N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion.
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.
Dhaunta
Neeraj
N
Protein Science & Engineering, Institute of Microbial Technology, Sector 39-A, Chandigarh 160 036, India.
Fatima
Uzma
U
Guptasarma
Purnananda
P
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D005450:Fluorescamine / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000737:chemistry*; D020539:Sequence Analysis, Protein; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D007202:Indicators and Reagents; D010455:Peptides; D005450:Fluorescamine; D009113:Muramidase; D014357:Trypsin