We have developed a method for mass-tagging of the N-terminus of any protein without tagging lysine or arginine sidechains. Briefly, we use fluorescamine at pH 6.0. Fluorescamine has been thought to act only in alkaline pH, but we show that at mildly acidic pH it selectively forms adducts with alpha amino groups but not with epsilon amino groups in proteins. We use this discovery to develop a method for identifying and sequencing the tryptic fragment that lies at the amino terminus of any protein. Briefly, we carry out peptide mass fingerprinting (PMF) with and without fluorescamine treatment. Of all the tryptic peptides, only one peptide (the N-terminal tryptic peptide) shows up in two forms, with a difference in mass of 280 Daltons, corresponding to the fluorescamine-adducted and non-adducted populations of the N-terminal tryptic peptide. This allows identification of the N-terminal tryptic peptide, which can be followed by regular MS-MS-based amino acid sequencing. Thus, we have developed a mass spectrometry based N-terminal sequencing method which can substitute for Edman degradation-based chemical (cycle) sequencing of protein N-termini.

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