Immediately before use, we equilibrated the TMT Label Reagents (Cat#90111, Thermo Scientific) to room temperature. To 0.8‐mg vials, we added 84 μL anhydrous acetonitrile and dissolved the reagent for 5 min with occasional vortexing. Each post‐IP peptide sample was dissolved in 20 μL 100 mmol/L tetraethylammonium bromide (pH = 8.0), and 8 μL TMT Label Reagent was carefully added. The reaction was incubated for 1 h at room temperature. Subsequently, 2 μL 5% hydroxylamine was added to each sample, and the sample was incubated for 15 min to quench the reaction. The supernatant was desalted using a homemade C18 stage tip and dried by vacuum centrifugation.
Tmt label reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TMT label reagent is a multiplex isobaric labeling technology used for quantitative proteomics analysis. It enables the simultaneous identification and relative quantification of proteins across multiple samples.
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Lab products found in correlation
Proteome discoverer 2, by Thermo Fisher Scientific (3 mentions)
Tmt mass tagging kits and reagents, by Thermo Fisher Scientific (2 mentions)
Orbitrap fusion lumos, by Thermo Fisher Scientific (1 mentions)
Teab buffer, by Merck Group (1 mentions)
Easy nlctm 1200 system, by Thermo Fisher Scientific (1 mentions)
Nanospray flex source, by Thermo Fisher Scientific (1 mentions)
Q exactive mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Eif3a, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg hrp affinity purifiedpab, by R&D Systems (1 mentions)
Ece 1, by Santa Cruz Biotechnology (1 mentions)
Q exactive, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Orbitrap fusion lumos tribrid, by Thermo Fisher Scientific (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
11 protocols using tmt label reagent
1
Protein Labeling with TMT Reagents
2
Proteomic analysis of cell activation
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RAW264.7 cells were activated with LPS for 3 h, incubated another 30 min with or without Cel, then treated with Cel-P or DMSO for 2 h. Total protein was extracted, estimated using a BCA kit, and subjected to click chemistry for 2 h at room temperature [37 (link)]. Proteins were precipitated using acetone pre-chilled to −20 °C, resolubilized in PBS containing 1.5% SDS, and incubated with streptavidin beads for 4 h at room temperature. Streptavidin beads were washed gently with 5 ml PBS containing 1% SDS (twice), 0.1% SDS (once), 6 mol/L urea (three times) and PBS (twice).
Proteins bound to the streptavidin beads were eluted and separated by SDS-PAGE. Bands were excised, minced, reduced with dithiothreitol (DTT) and alkylated using iodoacetamide (IAA), and digested with trypsin overnight at room temperature. The resulting peptides were desalted on a C18 column, labeled using TMT label reagents (Thermo Scientific, MA, USA), and identified by liquid chromatography – tandem mass spectrometry (LC–MS/MS, Orbitrap Fusion Lumos, Thermo Scientific, MA, USA).
Proteins bound to streptavidin beads were fractionated by SDS-PAGE as described above and analyzed by Western blotting.
Proteins bound to the streptavidin beads were eluted and separated by SDS-PAGE. Bands were excised, minced, reduced with dithiothreitol (DTT) and alkylated using iodoacetamide (IAA), and digested with trypsin overnight at room temperature. The resulting peptides were desalted on a C18 column, labeled using TMT label reagents (Thermo Scientific, MA, USA), and identified by liquid chromatography – tandem mass spectrometry (LC–MS/MS, Orbitrap Fusion Lumos, Thermo Scientific, MA, USA).
Proteins bound to streptavidin beads were fractionated by SDS-PAGE as described above and analyzed by Western blotting.
3
Peptide Labeling with TMT Reagents
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The dried and desalted peptides were labeled by TMT Label Reagents (Thermo Scientific USA) according to the manufacturer’s protocol [17 (link)]. The resultant peptide mixture was labeled with TMT reagent as follows: ATCC26695-126 isobaric tag, H879-127 isobaric tag and MALT1-128 isobaric tag.
4
Peptide Labeling with TMT Reagents
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Desalted peptides were reconstituted in 100 mM TEAB pH 8.5 and labeled using tandem mass tag (TMT) label reagents (Thermo Fisher Scientific). Each prepared TMT reagent was transferred to the peptide sample, the mixture was incubated for 1 hour, quenched by the addition of 5 mL of 5% hydroxylamine, and incubated for 15 minutes at room temperature. Differently labeled peptides was pooled and dried using vacuum concentrator.
5
Phosphoproteome Analysis of MAL2 Overexpression
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Lysates of NCI-H23 cells infected with control lentivirus or overexpressing MAL2 were further lysed with sonication at 4 °C for 3 min (80 W, on 1 s and off 1 s) and centrifuged at 12,000×g at 4 °C for 10 min to remove insoluble particles, then centrifuged one more time and the supernatant was collected. The samples were fractionated using sequencing-grade trypsin in 100 mM TEAB buffer (Sigma). Then, the samples were labeled using the TMT label reagent (Thermo scientific) according to the manufacturer’s instructions. Phosphorylated peptides were enriched by using titanium dioxide beads (TiO2). Mass spectrometry analysis was performed by Shanghai OE-biotech (China) with an EASY-nLCTM 1200 system (Thermo, USA) in Q-Exactive mass spectrometer equipped with a Nanospray Flex source (Thermo, USA). The data were processed with Proteome Discoverer™ 2.2 (Thermo, USA) software against the Homo sapiens Uniprot database. The phosphorylatedproteins with an average fold change (FC > 1.2; P < 0.05) in the experimentally treated groups were considered to be the differentially accumulated proteins.
6
Antibody Detection and Protein Quantification
7
Proteomic Analysis of SCMEC and BMEC Cells
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Protein extraction from SCMEC and BMEC samples labeled SP1, SP2, SP3 and BP1, BP2, BP3, respectively, was carried out using lysis buffer (8 M urea, 2 M thiourea, 4% CHAPS, 20 mM Tris-base, 30 mM dithiothreitol (DTT), 1 mg/10 μL) and protease inhibitors (Roche, Basel, Switzerland) on ice. Peptides were obtained using trypsin (sequencing grade) digestion, labeled with TMT Label Reagent and quenched with hydroxylamine, according to the manual of TMT Mass Tagging Kits and Reagents (Thermo Fisher Scientific, USA). Proteins were analyzed using the nanoscale liquid chromatography system U3000 nano (Thermo Fisher Scientific, USA) and the mass spectrometer Q-Exactive (Thermo Fisher Scientific, USA) at CapitalBio Technology Inc. Raw data analysis was processed using the Proteome Discoverer 2.3 software (Thermo Fisher Scientific, USA). Samples for RNA-seq and proteomics analysis were isolated from cells in the same culture passage.
8
Quantitative Proteomics by TMT Labeling
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The collected proteins (100 μg) from each sample were digested by trypsin enzyme overnight at 37 °C followed by reductive alkylation to generate the peptides. Then, the peptides were labeled with TMT label reagent (ThermoFisher, 90111, USA) at room temperature for 2 h. Three repeated trials of the control group were labeled with TMT10-126, TMT10-127 N, and TMT10-127C; the model group was labeled with TMT10-128 N, TMT10-128C, and TMT10-129 N; and the KHJSC group was labeled with TMT10-130 N, TMT10-130C, and TMT10-131.
9
Quantitative Proteome Profiling via TMT-MS3
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Digested samples were labeled with TMT Label Reagent (ThermoFisher Scientific, USA). Peptides were purified, lyophilized (Speedvac, ThermoFisher Scientific, USA), and stored at −80 °C. Samples were analyzed on an Orbitrap Fusion Lumos Tribrid mass spectrometer equipped with nanospray source and nano-liquid chromatography (nano-LC) system (ThermoFisher, San Jose, CA, USA). MS data were acquired using MultiNotch synchronous precursor selection MS3 scanning for TMT tags. Proteome Discoverer 2.2 (ThermoFisher Scientific, USA) was used to analyze raw files and Uniprot human database (Uniprot-Human-Jan 15, 2018.fasta, Sequest HT) was searched for fragment lists, with parent and fragment mass tolerances set to 10 ppm and 0.6Da, respectively. Complete tryptic peptides with a maximum of two missed cleavages were accepted. Search results were filtered at the peptide spectral match level using a strict false discovery rate (FDR) q-value of 0.01 and relaxed FDR q-value of 0.05 [23 (link)]. TMT reporter ions were quantified.
10
Peptide Labeling for Quantification
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Di-Gly–modified peptides enriched from each sample were dissolved in 20 μl of 100 mM TEAB and labeled with 0.8 mg 10plex TMT Label Reagent (ThermoFisher) in 50 μl of ACN. The reaction was proceeded at room temperature for 1 h and stopped by 2 μl of 5% hydroxylamine. Supernatant was desalted by homemade C18 stage tip and dried by vacuum centrifugation.
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