The Proteome of chicken cecum inoculated with Salmonella enterica serovar Enteritidis
Ontology highlight
ABSTRACT:
ORGANISM(S): Gallus Gallus
SUBMITTER: 
Xianyao Li
PROVIDER: PXD047433 | iProX | Fri Dec 01 00:00:00 GMT 2023
REPOSITORIES: iProX
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Project description:Carfilzomib-sensitive (AMO1) and resistant (AMO1-CFZ) MM cells were grown in RPMI-1640 medium supplemented with 10 % FBS and 0.68 mM L-glutamine, in a humidified incubator at 5% CO2 and 37 °C. The AMO1-CFZ medium was additionally added 90 nM CFZ, while the CFZ sensitive AMO1 cells were added vehicle (0.009 % DMSO) only. AMO1-CFZ was either harvested directly or grown for 1 week in CFZ free medium (vehicle only) prior to harvest. AMO1 and carfilzomib-resistant AMO1-CFZ cells were resuspended in 100 µl 1% sodium deoxycholate, 100 mM Tris-HCl pH 8.5, 10 mM tris(2-carboxyethyl)phosphine (TCEP), 40 mM chloroacetamide (CAA), heated at 90 °C for 45 min and sonicated for 10 cycles (30 s ON/30 s OFF) using a Bioruptor pico sonicator. After centrifugation at 16000 × g for 10 min, 50 µg soluble protein from each sample was added 100 µl 0.1M ammonium bicarbonate, 0.5 µg trypsin and digested overnight at 37°C. Peptides were desalted using C18 spin columns, dried in a speedvac centrifuge and resuspended in 0.1% formic acid prior to MS analysis. Label-free quantitatative (LFQ) LC-MS/MS was performed on a timsTOF Pro (Bruker Daltonics) connected to a nanoElute (Bruker Daltonics) HPLC. Peptides were separated over a Bruker PepSep C18 (75 µm × 15 cm) column with running buffers A (0.1 % formic acid) and B (0.1 % formic acid in acetonitrile) using a 100 min gradient from 2 % B to 40 % B. The timsTof instrument was operated in the DDA PASEF mode with 10 PASEF scans per acquisition cycle and accumulation and ramp times of 100 ms each. The ‘target value’ was set to 20000 and dynamic exclusion was activated and set to 0.4 min. The quadrupole isolation width was set to 2 Th for m/z < 700 and 3 Th for m/z > 800.
2024-06-07 | PXD045439 | Pride
Project description:Bacteria were washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 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 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da; Oxidation (M) and Carbamidomethyl (C) specified as variable modifications, Enzyme specificity was trypsin, 1 missed cleavage was possible
2014-04-28 | PXD000504 | Pride
Project description:Washed three times in NaCl (150 mM). Cells were resuspended in 1 ml TSU buffer (50 mM Tris pH 8.0, 0.1% SDS, 2.5 M urea) and lysed by two freeze-thaw cycles in liquid nitrogen. Lysate proteins (40 ug) were separated and digested. Digested peptides were separated by nano-LC using an Ultimate 3000 HPLC and autosampler system (Dionex; Amsterdam, Netherlands). Samples (1 ul) were concentrated and desalted onto a micro C18 pre-column (500 um×2 mm, Michrom Bioresources; Auburn, CA, USA) with H2O:CH3CN (98:2, 0.05% trifluoroacetic acid) at 15 ul min−1. After a 4 min wash the pre-column was switched (Valco 10 port valve; Dionex) into line with a fritless nano column (75 u×~10 cm) containing C18 media (5 u, 200 A Magic; Michrom) manufactured according to Gatlin. Peptides were eluted using a linear gradient of H2O:CH3CN (98:2, 0.1% formic acid) to H2O:CH3CN (64:36, 0.1% formic acid) at 250 nl min−1 over 30 min. High voltage (2000 V) was applied to low volume tee (Upchurch Scientific) and the column tip positioned ~0.5 cm from the heated capillary (T = 280 degrees C) of an Orbitrap Velos (Thermo Electron; Bremen, Germany) mass spectrometer. Positive ions were generated by electrospray and the Orbitrap operated in data dependent acquisition mode (DDA). A survey scan m/z 350–1750 was acquired in the Orbitrap (Resolution = 30,000 at m/z 400, with an accumulation target value of 1,000,000 ions) with lockmass enabled. Up to the 10 most abundant ions (>5,000 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 s. Peak lists were generated using Mascot Daemon/extract_msn (Matrix Science, Thermo; London, England) using the default parameters, and submitted to the database search program Mascot (version 2.1, Matrix Science). Search parameters were: Precursor tolerance 4 ppm and product ion tolerances +/-0.4 Da Oxidation (M) and Carbamidomethyl (C) specified as variable modifications Enzyme specificity was trypsin, 1 missed cleavage was possible
2014-04-28 | PXD000505 | Pride
Project description:Samples were subjected to LC–MS analysis using a dual pressure LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) connected to an electrospray ion source (Thermo Fisher Scientific) as recently described (PMID: 23017020). Peptide separation was carried out on an EASY nLC-1000 system (Thermo Fisher Scientific) equipped with a RP-HPLC column (75 μm × 30 cm) packed in-house with C18 resin (ReproSil-Pur C18–AQ, 1.9 μm resin, Dr. Maisch GmbH). A step-wise gradient from 95% solvent A (0.1% formic acid) and 5% solvent B (80% acetonitrile, 0.1% formic acid) to 50% solvent B over 60 min at a flow rate of 0.2 μl/min was used. Data acquisition mode was set to obtain one high resolution MS scan in the FT part of the mass spectrometer at a resolution of 240,000 full width at half-maximum (at m/z 400) followed by 20 MS/MS scans (TOP20) in the linear ion trap of the most intense ions using rapid scan speed. Unassigned and singly charged ions were excluded from analysis. Dynamic exclusion duration was set to 30 seconds.
2022-02-24 | PXD012490 | Pride
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.
2012-10-30 | PXD000056 | Pride
Project description:N6,2'-O-dimethyladenosine (m6Am) is a critical and reversible RNA modification that plays a significant role in regulating RNA stability and gene expression. However, the lack of precise tools for manipulating m6Am modifications on specific transcripts has hindered the ability to elucidate the relationship between m6Am-modified transcripts and their phenotypic outcomes. Here, we present a CRISPR-Cas13d-based tool for the targeted installation of m6Am modifications on specific RNA transcripts. This tool is engineered by fusing a catalytically inactive variant of RfxCas13d (dCasRx) with the m6Am methyltransferase PCIF1. The dCasRx-PCIF1 fusion protein enables precise methylation of target m6Am-modified RNAs. We demonstrate that INSTALLER can effectively install m6Am modifications on GAPDH and ACTB RNAs, significantly enhancing their stability. MTD keywords Human, HEK293T cells MTD sample_processing_protocol Protein extraction and tryptic digestion. Cell lysates were lysed in lysis buffer (RPIA) supplemented with protease inhibitors and phosphatase inhibitors for 30 min. Then they were sonicated by grinding instrument at 4 ℃ and centrifuged at 12000 rpm for 15 min to remove the tissue debris. The protein in supernatants were collected and measured by using BCA protein assay. Next, 20 mM Tris (2-chloroethyl) phosphate (TCEP) was added to the protein solution to react at 60℃ for 1 h. After that, 200 mM Chloroacetamide (CAA) was added to the mixture and react at room temperature for 30 min in darkness. After the reaction, ice-cold acetone was used to precipitate the protein at -20℃ for 4 h. The pellet was air-dried and then resuspended in 150 μl 0.1M ABB. Protein samples underwent trypsin digestion (enzyme-to-substrate ratio of 1:25 at 37 ℃for 16 hours) followed by desalting through sola HRP cartridges and vacuum- dried by Speed Vac. Nano-LC-MS/MS analysis. Peptide samples were analyzed by an EASY-nLC 1200 LC system coupled with Orbitrap mass spectrometry (Thermo Fisher). The column was C18 nano-capillary analytical column (25 cm*75 mm, 1.9 μm). Peptides were re-dissolved in mobile phase A (Water/CAN/formic acid, 98/2/0.1, v/v). The gradient was 0-5 min, 5-10% B; 5-55 min, 10-28% B; 55-58 min, 28-40% B，58-63 min, 40-95% B; 63-75 min; 95% B with flow rate of 350 nL/min. Mass spectrometry was operated under a data independent acquisition mode (DIA). The m/z range was 350 to 1500 Da in MS1 with the resolution of 60,000. The automatic gain control (ACG) was set as 3e6. The maximal ion injection time was 50 ms. MS2 acquisition was higher energy collision dissociation (HCD) with the collision energy of 30 eV. The resolution was 30,000, with loop count set to: 25, MSX count set to: 1, isolation window set to: 8.3m/z, fixed maximum mass set to: 200m/z, respectively.
2024-09-07 | PXD055286 | Pride
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.
2018-01-10 | PXD000118 | Pride
Project description: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.
2014-04-23 | PXD000133 | Pride
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:Proteomic analysis Epilepsy was induced using perforant pathway stimulation (PPS) in rats as described (Norwood BA, et al. (2010) Classic hippocampal sclerosis and hippocampal-onset epilepsy produced by a single "cryptic" episode of focal hippocampal excitation in awake rats. J Comp 803 Neurol 518(16):3381-3407). Rats were killed under deep anesthesia (xylazine+ketamine) by transcardial perfusion with ice-cold 0.9 % NaCl solution at the following time points: 1 h, 24 h, 72 h, 10 d and 16 d after PPS (epileptogenesis); within 1 d of first spontaneous seizure (early epilepsy); 1 month after first spontaneous seizure (chronic epilepsy). Control rats were killed on day 17 after surgery (corresponding to day 10 after PPS). Hippocampi were removed and snap frozen at -80°C. Frozen rat hippocampi were resuspended in 200 ul lysis buffer (6M GdmCl, 10mM TCEP, Complete protease inhibitor cocktail, PhosSTOP inhibitor, 100 mM TEAB, benzonase) and dounced 30 times on ice. Samples were boiled for 5 min, sonicated on ice for 2min and centrifuged at 13000 rpm for 3 min. The supernatant was added chloroacetamide to a final concentration of 20 mM followed by incubation in the dark for 30 min at room temperature (RT). Proteins were digested with LysC for 30 min at RT and then diluting 10x— using 100 mM TEAB followed by adding trypsin and incubating overnight at 37°C with shaking. LysC and trypsin were added in a LysC/trypsin:protein ratio of 1:100. Samples were centrifuged at 13000 rpm for 5 min and from the supernatant a volume corresponding to 50 ug peptide was transferred to a new eppendorff tube. Sets of 6 samples were labelled using the TMTsixplex isobaric label reagent. (ThermoFisher Scientific). The labeling reagent was prepared using the manufactures instructions to first equilibrate to RT, then dissolving the labeling reagent in 41 ul anhydrous acetonitrile. The labeling reagent was added to each sample and incubated for 1 hour at RT. The reaction was quenched by incubating with 0.76 M lysine for 15 min. A small amount was taken from each sample, mixed and tested by mass spectrometry for labeling efficiency and mixing ratio. Remaining samples were mixed in a 1:1 ratio. The Stage-tips for the high-pH reverse phase fractionation were prepared by placing first two C18 disks in a p200 pipette tip, then adding a slurry of C18 beads (ReproSil-Pur, 3 um) in methanol and adding one additional C18 disk in the top. The column was activated and equilibrated by washing one time in 150 ul buffer B (50% buffer A, 50% ACN) and then two times in 150 ul buffer A (20mM Ammonium hydroxide, pH 10). The labeled peptide sample was adjusted to pH 10 using buffer A and then loaded onto the activated and equilibrated stage-tip by spinning the sample through the column and collecting 26 the flow-through. The column was washed 2 times with 150 ul buffer 585 A and the washes were collected and combined with the flow-through. Next, peptides were eluted stepwise in 17 fractions by spinning 150 ul of buffer A containing increasing amounts of ACN for each step (3.5%, 4.5%, 6%, 8%, 10%, 10.5%, 11.5%, 13.5%, 15%, 16%, 17.5%, 18.5%, 19.5%, 21%, 27%, 50%, 80%) through the column. After collection, the liquid was evaporated completely in a Concentrator Plus (Eppendorf) and prepared for MS by resuspending in buffer A* (2% ACN, 0.1% TFA). Samples were analyzed using an Easy-nLC system coupled online to a Q Exactive HF mass spectrometer (Thermo Scientific) equipped with a nanoelectrospray ion source (Proxeon/Thermo Scientific). The peptides were eluted into the mass spectrometer during a chromatographic separation from a fused silica column packed in-house with 3 um C18 beads (Reprosil, Dr. Maisch) using a 120 min gradient of buffer B (80% ACN, 0.5% acetic acid) with a flow rate of 250 nL/min. The Q Exactive HF was operated in positive ion mode with a top 12 data-dependent acquisition method where ions were fragmented by higher-energy collisional dissociation (HCD) using a normalized collisional energy (NCE)/ stepped NCE of 28 and 32. The resolution was set to 60,000 (at 400m/z), with a scan range of 300-1700 m/z and an AGC target of 3e6 for the MS survey. MS/MS was performed at a scan range of 100 - 2000 m/z using a resolution of 30,000 (at 400 m/z), an AGC target of 1e5, an intensity threshold of 1e5 and an isolation window of 1.2 m/z. Further parameters included an exclusion time of 45 sec and a maximum injection time for survey and MS/MS of 15 ms and 45 ms respectively.
2020-06-12 | PXD019098 | Pride