Sequence Determination with the Help of Mass Spectra

Automatic sequencing with its numerous improvements is now routinely performed. A cycle requires an hour or less and the amount of peptide needed is well below the earlier applied 1 micromol. Yet, errors are not rare in the interpretation of results and the sequence of a peptide can be considered firmly established only if it is corroborated by a second, independent procedure. 

Sequence Determination with the Help of Mass Spectra 

Sequencing a peptide by the recording of a single spectrum is a very attractive proposition which stimulated considerable research. The classical process of mass spectrometry requires a volatile sample which when injected into an evacuated chamber evaporates. The vapors are then exposed to a beam of electrons. Under electron bombardment the molecular ion and fragment ions are produced. For this purpose most peptides are not sufficiently volatile. 

Substitution of polar groups yields a less polar and therefore more volatile material. Acetylation with acetic anhydride modifies the terminal amino group, the side chain amino group in lysine residues and also the hydroxyl groups in serine and threonine side chains in the desired sense. Volatility can be further increased by esterification of free carboxyls by treatment of the solution of the acetyl derivative in methanol with diazomethane  

The molecular ion of the derivatized peptide reveals only its molecular weight and has therefore limited importance. The charge propagates however along the chain and induces fragmentation. Fission of the C-terminal ester bond yields an ion with the mass of the molecular ion minus 31 (the methoxy group). This, usually abundant ion, 

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Sequence Determination with the Help of Mass Spectra 25 ment with substituted phenylisothiocyanate can be applied... 

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