Tmt kit itraq kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The TMT kit/iTRAQ kit is a set of reagents used for protein quantification and identification in mass spectrometry-based proteomics analyses. The kit provides isobaric labeling compounds that allow for the multiplexed analysis of up to 10 or 11 samples simultaneously. This enables the relative quantification of proteins across multiple experimental conditions.

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Lab products found in correlation

Strata x c18 spe column, by Phenomenex (6 mentions) Q exactive plus, by Thermo Fisher Scientific (3 mentions) C18 ziptip, by Merck Group (1 mentions) Antibody beads, by PTM Biolabs (1 mentions) Strata x c18 solid phase extraction column, by Phenomenex (1 mentions) Dithiothreitol (dtt), by Merck Group (1 mentions) Iodoacetamide, by Merck Group (1 mentions) High intensity ultrasonic processor, by Ningbo Scientz Biotechnology (1 mentions) Trypsin, by Phenomenex (1 mentions) Protease inhibitor cocktail, by Merck Group (1 mentions) Triethylammonium bicarbonate teab, by Merck Group (1 mentions) Trypsin, by Promega (1 mentions) Ultrasonic processor, by Ningbo Scientz Biotechnology (1 mentions) Thermo betasil c18 column, by Thermo Fisher Scientific (1 mentions) Pierce top 12 abundant protein depletion spin columns kit, by Thermo Fisher Scientific (1 mentions) Betasil c18 column, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

TMT kit (95 citations) ITRAQ kit (9 citations)

11 protocols using tmt kit itraq kit

Quantitative Proteome Analysis by TMT/iTRAQ

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After trypsin
digestion, the peptide was desalted by the Strata X C18 solid-phase
extraction column (Phenomenex, USA) and vacuum-dried. Peptides were
reconstituted in 0.5 M TEAB and processed in accordance with the manufacturer’s
protocol for the TMT kit/iTRAQ kit (Thermo Fisher Scientific). The
peptide mixtures were then incubated for 2 h at room temperature and
pooled, desalted, and dried by vacuum centrifugation.
The tryptic
peptides were fractionated into fractions by high pH reverse-phase
high-performance liquid chromatography using the Thermo Betasil C18
column (5 μm particles, 10 mm ID, 250 mm length). Briefly, peptides
were first separated with a gradient of 8–32% acetonitrile
(pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined
into eight fractions and dried by vacuum centrifuging.
For the
enrichment of the modified peptides, tryptic peptides were dissolved
in NETN buffer (containing 100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl,
and 0.5% NP-40; pH 8.0) and then incubated with prewashed antibody
beads (lot number 001, PTM Bio) at 4 °C overnight with gentle
shaking. Then, we washed the beads four times using NETN buffer and
twice using H₂O. The bound peptides were eluted from the
beads with 0.1% trifluoroacetic acid. For LC–MS/MS analysis,
the resulting peptides were desalted with C18 ZipTips (Millipore)
according to the manufacturer’s instructions.

Lu Y.P., Zhang X.L., Zheng F., Yun C., Zhu C., Cai W., Liu D., Hong X., Li Q., Hu B., Tang D., Yin L.H, & Dai Y. (2020). Quantitative Proteomic Analyses To Reveal the Key Features of Proteins in New Onset Ankylosing Spondylitis Patients. ACS Omega, 5(32), 20153-20161.

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Epididymal Adipose Tissue Proteomic Analysis

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Epididymal adipose tissue samples were powdered and dissolved in lysis buffer (8 M urea, 2 mM ethylenediamine tetraacetic acid and 1% protease inhibitor cocktail [Sigma, St. Louis, USA]), followed by sonication using a high-intensity ultrasonic processor (Scientz, Ningbo, China). The remaining debris was removed by centrifugation at 4°C. Supernatants were collected to determine protein concentration. For digestion, the protein solution was reduced with 5 mM dithiothreitol (Sigma, St. Louis, USA) for 30 min at 56 °C and alkylated with 11 mM iodoacetamide (Sigma, St. Louis, USA) for 15 min at room temperature in darkness. The protein sample was then diluted by adding 100 mM triethylammonium bicarbonate (TEAB; Sigma, St. Louis, USA) to a urea concentration <2 M. Trypsin (Promega, Madison, Wisconsin, USA) was added at 1:50 Trypsin:protein mass ratio for the first digestion overnight and 1:100 Trypsin:protein mass ratio for a second 4-h digestion. After Trypsin digestion, peptides were desalted using a Strata X C18 SPE column (Phenomenex, Torrance, California, USA) and vacuum-dried. Peptide samples were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for the TMT kit/iTRAQ kit (Thermo Fisher Scientific).

Wang J., Yang L., Yang L., Zhou F., Zhao H., Liu J., Ma H, & Song G. (2022). Adipose Dysfunction in Adulthood Insulin Resistance of Low-Birth Weight Mice: A Proteomics Study. Diabetes, Metabolic Syndrome and Obesity: Targets and Therapy, 15, 849-862.

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Protein Extraction and TMT/iTRAQ Labeling

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Protein samples of cells were acquired by dissolving in protein lysis buffer (8 M urea, 1% Protease Inhibitor Cocktail) and homogenizing in an ultrasonic processor (Scientz, Ningbo, China). The supernatant was collected after centrifugation at 12,000×g and 4 °C for 10 min.
Tandem Mass Tags/isobaric Tagging for Relative and Absolute Quantification (TMT/iTRAQ) Labeling
Peptides acquiring from digestion went through desalting by Strata X C18 SPE column and vacuum-drying. After reconstituted in 0.5 M TEAB, peptides were processed as the protocol for TMT kit/iTRAQ kit (Thermo Fisher Scientific Inc.) providing by the manufacturer

Song Q., He X., Xiong Y., Wang J., Zhang L., Leung E.L, & Li G. (2021). The functional landscape of Golgi membrane protein 1 (GOLM1) phosphoproteome reveal GOLM1 regulating P53 that promotes malignancy. Cell Death Discovery, 7, 42.

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Proteomic Analysis of KYSE150 Cells

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KYSE150 (4.5 × 10⁶ cells) were seeded into a 15 cm dish and treated with 5 μM Nuplazid or DMSO for 24 h as control. For trypsin digestion, protein was extracted from cells using buffer with 8 M urea with 1% protease inhibitor cocktail. After reduction with 5 mM dithiothreitol at 56 °C for 30 min, the protein sample was alkylated with 11 mM iodoacetamide for 15 min RT in darkness. Then 100 mM TEAB was added to dilute samples to a urea concentration less than 2 M. Trypsin was added to the sample (according to mass ratio 1:50 trypsin-to-protein) for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h digestion. After digestion with trypsin, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried according to the manufacturer’s protocol for TMT kit/iTRAQ kit. After reconstitution in 0.5 M TEAB and processing by TMT kit/iTRAQ kit (Thermo, 90064), peptides were fractionated by high pH reverse-phase HPLC using Agilent 300Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo) coupled online to the UPLC.

Wei Y., Wu W., Jiang Y., Zhou H., Yu Y., Zhao L., Wu X., Lu X., Yuan Q., Wang Z., Dong Z., He L., Zhao J, & Liu K. (2021). Nuplazid suppresses esophageal squamous cell carcinoma growth in vitro and in vivo by targeting PAK4. British Journal of Cancer, 126(7), 1037-1046.

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Peptide Purification and Labeling Protocol

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The peptides were desalted using a Strata X C18 SPE column (Phenomenex, USA) and vacuum freeze-dried. The peptides were dissolved in 0.5 M TEAB and labeled with the TMT kit/iTRAQ kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Then, the TMT/iTRAQ reagent was reconstituted in acetonitrile, the peptides were mixed and incubated for 2 h, and the samples were pooled and vacuum freeze-dried. The peptides were eluted using high-pH reverse-phase HPLC using a Thermo Betasil C18 column (particle size, 5.0 μm; 250 mm × 10.0 mm i.d.; Thermo Fisher Scientific, Waltham, MA, USA). Briefly, the peptides were combined into six fractions and dried by vacuum centrifugation.

Xi Y., Zhang D., Liang Y., Shan Z., Teng X, & Teng W. (2022). Proteomic Analysis of the Intestinal Resistance to Thyroid Hormone Mouse Model With Thyroid Hormone Receptor Alpha Mutations. Frontiers in Endocrinology, 13, 773516.

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Quantitative Proteomics of Cortical Arterioles

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Proteins of cortical arterioles were prepared immediately after cortical vessel collection (Dongsheng et al., 2016 (link)). After trypsin digestion, the peptides were desalted with a Strata X C18 SPE column (Phenomenex) and vacuum-dried. The peptides were reconstituted in 0.5 M TEAB and processed according to the protocol of the TMT kit/iTRAQ kit (Thermo Fisher). The tryptic peptides were fractionated by high pH reverse-phase HPLC using a Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm long). The peptides were subjected to a nanospray ionization (NSI) source followed by tandem mass spectrometry (MS/MS) with a Q ExactiveTM Plus mass spectrophotometer (Thermo) coupled online to the UPLC. The resulting MS/MS data were processed using the MaxQuant search engine (v.1.5.2.8). Furthermore, bioinformatics analysis of overlapping differentially expressed proteins (DEPs) was conducted with InterProScan, the KEGG database and the STRING database version 10.1. Selected DEPs were validated by Q-PCR and Western blotting.

Meng H., Fan L., Zhang C.J., Zhu L., Liu P., Chen J., Bao X., Pu Z., Zhu M.S, & Xu Y. (2021). Synthetic VSMCs induce BBB disruption mediated by MYPT1 in ischemic stroke. iScience, 24(9), 103047.

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Serum Proteome Profiling by TMT-MS

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Relative proteins were quantified using Tandem Mass Tag mass spectrometry by PTM BIOLABS company. Firstly, the top 12 high abundance proteins were removed from serum samples by Pierce Top 12 Abundant Protein Depletion Spin Columns Kit (Thermo Fisher Scientific, Waltham, MA, USA). Trypsin was then used for the protein digestion. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex, CA, USA) and labelled according to the manufacturer’s protocol for TMT kit/iTRAQ kit (Thermo Fisher Scientific, Waltham, MA, USA). Then, the tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column. The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q ExactiveTM Plus (Thermo Fisher Scientific, Waltham, MA, USA) coupled online to the UPLC. Finally, the resulting MS/MS data were processed using Maxquant search engine (v.1.5.2.8). Tandem mass spectra were searched against the Uniprot database concatenated with reverse decoy database.

Guo L., Chen Y., Li H., Yin F., Ge M., Hu L., Zi M., Qin Z, & He Y. (2022). Telomere length is maternally inherited and associated with lipid metabolism in Chinese population. Aging (Albany NY), 14(1), 354-367.

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Quantitative Proteomic Analysis of cr-rz1al Mutant

Cited 1 time

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Proteomic analyses were carried out by PTM-BIO (Zhejiang, China) as previously described [65 (link)]. Briefly, the extracted total proteins were digested into peptides with trypsin, as described in the users manual for the TMT kit/iTRAQ kit (ThermoFisher). Then, the peptides were quantitatively analyzed by LC–MS/MS. Finally, the resulting MS/MS data were analyzed using the Maxquant search engine (v.1.5.2.8). Differential expression proteins between cr-rz1al and WT were identified based on the threshold of |fold change| > 1.2 combined with a P-value of <.05. Four biological replicates were used here in proteomic analyses for WT and cr-rz1al mutants.

Li X., Yang Y., Zeng N., Qu G., Fu D., Zhu B., Luo Y., Ostersetzer-Biran O, & Zhu H. (2022). Glycine-rich RNA-binding cofactor RZ1AL is associated with tomato ripening and development. Horticulture Research, 9, uhac134.

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TMT/iTRAQ Peptide Fractionation and Labeling

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After trypsin digestion, peptides were desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptides were reconstituted in 0.5 M TEAB and processed according to the manufacturer’s protocol for the TMT kit/iTRAQ kit (Thermo Fisher Scientific). Briefly, one unit of TMT/iTRAQ reagent was thawed and reconstituted in acetonitrile. The peptide mixtures were then incubated for 2 h at room temperature and pooled, desalted and dried by vacuum centrifugation.
The tryptic peptides were fractionated into fractions by high pH reverse-phase HPLC using Thermo Betasil C18 column (5 μm particles, 10 mm ID, 250 mm length). Briefly, peptides were first separated with a gradient of 8–32% acetonitrile (pH 9.0) over 60 min into 60 fractions. Then, the peptides were combined into 6 fractions and dried by vacuum centrifugation.

Yang Z., Zhang H., Tan X., Wei Z., Wen C., Sun Z., Sun B, & Chen J. (2022). Insights Into the Effect of Rice Stripe Virus P2 on Rice Defense by Comparative Proteomic Analysis. Frontiers in Microbiology, 13, 897589.

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Proteomic Profiling by Tandem Mass Spectrometry

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The proteins extracted from the strains by liquid nitrogen grinding method were enzymolized twice by trypsin to obtain the peptide. Then, the obtained peptides were desalted, labelled using a TMT kit/iTRAQ kit (Thermo Scientific), separated by high performance liquid chromatography, then assessed by tandem mass spectrometry (MS/MS) (Thermo Scientific). Finally, database search and quality control of the obtained data were carried out. All assays were repeated three or more times.

Li S., Liu Y., Weng L., Zhao Y., Zhang Y., Zhang Z., Yang Y., Chen Q., Liu X, & Zhang H. (2023). The F1Fo-ATP synthase α subunit of Candida albicans induces inflammatory responses by controlling amino acid catabolism. Virulence, 14(1), 2190645.

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