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Isobaric tmt reagents

Manufactured by Thermo Fisher Scientific
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Isobaric TMT reagents are a set of chemical labels used in mass spectrometry-based proteomics for quantitative analysis of protein expression. These reagents enable the simultaneous identification and relative quantification of peptides across multiple samples.

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Lab products found in correlation

Bca protein concentration assay kit, by Thermo Fisher Scientific (7 mentions) Sep pak cartridges, by Waters Corporation (6 mentions) Lys c protease, by Fujifilm (4 mentions) Mass spectrometry grade trypsin, by Thermo Fisher Scientific (4 mentions) Complete protease inhibitor, by Merck Group (3 mentions) Lys c protease, by Thermo Fisher Scientific (3 mentions) High select fe nta phospohpeptide enrichment kit, by Thermo Fisher Scientific (2 mentions) Complete protease and phosphatase inhibitors, by Merck Group (2 mentions) Ms grade trypsin, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Sep pak cartridges 100 mg, by Waters Corporation (1 mentions)

11 protocols using isobaric tmt reagents

1

TMT Isobaric Labeling Protocol

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Tandem mass tag (TMT) isobaric reagents (Thermo Fisher Scientific, 90110) were resuspended in anhydrous acetonitrile (final concentration of 20 μg/μL ). 10 μL of the labeling reagents plus 30 μL of acetonitrile was added to the peptides obtained by the previous digestions (~100 μg). After incubation at room temperature (90 minutes), the reaction was quenched using hydroxylamine to a final concentration of 0.3% (v/v). The TMT-labeled samples were pooled at a 1:1 ratio across all the samples. Fractions were fractionated off-line using basic pH reversed phase chromatography (BPRP). Fractions were pooled into 6 or 12 super-fractions which were acidified with formic acid to a final concentration of 1%. The pooled peptides were desalted using homemade StageTip, dried with vacuum centrifugation, and reconstituted in 5% acetonitrile and 5% formic acid for LC-MS/MS processing.
Rossio V., Paulo J.A., Chick J., Brasher B., Gygi S.P, & King R.W. (2021). Proteomics of broad deubiquitylase inhibition unmasks redundant enzyme function to reveal substrates and assess enzyme specificity. Cell chemical biology.
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2

Reagent Preparation for Proteomic Analysis

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TMT isobaric reagents were from Thermo Fisher Scientific (Waltham, MA). Water and organic solvents were from J.T. Baker (Center Valley, PA). Unless otherwise noted, all other chemicals were from Sigma-Aldrich (St. Louis, MO).
Paulo J.A., O’Connell J.D., Gaun A, & Gygi S.P. (2015). Proteome-wide quantitative multiplexed profiling of protein expression: carbon-source dependency in Saccharomyces cerevisiae. Molecular Biology of the Cell, 26(22), 4063-4074.
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3

Tandem Mass Tag Proteomic Analysis

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Tandem mass tag (TMT) isobaric reagents were from ThermoFisher Scientific (Waltham, MA). Water and organic solvents were from J.T. Baker (Center Valley, PA).
Paulo J.A., Navarrete-Perea J., Thakurta S.G, & Gygi S.P. (2018). TKO6: A Peptide Standard to Assess Interference for Unit-Resolved Isobaric Labeling Platforms. Journal of proteome research, 18(1), 565-570.
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4

Isobaric Peptide Labeling and Normalization

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Peptides were then labeled with TMT isobaric reagents (Thermo Fisher Scientific, Waltham, MA) as per the manufacturer's instructions. Labeling was quenched using 5% hydroxylamine. A small aliquot of the labelled peptides was pooled from all the samples and analyzed by LC-MS/MS to determine the normalized factors. For normalized experiments, peptides were pooled as per the normalization factors to account for the protein loading differences between samples. Alternatively, in the volume-normalized experiments, peptides from differing TMT-labelled samples were pooled using equal proportions of the eluted sample volume from each affinity chromatography column. Pooled peptides were further cleaned using C 18 stage tips, vacuum dried, and subjected to LC-MS/MS analysis.
Shortreed N.A., Panicker A.J., Mangalaparthi K.K., Zhong J., Pandey A, & Griffiths L.G. (2023). Optimization of a high-throughput shotgun immunoproteomics pipeline for antigen identification. Journal of proteomics, 281.
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5

Isobaric Labeling Proteomics Workflow

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The following reagents were employed: Isobaric TMT reagents (Thermo Fisher Scientific), BCA protein concentration assay kit (Thermo Fisher Scientific), Empore-C18 material for in-house made StageTips (3 M), Sep-Pak cartridges (100 mg, Waters), solvents for Liquid chromatography (LC) (J.T. Baker), mass spectrometry (MS)-grade trypsin (Thermo Fisher Scientific), Lys-C protease (Wako), and cOmplete protease inhibitors (Millipore Sigma). Unless otherwise noted, all other chemicals were purchased from Thermo Fisher Scientific.
Nabet B., Ferguson F.M., Seong B.K., Kuljanin M., Leggett A.L., Mohardt M.L., Robichaud A., Conway A.S., Buckley D.L., Mancias J.D., Bradner J.E., Stegmaier K, & Gray N.S. (2020). Rapid and direct control of target protein levels with VHL-recruiting dTAG molecules. Nature Communications, 11, 4687.
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6

Proteomics and Phosphoproteomics Materials

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Materials used in proteomics and phosphoproteomics assessments: Isobaric TMT reagents (Thermo Fisher Scientific), BCA protein concentration assay kit (Thermo Fisher Scientific), Empore-C18 material for in-house made StageTips (3 M), Sep-Pak cartridges (Waters), Mass spectrometry (MS)-grade trypsin (Thermo Fisher Scientific), Lys-C protease (Wako), High-Select Fe-NTA phospohpeptide enrichment kit (Thermo Fisher Scientific), and cOmplete protease and phosphatase inhibitors (Millipore Sigma). Unless otherwise noted, all solvents used for liquid chromatography (LC) were purchased from J.T. Baker all other chemicals were purchased from Thermo Fisher Scientific.
Ferguson F.M., Nabet B., Raghavan S., Liu Y., Leggett A.L., Kuljanin M., Kalekar R.L., Yang A., He S., Wang J., Ng R.W., Sulahian R., Li L., Poulin E.J., Huang L., Koren J., Dieguez-Martinez N., Espinosa S., Zeng Z., Corona C.R., Vasta J.D., Ohi R., Sim T., Kim N.D., Harshbarger W., Lizcano J.M., Robers M.B., Muthaswamy S., Lin C.Y., Look A.T., Haigis K.M., Mancias J.D., Wolpin B.M., Aguirre A.J., Hahn W.C., Westover K.D, & Gray N.S. (2020). Discovery of a selective inhibitor of Doublecortin Like Kinase 1. Nature chemical biology, 16(6), 635-643.
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7

Mass Spectrometry-Based Proteomic Workflow

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Reagents for tissue culture, including DMEM, fetal bovine serum (FBS), penicillin/streptomycin, and phosphate-buffered saline (PBS) were obtained from Gibco. Mass spectrometry-grade trypsin and Lys-C protease were purchased from ThermoFisher Scientific and Wako, respectively. Isobaric TMT reagents and the BCA protein concentration assay kit were from ThermoFisher Scientific. Empore-C18 material for in-house StageTips was acquired from 3 M and Sep-Pak cartridges were purchased from Waters. Sera-Mag Speed Beads (cat. nos. 45152105050350 and 65152105050350) were from GE Life Sciences (Marlborough, MA). All solvents used for liquid chromatography were purchased from VWR. Unless otherwise noted, all other chemicals were purchased from ThermoFisher Scientific.
Yu Q., Liu X., Keller M.P., Navarrete-Perea J., Zhang T., Fu S., Vaites L.P., Shuken S.R., Schmid E., Keele G.R., Li J., Huttlin E.L., Rashan E.H., Simcox J., Churchill G.A., Schweppe D.K., Attie A.D., Paulo J.A, & Gygi S.P. (2023). Sample multiplexing-based targeted pathway proteomics with real-time analytics reveals the impact of genetic variation on protein expression. Nature Communications, 14, 555.
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8

Isobaric Labeling and MS-based Proteomics

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The following reagents were employed: Isobaric TMT reagents (Thermo Fisher Scientific), BCA protein concentration assay kit (Thermo Fisher Scientific), Empore-C18 material for in-house made StageTips (3 M), Sep-Pak cartridges (100 mg, Waters), solvents for Liquid chromatography (LC) (J.T. Baker), mass spectrometry (MS)-grade trypsin (Thermo Fisher Scientific), Lys-C protease (Wako), and cOmplete protease inhibitors (Millipore Sigma). Unless otherwise noted, all other chemicals were purchased from Thermo Fisher Scientific.
Banh R.S., Biancur D.E., Yamamoto K., Sohn A.S., Walters B., Kuljanin M., Gikandi A., Wang H., Mancias J.D., Schneider R.J., Pacold M.E, & Kimmelman A.C. (2020). Neurons Release Serine to Support mRNA Translation in Pancreatic Cancer. Cell, 183(5), 1202-1218.e25.
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9

TMT Labeling and BCA Assay

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Isobaric TMT reagents and the BCA protein concentration assay kit were sourced from Thermo Fisher Scientific, Empore-C18 material for in-house StageTips was acquired from 3 M and Sep-Pak cartridges (100 mg) were purchased from Waters. All solvents used for liquid chromatography were purchased from J.T. Baker. Mass spectrometry-grade trypsin and Lys-C protease were purchased from Thermo Fisher Scientific and Wako, respectively, and the SRC NanoBRET assay was purchased from Promega. Unless otherwise noted, all other chemicals were purchased from Thermo Fisher Scientific.
Kuljanin M., Mitchell D.C., Schweppe D.K., Gikandi A.S., Nusinow D.P., Bulloch N.J., Vinogradova E.V., Wilson D.L., Kool E.T., Mancias J.D., Cravatt B.F, & Gygi S.P. (2021). Reimagining high-throughput profiling of reactive cysteines for cell-based screening of large electrophile libraries. Nature biotechnology, 39(5), 630-641.
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10

Comprehensive Proteomic Sample Preparation

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Isobaric TMT reagents and the BCA protein concentration assay kit were from ThermoFisher Scientific (Rockford, IL, USA). Empore-C18 material for in-house made Stage Tips was from 3 M (Saint Paul, MN, USA). Sep-Pak cartridges (100 mg size) were purchased from Waters (Milford, MA, USA). All solvents used for Liquid chromatography (LC) were purchased from J.T. Baker (Central Valley, PA, USA). Mass spectrometry (MS)-grade trypsin and Lys-C protease were purchased from ThermoFisher Scientific and Wako (Boston, MA, USA), respectively. Complete protease inhibitors were from Millipore Sigma (Saint Louis, MO, USA). Unless otherwise noted, all other chemicals were purchased from ThermoFisher Scientific.
Zmyslowska A., Kuljanin M., Malachowska B., Stanczak M., Michalek D., Wlodarczyk A., Grot D., Taha J., Pawlik B., Lebiedzińska-Arciszewska M., Nieznanska H., Wieckowski M.R., Rieske P., Mancias J.D., Borowiec M., Mlynarski W, & Fendler W. (2021). Multiomic analysis on human cell model of wolfram syndrome reveals changes in mitochondrial morphology and function. Cell Communication and Signaling : CCS, 19, 116.
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