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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.
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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).
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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