Short peptides containing an N-terminal primary or secondary amino group, an amino acid with a thiol group in the side chain and either a Gly-Gly-Trp or a Gly-Trp appendix at the C terminus, were used. The tryptophan residues facilitated HPLC analysis of the peptides and reaction products due to the strong absorption at 280 nm. Using a 0.5-ml reaction tube, the following reagents were added sequentially and incubated for 1 hour at 37°C: 78 μl of 60 mM NH4HCO3 (pH 8), 2 μl of peptide in H2O (5 mM), and 20 μl of linker reagent in ACN (4 mM). The final concentrations of peptide and linker reagent were 100 and 800 μM, respectively. The reactions were analyzed by LC-MS (LCMS-2020, Shimadzu) [injection volume of 4 μl; Kinetix C18 column, 2.6 μm, 100 Å, 50 mm by 2.1 mm (Phenomenex); gradient: solvent A (99.95% H2O and 0.05% formic acid) and 0 to 40% solvent B (99.95% ACN and 0.05% formic acid) for 5 min; flow, 1 ml/min; MS analysis, positive mode].

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