N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion |
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Authors: | Neeraj Dhaunta Uzma Fatima Purnananda Guptasarma |
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Affiliation: | Protein Science & Engineering, Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India |
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Abstract: | We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins. |
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Keywords: | Fluorescamine adduction Mass spectrometry Protein sequencing N-terminal sequencing pH effects α-Amino group reactivity |
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