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Equine Cerebrospinal Fluid - data analysis

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.

INSTRUMENT(S): LTQ

ORGANISM(S): Equus Caballus (horse)

SUBMITTER: Carolyn Broccardo

PROVIDER: PXD000133 | Pride | 2014-04-23

REPOSITORIES: Pride

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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 [10] (Thermo Scientific, Rockford, IL) and 30 g of each sample underwent in-solution proteolytic digestion as previously described [11]. 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 [15]. 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:2-cell (E1.5) stage embryos were cultured in normal KSOM+AA conditions until E3.5 +2h and thereafter 100 embryos each were moved to control or p38-MAPKi conditions and cultured for another 7 hours (E3.5 +9h). The embryos were then washed through pre-warmed (37˚C) Hank’s balanced salt solution (HBSS, Sigma-Aldrich; Cat. No. H9269) and lysed by moving to a 1.5ml centrifuge tube containing about 15µl of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT). Cell lysis was performed by incubating the tubes in a 95˚C thermoblock for 12 minutes, brief centrifugation at 750 rpm, cooling to room temperature and storage at -80˚C. Individual protein solutions were processed by filter-aided sample preparation (FASP) method with some modifications (see the associated publication for more details). Resulting peptides were cleaned by liquid-liquid extraction (3 iterations) using water saturated ethyl acetate. 1/10th of the FASP eluate was taken out for direct LC-MS measurements, evaporated completely in SpeedVac concentrator (Thermo Fisher Scientific). Peptides were further transferred into LC-MS vials using 50μL of 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100μL of pure ACN and with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator. Phosphopeptides were enriched from the remaining 9/10th of the cleaned FASP eluate after complete solvent evaporation (SpeedVac concentrator) using High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) according to manufacturer protocol. Phosphopeptide standards (0.1 pmol of MS PhosphoMix 1, 2, 3 Light; Sigma-Aldrich, St. Louis, Missouri, USA) was added to suspended sample in binding/equilibration buffer. Flow-through fraction was dried and used for second enrichment step using High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer protocol. Resulting phosphopeptides were extracted into LC-MS vials by 2.5% FA in 50% ACN and 100% ACN with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator). LC-MS/MS analyses of all peptide mixtures (2 peptide solutions for each sample – 1) not enriched; 2) enriched on phosphopeptides using TiO2 enrichment kit) were done using RSLCnano system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Prior to LC separation, tryptic digests were online concentrated and desalted using trapping column (100 μm × 30 mm, column compartment temperature of 40˚C) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow rate - 300nl/min) from the trapping column onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; column compartment temperature of 40˚C, Thermo Fisher Scientific) by using 50 or 100 minutes long nonlinear gradient program (1-56% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN) for analysis of phosphopeptide enriched fractions or not enriched peptide mixtures, respectively. Equilibration of the trapping column and the analytical column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 (New Objective) ion source with sheath gas option and SilicaTip emitter (New Objective; FS360-20-15-N-20-C12) utilization. ABIRD (ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy with cycle time for 3 seconds and with survey scan (350-2000 m/z). The resolution of the survey scan was 60000 (200 m/z) with a target value of 4×105 ions and maximum injection time of 50ms. HCD MS/MS (30% relative fragmentation energy, normal mass range) spectra were acquired with a target value of 5.0x104. The MS/MS spectra were recorded in Orbitrap at resolving power of 30,000 or 15,000 (200 m/z) and the maximum injection time for MS/MS was 500 or 22 ms for analysis of phosphopeptides enriched fraction or non-enriched peptide mixture, respectively. Dynamic exclusion was enabled for 60 seconds after one MS/MS spectra acquisition. The isolation window for MS/MS fragmentation was set to 1.6 m/z.

Project description:Cyanophora paradoxa muroplasts were isolated on a Percoll gradient. Muroplasts were fractionated into soluble (stroma), envelope, and thylakoid proteins. Protein samples from envelope membranes, thylakoids, stroma and total muroplasts were separated by 12% SDS-PAGE. Each Protein lanewas cut into 10 slices of variable size to match similar protein amounts. The gel slices were washed and diced to 1mm3 cubes. Gel pieces were destained by two consecutive washes with a 50:50 mixture of 200 mM ammonium bicarbonate and acetonitrile (ACN) for 20 min at 37°C. Proteins were reduced with 10mM DTT for 45 min at 56°C, followed by alkylation with 55mM iodoacetamide for 45 min at room temperature in the dark. After reduction and alkylation, gel pieces were washed twice with 100 mM ammonium bicarbonate, dehydrated with 100% ACN for 10 min, and dried in a SpeedVac. Gel pieces were fully covered in 50 mM ammonium bicarbonate containing 10ng/ml trypsin and rehydrated at 4°C. The samples were incubated overnight at 37°C and peptides in the supernatant were pooled after successive extraction steps using 50 mM ammonium bicarbonate, 100% ACN and 2.5% formic acid (FA), 50% ACN. The samples were dried in a SpeedVac and reconstituted in 5% ACN, 0.1% FA prior to MS analysis. Each of the samples were analysed in duplicates. Tryptic peptides of the thylakoid, envelope and stroma fractions were loaded onto a fritless 100 μm capillary packed in-house with 10cm of ProntoSIL C18 ace-EPS, 3µm. A gradient of 5-80% (v/v) ACN in 0.1% (v/v) FA was passed over the column with a duration of 115 min. The eluted peptides were directly electrosprayed into the LTQ orbitrap XL MS at a flow rate of 250 nl min-1 and a spray voltage of 1.3 kV. The instrument was operated in a 4-step data-dependent positive mode to identify the top three most abundant ions in a high-resolution MS scan (400 to 2000 m/z), which were then selected for fragmentation. MudPIT analyses were performed on the muroplast samples using an in-house packed analytical column consisting of 10 cm ProntoSIL C18 ace-EPS, 3µm (Bischoff) and 2 cm of a strong cationic exchange (SCX) material, 3µm in combination with an 6-step salt pulse gradient (0, 50, 100, 150, 200 and 500 mM ammonium acetate). Each salt-step peptides were eluted from the SCX to the C18-phase of the analytical column and eluated with a 130- min reverse-phase solvent gradient (5–80% ACN containing 0.1% FA). The eluate was directly electrosprayed into the LTQ orbitrap XL MS which was operated as described before. All MS/MS samples were analyzed using Sequest and X! Tandem. Sequest was set up to search the C. paradoxa nuclear and muroplast protein database with a total of 32,286 entries, assuming the digestion enzyme trypsin. X! Tandem was set up to search a subset of the C. paradoxa database also assuming trypsin. Sequest and X! Tandem were searched with a fragment ion mass tolerance of 0,80 Da and a parent ion tolerance of 10,0 ppm. Oxidation of methionine and iodoacetamide derivatives of cysteine were specified in Sequest and X! Tandem as variable modifications. Scaffold (version 3.5.1, Proteome Software Inc., Portland, OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95,0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99,0% probability and contained at least 2 identified peptides.

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:Cells were treated with DMSO (biological triplicate) or degrader at indicated dose and time (Meta Treatment Table) and cells were harvested by centrifugation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, Protease and Phosphatase inhibitors from Roche) was added to the cell pellets and homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 1 - 4 mg/mL. A micro-BCA assay (Pierce) was used to determine the final protein concentration in the cell lysate. 200 µg of protein for each sample were reduced and alkylated as previously described1. Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme:protein) for 12 hours at room temperature. The LysC digestion was diluted to 0.5 M Urea with 200 mM EPPS pH 8 followed by digestion with trypsin (1:50; enzyme:protein) for 6 hours at 37 °C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label. The 11 or 16-plex labeling reactions were performed for 1.5 hours at room temperature and the reaction quenched by the addition of hydroxylamine to a final concentration of 0.3% for 15 minutes at room temperature. Each of the sample channels were combined in a 1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio comparison. Samples were then combined using the adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was then resuspended in 1% formic acid and acidified (pH 2 - 3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions and these fractions were used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific) or an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with an UltiMate 3000 RSLCnano System. Peptides were separated on an EasySpray ES803a/ES803a.rev2 75 μm inner diameter microcapillary column (Thermo Fisher Scientific) or a 100 µm inner diameter microcapillary column packed with ~ 50 cm of Accucore C18 resin (2.6 µM, 100 Å, Thermo Fisher Scientific). Peptides were separated using a 190 min gradient of 6 - 27% acetonitrile in 1.0% formic acid with a flow rate of 300 or 350 nL/min. Each analysis used an MS3-based TMT method as described previously. The data were acquired using a mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 2 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.

Project description:The proximal convoluted tubule is the primary site of renal fluid, electrolyte and nutrient reabsorption, processes that consume large amounts of ATP. Previous proteomic studies have profiled the adaptions that occur in this segment of the nephron in response to onset of metabolic acidosis. Proximal convoluted tubule isolation and mitochondrial enrichment procedures were described previously. Briefly, rat renal cortex was digested with collagenase and proximal convoluted tubules were purified by Percoll density gradient centrifugation. Mitochondria were isolated by differential and sucrose density centrifugation, which produced an 8-fold enrichment in specific activity of cytochrome C oxidase . The isolated mitochondria were resuspended in H2O, incubated on ice for 20 min with gentle vortexing, and then centrifuged at 12,000xg for 5 min. The pellet and little remaining supernatant were centrifuged again at 12,000xg for 5 min to tighten the pellet and remove residual supernatant. The combined supernatants contain the mitochondrial outer membrane and intermembrane proteins while the final pellet contains mitoplasts. The mitoplasts were subsequently treated with 0.1 M Na2CO3, pH 11.5, incubated on ice for 20 min, and then centrifuged at 100,000xg for 30 min. The supernatant contains matrix proteins and the pellet is the enriched MIM fraction. Samples containing 50 µg of protein were precipitated with acetone and an in-solution tryptic digestion preformed with ProteaseMax (Promega). The peptides were fractionated on a reverse phase column (1200 nanoHPLC, Zorbax C18, 5 um, 75 um ID x 150mm column, Agilent) using a 90 minute linear gradient from 25%-55% buffer B (buffer B = 90% acetonitrile and 0.1% formic acid) at a flow rate of 300 nl/min. Peptides were eluted directly into the mass spectrometer (LTQ linear ion trap, Thermo Scientific) and spectra were collected over a m/z range of 200-2000 Da using a dynamic exclusion limit of 3 MS/MS spectra of a given peptide mass for 30 sec and an exclusion duration of 90 sec. The compound lists from the resulting spectra were produced using Xcalibar 2.2 software (Thermo Scientific) with an intensity threshold of 5,000 and 1 scan/group. Each biological sample was injected five times for LC-MS/MS analysis. MS/MS spectra were searched against the Uniprot-KB Rattus norvegicus protein sequence database (74,140 sequence entries) concatenated with a reverse database using both the Mascot (version 2.3.02, Matrix Science) and SEQUEST (version v.27, rev. 11, Sorcerer, Sage-N Research) database search engines. 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 four missed cleavages, variable modifications of methionine oxidation and lysine acetylation, and a fixed modification of cysteine carbamidomethylation. Peptide identifications from both of the search engine results were combined using protein identification algorithms in Scaffold 3 (Version 3.6.0, Proteome Software). Peptide and protein probability thresholds of 90% and 99% were applied with Peptide and Protein Prophet algorithms in Scaffold 3 [6] [7]. Proteins containing shared peptides were grouped by Scaffold 3 to satisfy the laws of parsimony. A peptide false discovery rate (FDR) was determined by the target decoy approach of searching the reversed database [8]. Only proteins that were identified by a minimum of 2 unique peptides in at least 1 biological replicate were considered confidently identified. Manual validation of MS/MS spectra was performed for protein identifications that met the initial thresholds that were based on two unique peptides and for all of the acetylated peptides. The analysis confidently identified 207 proteins in the combined samples. Further proteomic analysis identified 14 peptides that contain an N-epsilon-acetyl-lysine, 7 of which are novel sites. This study provides the first proteomic profile of the mitochondrial inner membrane proteome of this segment of the rat renal nephron.

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Equine Cerebrospinal Fluid - data analysis

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