IAV-infected 293 T cells were lysed in lysis buffer [150 mM NaCl, 20 mM HEPES, 1 mM EDTA (pH 7.4), 1% Triton X-100, 10% glycerol] supplemented with complete protease inhibitor cocktail and a phosphatase inhibitor phosSTOP. The NP was purified with protein G agarose beads pre-bound to a rabbit anti-NP polyclonal antibody for 3 h at 4°C. Proteins were separated by 12% Mn2+-Phos-tag SDS-PAGE as described previously (Wang et al., 2013 (link)). Briefly, normal polyacrylamide gel electrophoresis was conducted according to the TaKaRa protocol, with an acrylamide-pendant phosphate-tagged (Phos-tag) ligand (50 μM) and 0.1 mM MnCl2 (Sigma) added to the separating gel before polymerization. The gel was silver stained, and the separated bands were subjected to nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS) identification (LCQ Deca XP Plus; Thermo).
Lcq deca xp plus
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LCQ Deca XP Plus is a liquid chromatography-mass spectrometry (LC-MS) system designed for high-performance analysis. It provides sensitive and accurate mass detection for a wide range of applications. The system features a robust design and advanced technology to deliver reliable and consistent results.
Automatically generated - may contain errors
Lab products found in correlation
Surveyor lc system, by Thermo Fisher Scientific (2 mentions)
Surveyor autosampler, by Thermo Fisher Scientific (2 mentions)
Mncl2, by Merck Group (1 mentions)
C18 reverse phase column, by Agilent Technologies (1 mentions)
Mascot searchingalgorithm, by Matrix Science (1 mentions)
Coomassie brilliant blue r 250 solution, by Merck Group (1 mentions)
Agilent 1100 hplc, by Agilent Technologies (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
Pierce silver stain for mass spectrometry kit, by Thermo Fisher Scientific (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Thermo accela uplc system, by Thermo Fisher Scientific (1 mentions)
Inertsil ods 3, by GL Sciences (1 mentions)
Kieselgel 60, by Merck Group (1 mentions)
Accela uplc, by Thermo Fisher Scientific (1 mentions)
Fmoc protected amino acids, by Iris Biotech (1 mentions)
Turbosequest software, by Thermo Fisher Scientific (1 mentions)
Accunanobead streptavidin magnetic nanobeads, by Bioneer (1 mentions)
Aviii hd 400, by Bruker (1 mentions)
K2co3, by Merck Group (1 mentions)
29 protocols using lcq deca xp plus
1
Purification and Mass Spectrometry of IAV NP
2
NanoLC-ESI-MS/MS Protein Digestion Protocol
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NanoLC-ESI-MS/MS analysis of the digested protein samples was carried out using a high-performance liquid chromatography (HPLC) system (Agilent, Santa Clara, California) with a reverse-phase C18 column, 8 cm in length with a 75 µm inner diameter. The particle size of the C18 column was 3 μm, with a pore size of 300 Å, and the injection time was 20 minutes; HPLC solvent A consisted of 97.5% water, 2% acetonitrile, and 0.5% formic acid, and HPLC solvent B consisted of 9.5% water, 90% acetonitrile, and 0.5% formic acid. The gradient time was 60 minutes from 2% solvent B to 90% solvent B, plus 20 minutes for sample loading and 20 minutes for column washing. The column flow rate was approximately 800 nL per minute after splitting, with a typical injection volume of 3 μL.
The HPLC system was online coupled to an ion trap mass spectrometer (LCQ DECA XP PLUS; Thermo Fisher) in a manner such that a sample eluted from the HPLC column was directly ionized through an electrospray ionization process and entered the mass spectrometer. The mass spectrometer was set to data-dependent mode to acquire MS/MS data via a low-energy collision-induced dissociation process. One full scan with 1 microscan in the mass range of 550 to 1800 amu was acquired, followed by 1 MS/MS scan of the most intense ion with a full mass range and 3 microscans. The exclusion width was 4 Da.
The HPLC system was online coupled to an ion trap mass spectrometer (LCQ DECA XP PLUS; Thermo Fisher) in a manner such that a sample eluted from the HPLC column was directly ionized through an electrospray ionization process and entered the mass spectrometer. The mass spectrometer was set to data-dependent mode to acquire MS/MS data via a low-energy collision-induced dissociation process. One full scan with 1 microscan in the mass range of 550 to 1800 amu was acquired, followed by 1 MS/MS scan of the most intense ion with a full mass range and 3 microscans. The exclusion width was 4 Da.
3
Quantitative Ceramide Analysis via HPLC-MS/MS
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Ceramide was extracted and quantified by HPLC-MS/MS (Thermo, LCQ DECA XPplus) according to the principles described previously [43 (link)–46 (link)]. For extraction of cellular lipids, cells were lysed with distilled water and homogenized by sonication after incubation with inhibitors for 12 h. Protein concentrations were measured and the equal amounts of protein (500 μg) were adjusted to the volume of 800 μL in 1 M NaCl. C12-ceramide (10 ng) was added to lysates as an internal standard and the resulting samples were extracted with chloroform/methanol (1 : 2) 3 mL for 3 h. Samples were then centrifuged at 3000 g, 5 min. Supernatants were transferred to the other tubes within CCL4 and 1 M NaCl 1 mL, respectively. After centrifugation, the lower organic phase was obtained and evaporated to near dryness under a gentle stream of dry N2. Meantime, samples were reconstituted by 100 μL methanol to measure ceramides C14, C16, C24:1, and C24 by HPLC-MS/MS. The levels of each ceramide species were determined by their relative abundance normalized to C12-ceramide and the gross of ceramides was quantified based on the standard curve which was constructed on ceramide standards (Avanti). The gross of these ceramides was used for statistics.
4
Peptide Analysis by LC-MS/MS
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Peptides were analysed by LC-MS/MS using a Surveyor LC system and LCQ Deca XP Plus (Thermo Scientific). Briefly, peptides were resolved by reverse phase chromatography (Biobasic column, Thermo Scientific; 180 uM × 15 mm) over a 30 min ACN gradient at a flow rate of 3 μL/min. Peptides were ionised by electrospray ionisation and MS/MS was acquired on ions dependant on their charge state and intensity. Quality control checks for the optimal performance of the instrumentation were in place. Mass accuracy and sensitivity of the MS was confirmed with the direct infusion of glufibriopeptide (2.5 pmoles/μL) and LC-MS/MS performance was assessed with a digest of BSA. Sensitivity, retention time, peptides identified and protein sequence coverage were all within the specified ranges. BSA quality control checks were performed prior to the analysis of the sample and post-acquisition.
5
Mass Spectrometry Analysis of Trypsin-Digested Samples
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Following trypsin digestion, samples were freeze dried and resuspended with 25 ul of
5% ACN/0.1% formic acid for mass spectrometry (MS). Peptides were analysed by
LC/MS/MS using a Surveyor LC system and LCQ Deca XP Plus (ThermoScientific). The raw
data files were converted into mascot generic files using the MassMatrix File
Conversion Tool (Version 2.0;http://www.massmatrix.net ) for input into the Mascot searching
algorithm (Matrix Science).
5% ACN/0.1% formic acid for mass spectrometry (MS). Peptides were analysed by
LC/MS/MS using a Surveyor LC system and LCQ Deca XP Plus (ThermoScientific). The raw
data files were converted into mascot generic files using the MassMatrix File
Conversion Tool (Version 2.0;
algorithm (Matrix Science).
6
Quantitative Ceramide Analysis via HPLC-MS/MS
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Ceramide was extracted and quantified by HPLC-MS/MS (Thermo, LCQ DECA XPplus) according to the principles described previously [43 (link)–46 (link)]. For extraction of cellular lipids, cells were lysed with distilled water and homogenized by sonication after incubation with inhibitors for 12 h. Protein concentrations were measured and the equal amounts of protein (500 μg) were adjusted to the volume of 800 μL in 1 M NaCl. C12-ceramide (10 ng) was added to lysates as an internal standard and the resulting samples were extracted with chloroform/methanol (1 : 2) 3 mL for 3 h. Samples were then centrifuged at 3000 g, 5 min. Supernatants were transferred to the other tubes within CCL4 and 1 M NaCl 1 mL, respectively. After centrifugation, the lower organic phase was obtained and evaporated to near dryness under a gentle stream of dry N2. Meantime, samples were reconstituted by 100 μL methanol to measure ceramides C14, C16, C24:1, and C24 by HPLC-MS/MS. The levels of each ceramide species were determined by their relative abundance normalized to C12-ceramide and the gross of ceramides was quantified based on the standard curve which was constructed on ceramide standards (Avanti). The gross of these ceramides was used for statistics.
7
Proteomic Profiling of TLR4 Interactome
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Total protein was extracted from washed platelets and subjected to immunoprecipitation. The immunoprecipitates were eluted with SDS sample buffer, resolved on an SDS-PAGE gel, and stained with Coomassie Brilliant Blue R-250 solution (Sigma, St. Louis, MO, USA). In addition to the TLR4 band, the protein bands common to the anti-TLR4 antibody IP sample but not present in the mouse IgG IP control sample were excised from the gel. The gel pieces were then washed, reduced, alkylated, and digested with trypsin. The tryptic peptides were then analyzed by nano-LC/MS/MS on an LCQ Deca XP Plus ion trap mass spectrometer (Thermo Scientific, San Jose, CA, USA) coupled to an Agilent 1100 HPLC (Agilent Technologies, Inc., San Jose, CA, USA). The MS/MS spectra were searched using Sequest through the Bioworks Browser version 3.3.1 (Thermo Scientific, San Jose, CA, USA) against the NCBI nonredundant protein database.
8
Purification and Characterization Methodology
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Reagents and solvents
were used without further purification, although, if required, they
were distilled and stored on molecular sieves. Column chromatography
was performed on silica gel. The following instrumentation was used:
Stuart scientific SMP3 apparatus (melting point), FT-IR Thermo-Nicolet
Avatar, FT-IR Bruker Alpha II, Jeol ECP 300 MHz (1H NMR),
Bruker AVANCE Neo 400 MHz or Jeol ECP 300 MHz (13C NMR),
Thermo Finningan LCQ-deca XP-plus equipped with an ESI source and
an ion trap detector or mass spectrometry (Thermo Scientific Q-Exactive
Plus) equipped with a heated electrospray ionization source. Chemical
shifts are reported in parts per million (ppm). All lead compounds
displayed a purity of 95% or higher, determined by HPLC (see theSupporting Information ). Boronic acids, azides,
and alkynes are commercially available or were synthesized following
procedures reported in the literature, except for compounds73 , 75 , 77 , and 78 (intermediates
for the synthesis of18 , 21 , 9 , and 13 , respectively) that were synthesized as reported
in theSupporting Information .
were used without further purification, although, if required, they
were distilled and stored on molecular sieves. Column chromatography
was performed on silica gel. The following instrumentation was used:
Stuart scientific SMP3 apparatus (melting point), FT-IR Thermo-Nicolet
Avatar, FT-IR Bruker Alpha II, Jeol ECP 300 MHz (1H NMR),
Bruker AVANCE Neo 400 MHz or Jeol ECP 300 MHz (13C NMR),
Thermo Finningan LCQ-deca XP-plus equipped with an ESI source and
an ion trap detector or mass spectrometry (Thermo Scientific Q-Exactive
Plus) equipped with a heated electrospray ionization source. Chemical
shifts are reported in parts per million (ppm). All lead compounds
displayed a purity of 95% or higher, determined by HPLC (see the
and alkynes are commercially available or were synthesized following
procedures reported in the literature, except for compounds
for the synthesis of
in the
9
Proteomic Analysis of RNA Pulldown
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The RNA pulldown samples were separated by 10% SDS-PAGE, after which the gels were stained with a Pierce Silver Stain for Mass Spectrometry Kit (Thermo Fisher Scientific, USA). The densest bands were subjected to analysis using nanoscale liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS, LCQ Deca XP Plus; Thermo, USA). The data were analyzed by performing a MASCOT search against the SWISS-PROT human database and IAV database.
10
Identifying Proteins via NanoLC/MS/MS
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The four native gel bands from the stained lane corresponding to the highest activities were excised and submitted to ProtTech Inc. for independent analysis via NanoLC/MS/MS. The following steps were carried out by Protech: Peptides were digested in-gel using sequencing grade modified trypsin (Promega) in 100 mM ammonium bicarbonate [pH 8.5] buffer. DTT and iodoacetamide were added for reduction and alkylation of cysteine residues. The digested peptides were extracted with acetonitrile, dried using a Thermo SpeedVac, then redissolved in 2% acetonitrile, 97.5% water, and 0.5% formic acid. Peptides were separated using a high pressure liquid chromatography system (HPLC) fitted with a reversed-phase C18 column (75 μM ID × 8 cm). Samples eluted from the HPLC column were directly ionized by electrospray ionization and analyzed by an ion trap mass spectrometer (LCQ DECA XP Plus, Thermo). MS/MS spectra were acquired via low energy collision induced dissociation. The collected mass spectrometric data were searched against the NCBI protein database using ProtTech’s ProtQuest software. Peptides were reported with a mass range of 550 to 1800 Da and a signal to noise ratio greater than or equal to 5.
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