Samples were sent to the Mass-spec center at the University of Texas at Austin for analysis. Protein identification was performed via NS-LC-MS/MS using a Dionex Ultimate 3000 RSLCnano UPLC coupled to a Thermo Orbitrap Fusion. Prior to HPLC separation, the peptides were desalted using Millipore U-C18 ZipTip Pipette Tips following the manufacturer’s protocol. A 2 cm long × 75 μm I.D. C18 trap column was followed by a 75 μm I.D. ×25 cm long analytical column packed with C18 3 μm material (Thermo Acclaim PepMap 100). Run-time was 1 h. The FT-MS resolution was set to 120,000, and 3 s cycle time MS/MS were acquired in ion trap mode. Raw data was processed using SEQUEST HT embedded in Proteome Discoverer. Scaffold (Proteome Software) was used for validation of peptide and protein identifications with filtering to achieve 99% protein confidence or a 1% FDR.
Ultimate 3000 rslcnano uplc
Manufactured by Thermo Fisher Scientific
The Ultimate 3000 RSLCnano UPLC is a high-performance liquid chromatography system designed for nanoflow applications. It offers precise flow control and high-resolution separation capabilities for the analysis of small sample volumes.
Automatically generated - may contain errors
Lab products found in correlation
Orbitrap fusion, by Thermo Fisher Scientific (2 mentions)
S trap column, by Protifi (2 mentions)
Orbitrap exploris oe240, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer software, by Thermo Fisher Scientific (1 mentions)
Scaffold software, by Proteome Software (1 mentions)
Orbitrap fusion lumos, by Thermo Fisher Scientific (1 mentions)
Fused silica emitter, by New Objective (1 mentions)
Stainless steel emitter, by Thermo Fisher Scientific (1 mentions)
Integrafrit, by New Objective (1 mentions)
Hypercarb, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions)
9 protocols using ultimate 3000 rslcnano uplc
1
Proteomic Identification via Nano-LC-MS/MS
2
Quantitative Proteomics by PRM-LC-MS/MS
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15 micrograms of each of the samples were digested with trypsin on STRAP columns (PROTIFI, Farmingdale, NY), according to the protocol described by the manufacturer. Tryptic peptides were dried in a speed-vacuum system and resuspended at 200 ng/μl, according to QUBIT quantification (Thermofisher Scientific). 5 μL of each sample (equivalent to 1 μg) were loaded online on a C18 PepMap 300 μm I.D. 0.3 × 5 mm trapping column (5 μm, 100 Å, Thermo Scientific) and analyzed by LC-ESI MSMS using a Thermo Ultimate 3000 RSLC nanoUPLC coupled to a Thermo Orbitrap Exploris OE240 mass spectrometer. Peptides were separated on a 15 cm, 75 μm ID column, with a flow rate of 300 nl/min and a 60 min long gradient. The liquid chromatographic system was coupled via a nanospray source to the mass spectrometer. The mass-spec method used worked in PRM mode (parallel reaction monitoring) monitoring the six selected peptides (3 per protein) both in light and heavy format. Selection and extraction of each of the transition areas (see Supplementary Table 1), was carried out with the Skyline v21.2 software [41 (link)]. Heavy peptides were PRM-analyzed at different amounts (ranging from 63 attomol to 1000 fentomol) and used to raise a linear regression model that was used to estimate the actual amount of the internal (light) peptides in the samples.
3
UHPLC-MS Analysis of Hydrolyzed DNA
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The dried hydrolyzed DNA samples were brought to RT in 20 min, reconstituted in 20 μL of water (LC-MS grade, Fluka) and then analyzed by LC-MS. For each sample 3 μL were injected. The LC was performed using a nanoflow UPLC (Ultimate 3000 RSLCnano UPLC, Thermo Scientific, Waltham, MA). The UPLC was equipped with a 5 μL loop and reversed-phase chromatographic separation was performed using a hand-packed commercially available fused-silica emitter (230 × 0.075 mm ID, 15 μm orifice, New Objective, Woburn MA) with C18 stationary phase (5 μm, 100, Luna Phenomenex, Torrance, CA). The mobile phase consisted of an aqueous solution of 0.05 %v/v formic acid (phase-A) and CH3CN (phase-B). The elution program included an isocratic step (2 % of B for 5 min at 1 μL/min), followed by a linear gradient of B (1.5%/min for 25 min at 0.3 μL/min) and it concluded with a washing isocratic step, performed at 98% of B for 5 min at 0.3 μL/min. At the end of the elution program, the LC-system was equilibrated for 5 min at isocratic condition (2% of B, 1 μL/min). At 6 min., the injection valve was switched to remove the sample loop from the liquid flow path. The injection valve and needle were washed with 200 μl of acetonitrile between sample injections to avoid carryover.
4
Nanoflow UPLC-based DNA Hydrolysate Analysis
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An aliquot of 1 μL of DNA hydrolysate was injected in a nanoflow Ultra Performance Liquid Chromatography (UPLC) system (Ultimate 3000 RSLCnano UPLC, Thermo Scientific, Waltham, MA). The UPLC system was equipped with a 5-μL autosampler injection loop. Chromatographic separation was achieved using a hand-packed commercially available fused-silica emitter (230 × 0.075-mm ID, 15-μm orifice, New Objective, Woburn MA) with C18 stationary phase (5 μm, 100, Luna Phenomenex, Torrance, CA). The mobile phase consisted of (A) 0.05% (v/v) formic acid in H2O and (B) CH3CN. The eluent was held at 2% B for 2 min, brought to 20% B in 24 min, then to 60% B in 10 min, to 98% B in 1 min, and then maintained at 98% for 4 min. The column was re-equilibrated for 4 min. The injection valve position was switched at 6 min to take the injection loop out of the flow path.
5
Targeted Proteomics of ExsE Peptides
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Samples were digested with trypsin on STRAP columns (PROTIFI, Farmingdale, NY, USA), according to the protocol described by the manufacturer. Tryptic peptides were dried in a speed-vacuum system and resuspended at 100 ng/µL, according to QUBIT quantification (Thermofisher Scientific, Waltham, MA, USA). An amount of 500 ng of each sample was loaded online on a C18 PepMap 300 µm I.D. 0.3 mm × 5 mm trapping column (5 µm, 100 Å, Thermo Scientific) and analyzed by LC-ESI MSMS using a Thermo Ultimate 3000 RSLC nanoUPLC coupled to a Thermo Orbitrap Exploris OE240 mass spectrometer. Peptides were separated on a 15 cm × 75 µm RP C18 column in a 60 min long gradient at a 300 nL/min flow rate. The liquid chromatographic system was coupled via a nanospray source to the mass spectrometer. Targeted proteomics experiments were performed in PRM mode (parallel reaction monitoring) monitoring two ExsE specific peptides, IESISPVQPSQDAGAEAVGHFEGR (827.738 m/z, charge 3+) and LADGDGTPLEAR (607.804, charge 2+). Selection and extraction of each of the transition areas for quantitation, was carried out with the Skyline v21.2 software [62 (link)] following the usual criteria in the design of targeted proteomics experiments and using previous information obtained through shotgun proteomics assays.
6
Methyl-Proteome Profiling of MEFs and HeLa
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Immunoprecipitated proteins from MEFs or HeLa cells were loaded into SDS-PAGE gel and electrophorized for 20 min. The gel plug containing the proteins of interest was excised and subjected to in-gel digestion with trypsin. Peptides were eluted and analyzed by nanoflow LC-MS/MS using the Thermo Ultimate 3000 RSLCnano UPLC in line with the Orbitrap Fusion hybrid mass spectrometer. The resulting spectra were assigned to peptide sequences using Proteome Discoverer software (Thermo) with the Sequest-HT database search algorithm. The appropriate Uniprot (mouse or human) database was used along with a list of common protein contaminants. Scaffold software (Proteome Software) provided data validation. The protein identifications were filtered for minimum 2 peptide at 95% confidence and 99% protein confidence. Lists of credible methyl-peptide sequence assignments were generated for samples from MEFs and lists of credible peptide sequence assignments were generated for samples from HeLa and H69 cells.
7
LC-MS/MS Analysis of DNA Adducts
8
Porous Graphitic Carbon LC-MS Protocol
9
Identification of Differentially Expressed Proteins in EPZ015666-Treated 293T Cells
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