For labeling of peptides with tandem mass tag (TMT) reagents (Thermo), peptides on StageTips were washed once with HEPES buffer pH 8 and soaked with 4 µl of TMTsixplex™ (Thermo) isobaric label reagent dissolved in ACN for 1 h. Labeled peptides were eluted twice with 20 µl 80% (v/v) ACN, 0.1% FA and the reaction was quenched by adding 0.8 µl of 5% (v/v) Hydroxylamine for 15 min. Organic solvents were evaporated by vacuum centrifugation and peptides were loaded on StageTips as described above under “In-gel digestion”.
Tmtsixplex
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The TMTsixplex is a multiplexed isobaric labeling reagent used for quantitative proteomics analysis. It allows for simultaneous identification and quantification of up to six different protein samples in a single mass spectrometry experiment.
Automatically generated - may contain errors
Lab products found in correlation
Tandem mass tag 6 plex, by Thermo Fisher Scientific (2 mentions)
Tmt reagent, by Thermo Fisher Scientific (1 mentions)
Neuraminidase, by Merck Group (1 mentions)
Chymotrypsin, by Roche (1 mentions)
Tmt10plex, by Thermo Fisher Scientific (1 mentions)
Speedvac vacuum concentrator, by Thermo Fisher Scientific (1 mentions)
Sep pak c18 column, by Waters Corporation (1 mentions)
Trypsin, by Roche (1 mentions)
Glu c, by Roche (1 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Facsaria iiiu, by BD (1 mentions)
Orbitrap elite mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (1 mentions)
Hydroxylamine hydrochloride, by Merck Group (1 mentions)
3100 offgel fractionator, by Agilent Technologies (1 mentions)
8 protocols using tmtsixplex
1
Peptide Labeling with TMT Reagents
2
Protein Denaturation, Reduction, and Alkylation
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All extracted samples were adjusted to 50 mM ammonium bicarbonate, heated for 10 min at 80 °C, followed by reduction with 5 mM dithiothreitol (60 °C, 30 min) and alkylation with 10 mM iodoacetamide (30 min, RT, in the dark). Subsequently, samples were incubated with trypsin, chymotrypsin or GluC (Roche; 37 °C, overnight, 1 µg enzyme per 100 µg protein). The following day, the enzyme reaction was quenched and Rapigest was precipitated by acidifying with trifluoroacetic acid (TFA). The solution was cleared by centrifugation (10,000 × g, 10 min) and peptides were purified on C18 Sep-Pak columns (Waters), and dried down using a SpeedVac vacuum concentrator (Thermo Fischer Scientific). If not already desialylated, the dried peptides were resuspended in 1 mL 50 mM sodium acetate (pH 5.5) containing 0.1 U/mL neuraminidase (Sigma, N3001) followed by incubation at 37 °C for 1 h, purified by C18 Sep-Pak columns and dried down.
In the case of rat tissues, 200 µg digest was labeled with TMTsixplex or TMT10plex (Thermo Fischer Scientific) according to manufacturer’s instructions (Supplementary Data2 and 3 : Rapigest).
In the case of rat tissues, 200 µg digest was labeled with TMTsixplex or TMT10plex (Thermo Fischer Scientific) according to manufacturer’s instructions (Supplementary Data
3
Quantitative Proteomic Analysis of Colon Tissue
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Quantitative proteomic analysis of colon tissue extracts from CRL and IF1-KO mice was performed with isobaric tags for relative and absolute quantitation (iTRAQ, AB Sciex, 4383502) labeling method coupled to tandem mass spectrometry (MS/MS) as previously described [18 (link)]. Quantitative proteomic analysis of isolated colon mitochondria from CRL and IF1-KO mice (n = 3) was performed with Tandem Mass Tag™ 6-plex (TMTsixplex, Thermo Fisher, 90061) labeling method coupled to tandem mass spectrometry (MS/MS). In-gel digestion of protein extracts, iTRAQ/TMTsixplex labeling, reverse phase-liquid chromatography-MS/MS analysis and protein identification and quantitation were performed by the Proteomic Facility (CBMSO, Spain).
4
Proteomic Profiling of Cerebellar GNPs
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P7 cerebellar GNPs from Wdr4 A-cKO; Ai14 (tdTomato) and control mice were isolated using FACSAria IIIu (Becton Dickinson, NJ, USA). 5 × 105 GNPs were lysed with 8 M urea in 25 mM ammonium bicarbonate. After lysis and centrifugation, the extracted proteins were linearized with 2 mM dithioerythritol (DTE, #D8255, Sigma) at 37 °C for 1 h, and then with 10 mM iodoacetamide (IAM, #I6125, Sigma) at room temperature for 50 min in dark. Next, the linearized proteins were digested to peptides with Lys-C (0.3 µg for 15 µg substrate, #125-05061, Wako) at 37 °C for 3 h and then with Trypsin (0.3 µg for 15 µg substrate, #V5111, Promega) for 16 h. The digested peptides were purified using C18 ZipTip and then labeled by Tandem Mass Tag™ 6-plex (TMTsixplex™, #90064, Thermo Scientific) at room temperature for 1 h. Subsequently, equal amounts of labeled peptides from each group were pooled together and fractionated using high pH reversed-phase chromatography (#84868, Thermo Scientific) with 8 fractions eluted by increased acetonitrile (10, 12.5, 15, 17.5, 20, 22.5, 25, and 50%) buffer in 0.1% triethylamine, before analysis using Orbitrap Elite hybrid mass spectrometer (Thermo Electron, Bremen, Germany).
5
Proteomic Analysis of Ribosomal Proteins
6
Multiplexed Glycopeptide Profiling
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The enriched
glycopeptides from different samples were labeled with the TMT sixplex
(Thermo) reagents in 200 mM HEPES, pH = 8.6. The duplicate samples
with co-translational O-GlcNAcylated peptides were
labeled with the first two channels (126 and 127), and the duplicate
control experiments were labeled with the channels of 128 and 129.
The boosting sample was labeled with the channel of 131. The channel
of 130 was not used to avoid potential isotopic contamination from
the boosting sample. For the control experiment without the boosting
approach, no boosting sample was used, and all of the other experimental
setups were the same. The reaction lasted for 1 h at room temperature,
and then it was quenched by adding hydroxylamine hydrochloride (Sigma).
The labeled glycopeptides were purified using a stage-tip. Then, the
glycopeptides were fractionated through being sequentially eluted
with different concentrations of ACN with 1% of acetic acid before
being analyzed by liquid chromatography coupled to tandem mass spectrometry
(LC–MS/MS).
glycopeptides from different samples were labeled with the TMT sixplex
(Thermo) reagents in 200 mM HEPES, pH = 8.6. The duplicate samples
with co-translational O-GlcNAcylated peptides were
labeled with the first two channels (126 and 127), and the duplicate
control experiments were labeled with the channels of 128 and 129.
The boosting sample was labeled with the channel of 131. The channel
of 130 was not used to avoid potential isotopic contamination from
the boosting sample. For the control experiment without the boosting
approach, no boosting sample was used, and all of the other experimental
setups were the same. The reaction lasted for 1 h at room temperature,
and then it was quenched by adding hydroxylamine hydrochloride (Sigma).
The labeled glycopeptides were purified using a stage-tip. Then, the
glycopeptides were fractionated through being sequentially eluted
with different concentrations of ACN with 1% of acetic acid before
being analyzed by liquid chromatography coupled to tandem mass spectrometry
(LC–MS/MS).
7
Multiplexed Quantitative Proteomics of E. coli
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Each peptide sample of individual proteins extracted from three C2, C4, and C6 sample points of Escherichia coli was divided into two fractions and was individually labeled using a TMTSixplex (TMT stands for tandem mass tag) reagents kit (catalog no. 90064, Thermo Scientific, MA) according to the manufacturer’s protocol. TMT-126 and TMT-129 were used to label C2, TMT-128 and TMT-131 were used to label C4, and TMT-127 and TMT-130 were used to label C6. Aqueous hydroxylamine solution (5%, wt/vol) was added to quench the reaction. The six samples were then combined, speed vacuum dried, and then dissolved in 50 µl of Milli-Q−water containing 0.1% formic acid for liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis.
8
Tandem Mass Tag Peptide Profiling
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Purified and dried tryptic peptides were labeled with tandem mass tag (TMT™) six-plex (Thermo Scientific, Bremen, Germany) according to manufacturer’s instructions. Each biological replicate was labeled separately, pooled together and then fractionated using a 3100 OFFGEL fractionator (Agilent Technologies, Santa Clara, CA, USA) using 24-well high resolution immobilized pH gradient strips, pH 3–10, as described previously [68 (link)].
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