Synthesis of DiLeu tags was conducted according to protocol described in detail by Xiang et al.63 (link) DiLeu tag of 0.6 mg was activated in anhydrous DMF combined with 0.6 mg DMTMM, 0.2 mg NMM and vortexed at room temperature for 45 minutes. After centrifugation, the supernatant was used immediately for peptide labeling. DiLeu labeling was performed by addition of labeling solution to CSF peptide digest and vortex at room temperature for 2 h. The labeling reaction was quenched by addition of hydroxylamine to a concentration of 0.25%, and the labeled peptide samples were dried in vacuo. The samples were combined and cleaned with SCX SpinTips (Protea Biosciences, Morgantown, WV), and desalted with Omix C18 pipet tips (Agilent, Santa Clara, CA).
Protocol detail
Synthesis and Labeling of DiLeu Tags
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Corresponding Organization : University of Wisconsin–Madison
Other organizations : University of Wisconsin Carbone Cancer Center, Children's Hospital of Wisconsin, Medical College of Wisconsin
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Variable analysis
independent variables
- Concentration of DiLeu tag (0.6 mg)
- Concentration of DMTMM (0.6 mg)
- Concentration of NMM (0.2 mg)
- Reaction time (45 minutes)
- Concentration of hydroxylamine (0.25%)
- Yield of DiLeu-labeled peptides
- Anhydrous DMF as solvent
- Room temperature
- Vortexing during reactions
- Centrifugation and use of supernatant for peptide labeling
- Drying of labeled peptide samples in vacuo
- SCX SpinTips and Omix C18 pipet tips for sample cleanup and desalting
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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