Based on the fluorescent peptide assay, the volume for 20 μg of the most concentrated sample was determined, and equal volumes of each sample were diluted with 50 mM TEAB to 25 μl per replicate. Each sample was labeled with TMT 6 Plex Mass Tag Labeling Kit (Thermo Scientific). Briefly, 20 μl of each TMT label (126-131) was added to each digested peptide sample and incubated for an hour. The reaction was quenched with 1 μl of 5% hydroxylamine and incubated for 15 min. All labeled samples were then mixed together and lyophilized to almost dryness. The TMT-labeled sample was reconstituted in 2% acetonitrile 0 0.1% TFA and desalted with a zip tip.
Tmt 6 plex mass tag labeling kit
Manufactured by Thermo Fisher Scientific
The TMT 6 Plex Mass Tag Labeling Kit is a product offered by Thermo Fisher Scientific. It is a multiplex isobaric mass tagging reagent system used for quantitative proteomics analysis.
Automatically generated - may contain errors
3 protocols using tmt 6 plex mass tag labeling kit
1
Fluorescent Peptide Quantification via TMT Labeling
2
Quantitative Proteomics Sample Preparation
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Desalted peptides were reconstituted in 40 μl of 50 mM TEAB and quantified using Pierce Fluorometric Peptide Assay (Thermo Scientific). Each sample was diluted with 50 mM TEAB to 0.5 μg/μl for a total of 50 μg of peptide per replicate and labeled with TMT 6 Plex Mass Tag Labeling Kit (Thermo Scientific). Briefly, 41 μl of each TMT label (126–131) was added to each digested peptide sample and incubated for 1 h. The reaction was quenched with 8 μl of 5% hydroxylamine and incubated for 15 min. All labeled samples were then mixed together and lyophilized to almost dryness. TMT labeled samples were reconstituted in 0.1% trifluoroacetic acid (TFA) and the pH was adjusted to 2 with 10% TFA. The combined sample (20 μg) was separated into 8 fractions by Pierce High pH Reverse-Phase Peptide Fractionation Kit (Thermo Scientific) with an extra wash before separation to remove excess labels. The 8 fractions were dried almost to completion.
3
TMT Labeling of Peptide Samples
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Based on the Fluorescent Peptide assay, the volume for 20 μg of the most concentrated sample was determined, and equal volumes of each sample were diluted with 50mM TEAB to 25 μl per replicate. Each sample was labeled with TMT 6 Plex Mass Tag Labeling Kit (Thermo Scientific).
Briefly, 20 μl of each TMT label (126-131) was added to each digested peptide sample and incubated for an hour. The reaction was quenched with 1μl of 5% Hydroxylamine and incubated for 15 minutes. All labeled samples were then mixed together and lyophilized to almost dryness.
The TMT labeled sample was reconstituted in 2% Acetonitrile 0 .1% TFA and desalted with a zip tip.
Briefly, 20 μl of each TMT label (126-131) was added to each digested peptide sample and incubated for an hour. The reaction was quenched with 1μl of 5% Hydroxylamine and incubated for 15 minutes. All labeled samples were then mixed together and lyophilized to almost dryness.
The TMT labeled sample was reconstituted in 2% Acetonitrile 0 .1% TFA and desalted with a zip tip.
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