 nl per minute using a gradient of 2% ACN to 50% ACN in 4 hours. Eluate was analyzed using an LTQ Velos Pro mass spectrometer with 20 MS/MS scans of the 20 most intense peaks from each MS scan. Dynamic exclusion was enable for 3 minutes for each m/z with a repeat count of 1. LTQ FT Ultra: Two cell pellets for high resolution analysis were lysed and trypsin digested. Digested peptides were dried, resuspended in 20 mM KH2PO4, 20% ACN, pH 3 (Buffer A) in 2.5 µL and transferred to low retention vials in preparation for separation using an Ultimate 3000 configured for 2D-LC. Each sample was loaded at 15 µl/min onto an SCX microtrap for the first dimension of separation, involving SCX steps of Buffer A + 0, 5, 10, 15, 20, 25, 30, 40, 50, 100, 250, 500, and 1000 mM KCl. For the second dimension of separation, each eluted salt step was desalted with an inline peptide microtrap with 2% ACN, 0.1% FA at 5 µl/min. Once desalted, the microtrap was switched into line with a fritless nano column (75µm x ~10cm) containing C18 media (5µ, 200 Å Magic, Michrom). Peptides were eluted using a gradient of 2% to 36% ACN, 0.1% FA at 350 nl/min over 60 min and electrospray ionized for analysis using an LTQ FT Ultra mass spectrometer. A survey scan m/z 350-1750 was acquired in the FT ICR cell (Resolution = 100,000 at m/z 400, with an accumulation target value of 1,000,000 ions). Up to the 6 most abundant ions (>3,000 counts) with charge states > +2 were sequentially isolated and fragmented within the linear ion trap using collisionally induced dissociation with an activation q = 0.25 and activation time of 30 ms at a target value of 30,000 ions. M/z ratios selected for MS/ MS were dynamically excluded for 30 seconds. Analysis: X!tandem searches were performed using the Mycoplasma hyopneumoniae strain 232 reference protein set from NCBI. The only difference between the searches for the LTQ Velos and LTQ FT was the precursor mass tolenance being set to +-1500ppm and +-24ppm respectively. Decoy searches were performed and the data filtered at e-value <= 0.01 with single peptide proteins discarded. These results are included in the submission as two tab-delimited text files.
Project description:Multiple sclerosis (MS) is a demyelinating disease causing major neurological disability in young adults. We characterized the transcriptome of the choroid plexus (CP), which is part of the blood-brain barriers and the major site of cerebrospinal fluid (CSF) production, in the experimental autoimmune encephalomyelitis mouse model of MS. Genes encoding for adhesion molecules, chemokines and cytokines displayed the most altered expression, supporting the CP as a site of immune-brain interaction in MS. The gene encoding for lipocalin 2 (LCN2) was the most up-regulated, and CSF LCN2 levels coincided with the phases of active disease. Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. After quality assessment using the Agilent Bioanalyzer (Agilent Technologies, CA, USA), 100ng of total RNA from 3 pooled controls and 3 or 2 pooled samples of each experimental autoimmune encephalomyelitis (EAE) phase were amplified and labelled with the Illumina TotalPrep RNA Amplification Kit (Illumina Inc., San Diego, CA, USA). The labeled cRNA was then hybridized using the recommended protocol in a total of two Illumina Whole-Genome MouseRef-8 expression Beadchips (Illumina Inc., San Diego, CA, USA).
Project description:MIADB: a cumulative collection of 172 tandem mass spectrometry (MS/MS) of a vast array of monoterpene indole alkaloids. Samples were analyzed using an Agilent LC-MS system composed of an Agilent 1260 Infinity HPLC coupled to an Agilent 6530 ESI-Q-TOF-MS operating in positive mode. A Sunfire analytical C18 column (150 × 2.1 mm; i.d. 3.5 μm, Waters) was used, with a flow rate of 250 μL/min and a linear gradient from 5% B (A: H2O + 0.1% formic acid, B: MeOH) to 100% B over 30 min. ESI conditions were set with the capillary temperature at 320 °C, source voltage at 3.5 kV, and a sheath gas flow rate of 10 L/min. The divert valve was set to waste for the first 3 min. There were four scan events: positive MS, window from m/z 100−1200, then three data-dependent MS/MS scans of the first, second, and third most intense ions from the first scan event. MS/MS settings were three fixed collision energies (30, 50, and 70 eV), default charge of 1, minimum intensity of 5000 counts, and isolation width of m/z 2. In the positive-ion mode, purine C5H4N4 [M + H]+ ion (m/z 121.050873) and the hexakis(1H,1H,3H-tetrafluoropropoxy)-phosphazene C18H18F24N3O6P3 [M + H]+ ion (m/z 922.009 798) were used as internal lock masses. Full scans were acquired at a resolution of 11 000 (at m/z 922). A permanent MS/MS exclusion list criterion was set to prevent oversampling of the internal calibrant.
Project description:Proteomics section: 300ug of rotein extract from Ostreococcus tauri cells were reduced alkylated and trypsin digested. Phosphopeptide enrichment done on a Titanium dioxide column. LC-MS analysis performed on a cap-HPLC-MS (Orbitrap XL). Data analysis: Data were searched using Mascot. Peptide quantitation performed with Progenesis. Position of the phosphorylated amino acid confirmed with Maxquant and manual validation.
Project description:Intense immunosuppression followed by autologous hematopoietic stem cell transplantation (aHSCT) is a potential treatment for patients suffering from aggressive multiple sclerosis (MS). However it remains unresolved whether autologous CD34+ hematopoietic progenitor cells of MS patients show gene expression differences prior to aHSCT that indicate a preset proinflammatory state, which would then also predispose to or predetermine recurrence of the autoimmune disease. To approach this key point we compared the gene expression signature of CD34+ and CD34- cells collected from MS patients and healthy donors (HD). Whole genome gene expression of CD34+ and CD34- cells was analysed with the Human 4x44K Design Array (Agilent-Technologies). As main observation we found only minor differences in the gene expression signature of MS patients compared to HD. Only a single gene, troponin-type-1 (TNNT1), reached statistical significance after correction for multiple comparisons (logFC=3.1, p<0.01). There was a decreased expression of several protease genes, myeloperoxidase (MPO), neutrophil-elastase (ELA2), cathepsin-G (CTSG) and serine-protease 21 (PRSS21) in HPCs of MS patients, albeit not reaching statistical significance. In summary we did not detect substantial alterations in the gene expression profile of CD34+ HPCs in MS. Our data support the use of autologous hematopoietic stem cell transplantation for treatment of an autoimmune disease. Overall design: Samples of CD34+ cells were obtained from 4 female MS patients and 4 age matched healthy donors (3 female) mobilized by G-CSF (2x5μg/kg/day) and 4 MS patients (2 female) mobilized by G-CSF (5μg/kg/day) plus Cyclophosphamide (Cy, 4g/m2). White blood cells, containing the CD34+ cell fraction, were collected by leucocytapheresis from peripheral blood, frozen and stored in liquid nitrogen. All samples were thawed and processed at one center and CD34+ HPCs purified by magnetic bead separation using the autoMACS system (Miltenyi). Purity and viability of CD34+ cells was analyzed by Fluorescence Activated Cell Sorter (FACS). Total cellular RNA were extracted with TRIzol reagent and analyzed with the Human 4x44K Design Array (Agilent-Technologies).
Project description:Cervicovaginal lavage (CVL)supernatants were heat inactivated for 30 minutes at 56°C before further processing. Total protein concentrations were determined using the Pierce Coomassie Plus (Bradford) Protein Assay (Thermo Scientific, Rockford, IL, USA). Sample protein content and volume were normalised with 25mM ammonium bicarbonate (ABC). Soluble proteins were precipitated using an equal volume of ice cold 30% (w/v) TCA in acetone and incubated at -20⁰C for 2 hours. Samples were centrifuged at 12,000g for 10 minutes (4⁰C) to pellet proteins. Pellets were washed three times with ice cold acetone and allowed to air dry. Further sample processing was performed as previously described (Armstrong et al, 2014) with minor modifications. Briefly, protein pellets were resuspended in 25 mM ABC, 0.05%(w/v) rapigest (Waters), reduced and alkylated. Digestion was performed with proteomic-grade trypsin (Sigma-Aldrich, St. Louis, MO, USA) at a protein:trypsin ratio of 50:1. Rapigest was precipitated by addition of trifluoroacetic acid to a final concentration of 0.5% (v/v). Peptide mixtures were analyzed by on-line nanoflow liquid chromatography using the nanoACQUITY-nLC system (Waters MS technologies) coupled to an LTQ-Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) mass spectrometer equipped with the manufacturer’s nanospray ion source. The gradient of the analytic column (nanoACQUITY UPLCTM BEH130 C18 15cm x 75µm, 1.7µm capillary column) consisted of 3-40% acetonitrile in 0.1% formic acid for 90 min then a ramp of 40-85% acetonitrile in 0.1% formic acid for 5min.
2016-03-15 | PXD003176 | Pride
Project description:Microbiome of CSF from MS patients
Project description:1.6 mL CSF from a pool of 21 neurologically healthy donors was separated into a bound and a depleted fraction using a MARS Hu-14 column. This experiment represents the depleted fraction. Sample was in-solution trypsin digested and resulting peptides were fractionated into 66 fractions by mixed-mode reversed phase anion exchange (MM(RP-AX) using a Promix MP column. Each fraction was analyzed separately by LC-MS/MS on an Orbitrap Velos Pro mass spectrometer. Resulting data was searched using SeachGui, summarized in PeptideShaker and exported to the CSF-PR.