Efficient peptide ladder sequencing by MALDI‐TOF mass spectrometry using allyl isothiocyanate

A new modification of the peptide ladder sequencing technique is described in which allyl isothiocyanate (AITC) replaces trifluoroethyl isothiocyanate as the volatile amine-modification reagent. AITC is commercially available, readily purified, stable up to 80 degrees C and reacts cleanly and rapidly with all amino groups of polypeptides. Several model peptides and two side chain-modified peptides were sequentially degraded using AITC and the cleavage reagent heptafluorobutyric acid (HFBA) up to seven amino acids from the N-terminus. Matrix-assisted laser-desorption and ionization coupled with time-of-flight (MALDI-TOF) mass spectroscopy of the peptide mixture provided a clear ladder-like mass profile with consecutive molecular ions corresponding to each shortened peptide at picomole range. The results indicate the general utility of this analytical protocol by the use of AITC as the amine-coupling reagent.