The MS data was acquired on a Q-ExactiveTM plus instrument (Thermo ScientificTM) using a data-dependent top 20 method, dynamically choosing the most abundant precursor ions from the survey scan (350–1600 m/z) for Higher energy Collisional Dissociation (HCD) fragmentation. Dynamic exclusion duration was 60 s. Isolation of precursors was performed with a 1.6 m/z window and MS/MS scans were acquired with a starting mass of 80 m/z. Survey scans were acquired at a resolution of 70,000 at m/z 400 (AGC set to 106 ions with a maximum fill time of 100 ms). Resolution for HCD spectra was set to 70,000 at m/z 200 (AGC set to 105 ions with a maximum fill time of 200 ms). Normalized collision energy was 28 eV. The underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.4%. The instruments was run with peptide recognition mode (i.e., from 2 to 8 charge), exclusion of singly charged and of unassigned precursor ions enabled.
Q exactive plus instrument
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Q-Exactive Plus is a high-resolution, accurate-mass (HRAM) mass spectrometer designed for a wide range of applications in life science research. It features a high-performance quadrupole and Orbitrap mass analyzer, enabling precise and sensitive detection of molecular compounds.
Automatically generated - may contain errors
Lab products found in correlation
Easy nlc 2 system, by Thermo Fisher Scientific (2 mentions)
Ultimate 3000 uplc system, by Thermo Fisher Scientific (2 mentions)
Easy nlc 1000 uplc system, by Thermo Fisher Scientific (1 mentions)
Esi source, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 liquid chromatography system, by Thermo Fisher Scientific (1 mentions)
Reprosil pur c18 aq 3 μm resin, by Dr. Maisch (1 mentions)
Betasil c18 column, by Thermo Fisher Scientific (1 mentions)
Spectronaut software, by Biognosys (1 mentions)
Easy nanolc, by Thermo Fisher Scientific (1 mentions)
Q exactive hf instrument, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200 chromatography system, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200 uplc system, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1000 uhplc system, by Thermo Fisher Scientific (1 mentions)
Ion pi chip kit v3, by Thermo Fisher Scientific (1 mentions)
Ion proton sequencer, by Thermo Fisher Scientific (1 mentions)
Ion pi hi q sequencing 200 kit, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslcnano, by Thermo Fisher Scientific (1 mentions)
Nanospray flex ion source, by Thermo Fisher Scientific (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Easy nlc 1000, by Thermo Fisher Scientific (1 mentions)
20 protocols using q exactive plus instrument
1
Comprehensive Proteomic Analysis by Q-Exactive
2
Capillary Reversed-Phase LC-MS/MS Analysis
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Digested peptides were separated using an Acclaim PepMap RSLC C18 capillary reversed-phase analytical column (Thermo) with a 60 min 4-85% ACN/water gradient containing 0.1% formic acid (FA) at a constant flow rate of 700 nL/min with an EASY-nLC 1000 UPLC system (Thermo).
The eluted peptides were further ionized and sprayed into the nanospray-ionization (NSI) source followed by tandem mass spectrometry (MS/MS) with a Q ExactiveTM Plus instrument (Thermo) coupled online to UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using an NCE setting of 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. Automatic gain control (AGC) was set at 5E4.
The eluted peptides were further ionized and sprayed into the nanospray-ionization (NSI) source followed by tandem mass spectrometry (MS/MS) with a Q ExactiveTM Plus instrument (Thermo) coupled online to UPLC. The electrospray voltage applied was 2.0 kV. The m/z scan range was 350 to 1800 for full scan, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using an NCE setting of 28 and the fragments were detected in the Orbitrap at a resolution of 17,500. Automatic gain control (AGC) was set at 5E4.
3
Quantifying Protein Abundance via iBAQ
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For iBAQ, NGP cells were collected, pelleted and flash-frozen before precipitation of proteins with equal volumes of chloroform, methanol and water69 (link). Proteins were solubilized in 6 M urea/2 M thiourea buffer containing 10 mM HEPES and treated with benzonase to digest DNA and reduce viscosity. An amount of 50 μg lysate was subjected to reduction with TCEP (tris(2-carboxyethyl)phosphine) and alkylation with CAA (2-chloroacetamide) before digestion with trypsin. Peptides were extracted and desalted with a C18 StageTip before elution and reconstitution in 3% ACN/0.1% formic acid, followed by separation on a reversed-phase column (20 cm length, 75 μm ID, 3 μm Dr Maisch C18) with a gradient from 5 to 45% ACN in 120 min, while MS and MS/MS spectra were acquired in data-dependent mode on a Q-Exactive Plus instrument (Thermo). Raw data files were analysed with MaxQuant 1.5.2.8.
To calculate protein abundances, we applied the ‘iBAQ' algorithm29 (link). In short, we first calculated the number of theoretically observable peptides per protein by in silico trypsin digesting the Uniprot data base. We then divided the protein intensities by the theoretical observable peptide count.
To calculate protein abundances, we applied the ‘iBAQ' algorithm29 (link). In short, we first calculated the number of theoretically observable peptides per protein by in silico trypsin digesting the Uniprot data base. We then divided the protein intensities by the theoretical observable peptide count.
4
Comprehensive Bile Acid Profiling in Feces
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The bile acid quantification included four primary bile acids (α-MCA, β-MCA, CA, and UDCA), four secondary bile acids (ω-MCA, chenodeoxycholic acid [CDCA], deoxycholic acid [DCA], and lithocholic acid [LCA]), and seven conjugated bile acids (tauro-β-muricholic acid [T-β-MCA], tauroursodeoxycholic acid [TUDCA], taurocholic acid [TCA], glycocholic acid [GCA], taurochenodeoxycholic acid [TCDCA], taurodeoxycholic acid [TDCA], and taurolithocholic acid [TLCA]). Briefly, 20 mg of raw feces was dissolved in 200 μL of 70% d3-cholic acid aqueous solution containing 2 ppm d4-cholic acid as an internal standard, and it was homogenized using an ultrasonicator for 30 min. The sample solutions were then centrifuged at 18,000 × g for 5 min. LC-MS analysis was performed using an UltiMate 3000 liquid chromatography system (Thermo Fisher Scientific, Dreieich, Germany) coupled with a high-resolution Q Exactive Plus instrument equipped with an ESI source (Thermo Fisher Scientific) and an Acquity HSS T3 (2.1 by 100 mm, 1.7 μm) column (Waters, Milford, MA, USA).
5
Peptide Identification Protocol via MS
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The enriched tryptic peptides were introduced into the NSI source using a Q Exactive™ Plus instrument (Thermo). The ensuing MS data were subsequently processed via Skyline (v3.6). Regarding peptide settings, the specified enzyme was trypsin (KR/P), with a maximum allowed missed cleavage set at 2. Peptide lengths were limited to the range of 8–25 amino acids. Variable modifications included carbamidomethyl on cysteine (Cys) and oxidation on methionine (Met), with a maximum of three variable modifications allowed. In the context of transition settings, precursor charges were constrained to 2 and 3, with ion charges limited to 1 and 2. Mass tolerance of product ions was 0.02 Da.
6
Mass Spectrometric Analysis of Lipid A
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Lipid A from 10 µg of STD Pg LPS or UP Pg LPS were isolated by mild acid hydrolysis as previously36 (link). The lyophilized Lipid A was suspended in 1000 µL of 1:100 CHCl3–CH3OH. ESI-MS, performed with a Q exactive plus instrument (Thermofischer), was used to evaluate negative ions of P. gingivalis lipid A for the 1417 m/z specie. Syringe pump was used to infuse the lipid A sample. Typical conditions were: infusion rate, 5 µL/min, spray voltage, 3.8 kV; capillary temperature, 320 °C. The 1417 m/z specie first isotope was normalized at 100% of abundance and intensity (NL) of this species was determined for each type of lipid A. This NL value represents the abundance 1417 m/z specie.
7
Peptide Separation and Quantification
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Desalted peptides were separated on a 5%–45% acetonitrile gradient (240 min) with 0.1% formic acid at a flow rate of 200 nL/min using the EASY-nLC II system (Thermo Fisher Scientific) on in-house manufactured 25 cm fritless silica micro-columns with an inner diameter of 75 μm. Columns were packed with the ReproSil-Pur C18-AQ 3 μm resin (Dr. Maisch GmbH, Entringen, Germany). A Q Exactive plus instrument (Thermo Fisher Scientific) was operated in the data dependent mode with a full scan in the Orbitrap followed by top 10 MS/MS scans using higher-energy collision dissociation (HCD). The full scans were performed with a resolution of 70,000, a target value of 3 × 106 ions and a maximum injection time of 20 ms. The MS/MS scans were performed with a 17,500 resolution, a 1 × 106 target value and a 60 ms maximum injection time.
8
Proteomic Analysis via High-pH HPLC and Q Exactive Plus MS
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After fractionation by high-pH reverse-phase HPLC using a Thermo Betasil C18 column, the tryptic peptides were dissolved in 0.1% formic acid (solvent A) and directly loaded onto a homemade reverse-phase analytical column (15-cm length, 75 μm i.d.). The gradient comprised an increase from 6% to 23% solvent B (0.1% formic acid in 98% acetonitrile) over 26 min, a further increase from 23% to 35% over 8 min and a climb to 80% in 3 min; the concentration was held at 80% for the last 3 min. All steps were performed at a constant flow rate of 400 nL/min with an EASY-nLC 1000 ultra-performance liquid chromatography (UPLC) system. The peptides were subjected to a nanoelectrospray ionization (NSI) source followed by tandem mass spectrometry (MS/MS) with a Q Exactive™ Plus instrument (Thermo) that was coupled online to the UPLC. The applied electrospray voltage was 2.0 kV. The m/z scan range was 350 to 1800 for full scans, and intact peptides were detected in the Orbitrap at a resolution of 70,000. Peptides were then selected for MS/MS using a normalized collision energy (NCE) setting of 28, and the fragments were detected in the Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan and 20 MS/MS scans with 15.0 s of dynamic exclusion was used. The automatic gain control (AGC) was set at 5E4. The fixed first mass was set as 100 m/z.
9
Proteomic Analysis of CD8+ T Cells
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Cell samples for proteome analysis were prepared from CD8+ T cells of three donors, including the conditions non-treated (nt), mock electroporated, and BCL11B KO 22 days after activation with TransAct. Whole protein was extracted, protein concentration was determined, and peptide solutions were prepared by an SP3 bead-based protocol26 (link) and digestion with trypsin. LC-MS/MS analysis was performed on the peptides in data-independent acquisition (DIA) mode using an Ultimate 3000 UPLC system coupled to a QExactive Plus instrument (Thermo Scientific, USA). Further details on protein preparation and LC-MS/MS analysis can be found in Supplementary Table 1, and data acquisition details are compiled in Supplementary Table 3.
Analysis of mass spectrometric raw data was carried out using Spectronaut software (v14.9., Biognosys, Germany). Statistical data analysis was conducted using an in-house developed R tool. Further details on quantitation algorithms are provided in Supplementary Table 3 and on raw data analysis and statistical analysis in Supplementary Table 1.
Finally, differential abundant proteins (absolute fold change ≥ 1.5 and p ≤ 0.05) were identified by the statistical analysis using the ROPECA algorithm27 (link) applied on peptide level.
Analysis of mass spectrometric raw data was carried out using Spectronaut software (v14.9., Biognosys, Germany). Statistical data analysis was conducted using an in-house developed R tool. Further details on quantitation algorithms are provided in Supplementary Table 3 and on raw data analysis and statistical analysis in Supplementary Table 1.
Finally, differential abundant proteins (absolute fold change ≥ 1.5 and p ≤ 0.05) were identified by the statistical analysis using the ROPECA algorithm27 (link) applied on peptide level.
10
LC-MS/MS Peptide Quantification Protocol
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For LC–MS/MS, peptides were dissolved in 5% ACN/0.1% FA to a concentration of 0.25 µg/µL. Mass spectrometry experiments were performed on a Q Exactive Plus instrument (Thermo Scientific, San Jose, CA, USA) equipped with an Easy-nanoLC (Thermo Scientific, San Jose, CA, USA). Peptides were trapped on 2 cm × 75 µm columns (Thermo Scientific, San Jose, CA, USA). The analytical separation was run for 60 min using a gradient of 99.9% H2O/0.1% (solvent A) and 99.9% ACN/0.1% FA (solvent B) at a flow rate of 300 nl min−1. For MS survey scans, the OT resolution was set to 140,000 and the ion population was set to 3 × 106 with an m/z window from 350 to 2000. Twenty precursor ions were selected for higher-energy collisional dissociation (HCD) with a resolution of 35,000, an ion population set to 1 × 105 (isolation window of 0.5 m/z) and a normalized collision energy set to 30%.
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