Practical Protein Chemistry - A. Darbre 1989

Application of Electron Impact Mass Spectrometry for Determining the Amino Acid Sequence of Peptides and Proteins
Special Cases of Method Application
Determination of the N-Terminal Amino Acid Sequence

Typically, a protein Sequencer is used to determine the N-terminal Amino Acid Sequence of a protein. However, in certain cases—for instance, when the protein N-terminus is blocked or when it is necessary to determine the N-terminal sequences of several protein subunits simultaneously—mass spectrometry yields the required information much more successfully. To achieve this, 50–100 nmol of protein is dissolved in 200 µl of 98% formic acid, and 100 µl of acetic anhydride is added. After standing at room Temperature for 30 min, the solution is evaporated under reduced pressure. The protein formylated in this manner is then dissolved in 20 mM NH₄HCO₃ solution at pH 8.5 and hydrolyzed with Chymotrypsin or Elastase. The hydrolyzate is lyophilized and permethylated. Upon Permethylation, Peptides with free N-terminal amino groups form the corresponding quaternary ammonium salts, which are readily removed from the reaction mixture by washing with Water. Following this wash, only the peptide formylated at the N-terminal amino group remains in the reaction mixture. The described

method is used to obtain a mass spectrum, which is then used to establish The amino acid sequence of the N-terminal peptide. Clearly, for the successful application of this method, it is essential that, at a specific distance from the N-terminal amino acid residue, the protein contains peptide bonds cleavable by the specified Enzymes to yield peptides suitable for mass spectrometric analysis.