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A platform for distributed production of synthetic nitrated proteins in live bacteria

ABSTRACT: For LC-MS/MS measurements, protein samples (20 µg/lane) were first submitted to SDS-PAGE. The gel was then stained with Bio-SafeTM Coomassie stain (Bio-Rad), and de-stained with water. Then, protein bands corresponding to the molecular weight of the protein of interest were cut from the polyacrylamide gel and the gel fragments were digested using an in-gel tryptic digestion kit (Thermo Scientific: p/n 89871). Following digestion, extracted peptides were then desalted using PierceTM pipette tips (ThermoFisher) and dried, resuspended in 20 µL of 0.5% acetic acid (pH4.5) and then subjected to LC-MS/MS using an Thermo Scientific Orbitrap Eclipse Tribrid Mass Spectrometer (MS; Thermo Scientific) with an Ultimate 3000 nano-liquid chromatography (nano-LC) and a FAIMS Pro Interface (Thermo Scientific). The LC-MS/MS analysis was performed using an Orbitrap Eclipse MS (Thermo Scientific). Peptides were first loaded onto a trap column (PepMap C18) and then separated by an analytical column (PepMap C18, 2.0 um; 15 cm x 75mm I.D.; Thermo Scientific) at 300 nl/min flow rate using a binary buffer system (buffer A, 0.1% formic acid in water; buffer B, 0.1% formic acid in acetonitrile) with a 165-min gradient (1% to 10% in 8 min; then to 25% buffer B over 117 min; 25% to 32% buffer B in 10 min, then to 95% buffer B over 3 min; back to 1% B in 5 min, and stay equilibration at 1% B for 20 min). Multiple CVs (-40, -55 and -75) were applied for FAIMS separation. For all experiments, the survey scans (MS1) were acquired over a mass range of 375-1500 m/z at a resolution of 60,000 in the Orbitrap. The maximum injection time was set to Dynamic, and AGC target was set to Standard. Monoisotopic peak selection was set to Peptides, and the charge state filter was set to 2-7. For MS/MS acquisition, precursors were isolated with a width of 1.6 m/z, fragmented with HCD using 30% collision energy with a maximum injection time of 100 ms, and collected in Orbitrap at 15,000 resolution. The dynamic exclusion was set to 60 s, and can be shared across different FAIMS experiments. LC-MS/MS data was collected in n=3 independent biological replicates. Proteomic analysis was performed in the MaxQuant-Andromeda software suite (version 1.6.3.4) with most of the default parameters51. An E. coli reference proteome (strain BL21-DE3; taxonomy_id:469008) was used for database search. Other parameters include: trypsin as an enzyme with maximally two missed cleavage sites; protein N-terminal acetylation, methionine oxidation, and pA-Phe and pN-Phe substitution for tyrosine were entered as variable modifications; cysteine carbamidomethylation as a fixed modification; and peptide length was set to a minimum of 7 amino acids. The false-discovery rate of high-confidence protein and peptide identification was 1%. Peptide intensity values derived from MaxQuant were used for quantification. To obtain the MS/MS spectra of peptides, additional analysis was performed using Proteome Discoverer software (version 3.0; Thermo Fisher). Raw data was searched against the E. coli reference proteome (strain BL21(DE3)) with NCBI reference Proteome (469008) using Sequest HT Processor and CWF Basic analysis workflows. Iodoacetamide-mediated cysteine carbamidomethylation was set as a static modification, while methionine oxidation and pA-Phe and pN-Phe substitution for tyrosine were entered as dynamic modifications. Precursor mass tolerance was set at 10?ppm while allowing fragment ions to have a mass deviation of 0.02?Da for the HCD data. Validation of peptide-spectrum matches (PSM) based on q value was done using Percolator, with target false-discovery rates (FDR) of 1% and 5% for stringent and relaxed validations, respectively.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Escherichia Coli (ncbitaxon:562)

SUBMITTER: Aditya Kunjapur

PROVIDER: MSV000091379 | MassIVE | Tue Feb 28 11:45:00 GMT 2023

REPOSITORIES: MassIVE

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Project description:The cells were grown on Fe(II)-citrate as the aqueous Fe(II) source and Fe(II)-smectite as the solid Fe(II) source. Cells were collected on a 0.2 um filter membrane (Millipore GPWP) by filtration. Then cells on the membrane were lysed by vortex in the lysis buffer containing 100 mM Tris/HCl, 10 mM Ethylenediaminetetraacetic acid (EDTA), pH 8.0, 0.05% Tween-20 and 1% w/v sodium dodecylsulfate (SDS). The proteins were digested with trypsin using in-house packed glass fiber filters following the suspension trapping (STrap) protocol, and desalted using C18-based StageTips. The LC-MS/MS analysis was performed using an Orbitrap Eclipse MS (Thermo Scientific) coupled with an Ultimate 3000 nanoLC system and a FAIMS Pro Interface (Thermo Scientific). Peptides were first loaded onto a trap column (PepMap C18; 2 cm x 100 um I.D.) and then separated by an analytical column (PepMap C18, 3.0 um; 10 cm x 75 um I.D.; Thermo Scientific) at 300 nl/min flow rate using a binary buffer system (buffer A, 0.1% formic acid in water; buffer B, 0.1% formic acid in acetonitrile) with a 165-min gradient (1% to 10% in 8 min; then to 25% buffer B over 117 min; 25% to 32% buffer B in 10 min, then to 95% buffer B over 3 min; back to 1% B in 5 min, and stay equilibration at 1% B for 20 min). Multiple CVs (-50, -65 and -80) were applied for FAIMS separation. For all experiments, the survey scans (MS1) were acquired over a mass range of 375-1500 m/z at a resolution of 60,000 in the Orbitrap. The maximum injection time was set to Auto, and AGC target was set to Standard. Monoisotopic peak selection was set to Peptides, and the charge state filter was set to 2-7. For MS/MS acquisition, precursors were isolated with a width of 1.6 m/z, fragmented with HCD using 30% collision energy with a maximum injection time of 100 ms, and collected in Orbitrap at 15,000 resolution. The dynamic exclusion was set to 60 s, and can be shared across different FAIMS experiments. Protein identification and quantitation were performed using the MaxQuant-Andromeda software suite (version 1.6.3.4) with most of the default parameters. An UniProt database (Sideroxydans lithotrophicus ES-1; Taxonomy ID, 580332; 2,978 sequences) was used for the protein identification. Other parameters include: trypsin as an enzyme with maximally two missed cleavage sites; protein N-terminal acetylation and methionine oxidation as variable modifications; cysteine carbamidomethylation as a fixed modification; peptide length must be at least 7 amino acids. False discovery rate was set at 1% for both proteins and peptides. Perseus (version 1.6.7.8) was used for downstream bioinformatics analysis such as clustering, correlation, t-test and ANOVA.

Project description:Glands were frozen at -80 ºC immediately after being retrieved and stored under this condition until initiating the proteomics analysis protocol. Glands were washed with cold PBS and immersed in 120 l of homogenization buffer (50 mM Tris-HCl, pH 6.8, 10 mM DTT, 4% (w/v) SDS). Glands were boiled for 5 min, incubated overnight at 4 ºC and centrifuged at 4 ºC and 13000 rpm for 10 min. The whole gland proteome of each gland was concentrated as described elsewhere (Bonzon-Kulichenko, F. Garcia-Marques, M. Trevisan-Herraz and J. Vazquez, Journal of Proteome Research, 2015, 14, 700-710 (DOI:10.1021/pr5007284).Briefly, samples were loaded into an SDS-PAGE gel (0.5 mm-thick, 4% stacking, and 10% resolving) and the electrophoresis process was stopped when the front entered 2 mm into the resolving gel. The unseparated protein bands were then visualized by Coomassie staining, excised and digested at 37 ºC overnight with 500 l of 25 g/l chymotrypsin (Promega) in 100 mM Tris-HCl, pH 7.8 containing 10 mM CaCl2. The cleaved peptides were extracted by incubation for 2 h in 100 mM Tris-HCl, pH 7.8 on shaking, acidized with 1% (v/v) trifluoroacetic acid, desalted onto C18 cartridges (Oasis, Waters Corporation, Milford, MA, USA), and dried down.The analysis of the samples proceeded through liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an Easy nLC 1000 nano-HPLC coupled to a Q-Exactive Orbitrap mass spectrometer (Themo Scientific). Peptides were injected onto a C18 reverse-phase precolumn (Acclaim PepMap100, 75-m i.d., 3-m particle size, 2-cm length, Thermo Scientific), and a reverse-phase analytical column (Acclaim PepMap 100, 75-m, i.d., 3-m particle size, 50-cm length, Thermo Scientific) in buffer A [0.1% formic acid (v/v)] and eluted with a 60 min linear gradient of buffer B [90% acetonitrile, 0.1% formic acid (v/v)], at 200 nL/min. Mass spectrometry (MS) runs consisted of 70000 FT-resolution spectra in the 390-1200 m/z wide range, followed by data-dependent MS/MS spectra of the 15 most intense parent ions acquired along the chromatographic run. High-energy collisional (HCD) fragmentation was performed at 27% of normalized collision energy and the isolation window for the parent ion mass was established at 2 Da.

Project description:HEK293 cells were cultured in two 10-cm dishes and transfected with BAG3-FLAG as well as Myc vector, CaMKIIβ(D-S)-Myc, and CaMKIIδ(D-S)-MYC, respectively. After the cells were transfected for 24 h, they were treated with the indicated regent. The cells were lysed with NP-40 alternative cell lysis buffer containing protease (Thermo Fisher Scientific, 87785) and phosphatase inhibitor mixtures (Thermo Fisher Scientific, 78427) on ice for 30 min and centrifuged at 14 000 g for 15 min at 4°C. For the supernatant, BAG3-FLAG was immunopurified with anti-FLAG Magnetic Beads (MCE, HY-K0207). Immunoprecipitated proteins were separated by SDS-PAGE and identified by staining with 0.25 % Coomassie brilliant blue R250. The BAG3-FLAG protein bands were cut out and digested with trypsin at 37°C overnight. The tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reversed-phase analytical column (15 cm length, 75 μm i.d.). The gradient increased from 6-23% solvent B (0.1% formic acid in 98% acetonitrile) over 16 min, 23-35% in 8 min and climbing to 80% in 3 min, and then holding at 80% for the last 3 min. The flow rate remained constant at 400 nl/min on an EASY-nLC 1000 UPLC system. The peptides were subjected to an NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for a full scan, and the intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using NCE setting at 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure alternated between one MS scan and 20 MS/MS scans with 15.0s dynamic exclusion. Automatic gain control (AGC) was set at 5E4. The resulting MS/MS data were processed using Proteome Discoverer 2.4. Tandem mass spectra were searched against the Uniport protein database. Trypsin was specified as a cleavage enzyme allowing up to 2 missing cleavages. The mass error was set to 10 ppm and 0.02 Da for precursor and for fragment ions, respectively. Phospho-(S/T/Y) were chosen as variable modifications. Peptide confidence was set at high, and peptide ion score was set at > 20.

Project description:Serum samples were inactivated and sterilized at 56 ℃ for 30 min and processed as previous study with some modifications (Shen et al., 2020). Briefly, 4 μL serum from each sample was depleted using High Select Top-14 Abundant Protein Depletion MiniSpin Columns (Thermo Fisher Scientific, San Jose, USA). Then the elutes were denatured, reduced and alkylated to derive protein lysates. The solutions were diluted with 200 μL 100 mM triethylammonium bicarbonate (TEAB) followed by a double-step trypsinization (enzyme-to-substrate ratio kept at 1:40 in each step). 10% trifluoroacetic acid (TFA) was added to each sample to quench the enzymatic reaction. Digested peptides were cleaned-up with SOLAμ (Thermo Fisher Scientific, San Jose, USA) according to the manufacturer’s instructions and labeled by TMTpro 16 plex reagents (Thermo Fisher Scientific, San Jose, USA). 660 samples in total (633 serum samples and 27 technical replicates) were divided into 44 batches for labelling experiment, each batch containing fifteen samples and one pool. The labeled samples in each batch were combined and fractionated. Each sample was separated into 30 fractions and then consolidated into 10 fractions. Dried and re-dissolved fractions with 2% acetonitrile (ACN)/0.1% formic acid (FA) of MS grade. The re-dissolved peptides were analyzed with a U3000 HPLC system coupled to a Orbitrap Exploris 480 (Thermo Fisher Scientific, Bremen, Germany) in data-dependent acquisition (DDA) mode combined to a front-end high-field asymmetric waveform ion mobility spectrometry (FAIMS). Peptides of each fraction were loaded onto a pre-column (3 μm, 100 Å, 20 mm*75 mm i.d.) and separated with a 75 min LC gradient at a flow rate of 300 nL/min (analytical column: 1.9 mm, 120 Å, 150 mm*75 mm i.d.; Buffer A: 2% ACN, 98% H2O containing 0.1% FA; Buffer B: 98% ACN, 2% H2O containing 0.1% FA). FAIMS was operated at two different CV, -48 V and -68 V, respectively. For MS acquisition, RF level was set at 50% and ion transfer tube temperature at 320 °C. The turbo-TMT mode was enabled. Scan range of MS1 was 350–1800 m/z. Resolutions were set to 60000 for MS1 and 30000 for MS2. Normalized AGC target was set at 300% for MS1 and 200% for MS2. The maximum injection time was set as 50 ms for MS1 and 86 ms for MS2. Dynamic exclusion was on and mass tolerance was set to ±10 ppm. Intensity threshold was set at 20000 for MS1, and HCD collision energy was fixed to 38%. MS/MS data was recorded in centroid mode. Isolation window was set to 0.7 m/z.

Project description:The cell lysates were mixed with anti-FLAG-antibody-crosslinked Dynabeads. The beads-lysate mix was incubated at 4°C for 45 min. After washing with PBS-T, proteins were eluted with Laemmli’s SDS ample buffer by incubating at 37°C for 30 min. The collected supernatants were frozen at -80°C as samples for LC-MS. After reduction with 10 mM TCEP at 100°C for 10 min and alkylation with 50 mM iodoacetamide at ambient temperature for 45 min, the protein samples were subjected to digestion with Trypsin/Lys-C Mix (Promega) at 47°C for 2 h on S-Trap columns (ProtiFi, NY, USA). The resulting peptides were extracted from gel fragments and analysed using an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific) combined with UltiMate 3000 RSLC nano-flow HPLC (Thermo Scientific). The peptides were enriched with μ-Precolumn (0.3 mm i.d. × 5 mm, 5 μm, Thermo Scientific) and separated on an AURORA column (0.075 mm i.d. × 250 mm, 1.6 μm, Ion Opticks Pty Ltd, Australia) using the two-step gradient 2-40% acetonitrile for 110 min, followed by 40-95% acetonitrile for 5 min in the presence of 0.1% formic acid. The compensation voltages for the gas-phase fractionation by FAIMS Pro (Thermo Scientific) were set at -40, -60 and -80 V. The analytical parameters of the Orbitrap Fusion Lumos were set as follows: resolution of full scans = 50,000, scan range (m/z) = 350-1500, maximum injection time of full scans = 50 ms, AGC target of full scans = 4 × 10^5, dynamic exclusion duration = 30 s, cycle time of data dependent MS/MS acquisition = 2 s, activation type = HCD, detector of MS/MS = ion trap, maximum injection time of MS/MS = 35 ms, and AGC target of MS/MS = 1 × 10^4. The MS/MS spectra were searched against the Homo sapiens protein sequence database in SwissProt using Proteome Discoverer 3.0 software (Thermo Scientific), in which peptide identification filters were set at “false discovery rate < 1%”. Label-free relative quantification analysis for proteins was performed with the default parameters of Minora Feature Detector node, Feature Mapper node, and Precursor Ions Quantifier node in Proteome Discoverer 3.0 software.

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A platform for distributed production of synthetic nitrated proteins in live bacteria

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