QB3 Berkeley / Facilities / Core Research Facilities / Vincent J. Coates Proteomics/Mass Spectrometry Laboratory / Protocols / TMT Peptide Labeling

TMT Peptide Labeling

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  1. Plan cell growth or IP so that the target sample will be 100 ug or more of protein.
  2. Take digested peptides either from a large scale IP or a whole cell lysate and desalt using C18 Column and speed vac dry overnight (see separate peptide desalting and trypsin digestion protocols)
  3. Resuspend peptides in 200mM HEPPS pH 8
  4. Quantify peptides using Pierce Quantitative Colorimetric Peptide Assay (Pierce 23275) (Fisher accuSkan FC use Abs 490 in mass spec facility)
  5. Normalize peptides to be compared to equal concentration and volume in 200mM HEPPS pH 8 final volume 100ul for one reagent vial. (can use less with ratio of 8ug of TMT reagent to 1ug/peptide) (range recommended 25-100ug)
  6. Label using TMT labeling reagents (protocol TMT Mass Tagging Kits and Reagents) (used TMTduplex Label Reagent Set)
    1. Before use Equilibrate the TMT label reagents at room temp.
    2. Resuspend .8mg vial with 41ul anhydrous acetonitrile. Allow reagent to dissolve for 5minitues with occasional vortexing. Centrifuge tube briefly.
    3. Add 41ul of resuspended TMT reagent to tube of peptides (100ul).
    4. Mix gently and let react for 1 hour at room temp
    5. Quench reaction with 8ul 5% Hydroxylamine for 15min
    6. Combine samples at equal amounts in a new tube and store at -80
  7. Desalt using C18 Column and speed vac dry.
  8. Mass spec time!