Identification of asparagine, glutamine and the car☐yl‐terminal amino acids in polypeptides via sequence analysis by gas chromatography—mass spectrometry

Gas chromatography-mass spectrometry (CC-MS) has proved in recent years to be a valuable alternative and extension to the sequential degradation method of Edman for the determination of the amino acid sequence of polypeptides; for recent reviews see [ 1,2]. A particular polypeptide is first degraded, either by limited acid or by enzymatic hydrolysis, to a complex mixture of oligopeptides which is then suitably converted to a corresponding mixture of volatile derivatives. Analysis is performed with the aid of a CC-MS-Computer system. From the identified oligopeptides the sequence of the original polypeptide is finally deduced. Such approach has been successfully applied to the sequence determination of polypeptides between 20 and 39 amino acid residues long [2-61. The peptide derivatives used are obtained by LiAlD4reduction and 0trimethylsilylation ofN-perfluoroacyl oligopeptide methyl esters [7] and allowed the identification of all amino acid residues including the very polar ones (arginine, histidine, tryptophan). We found, however, that oligopeptides containing asparagine and glutamine residues are derivatised with rather low yields and also do not chromatograph as well as other derivatives. Even more important, the asparagine and glutamine residues are completely hydrolysed during acid hydrolysis to aspartic and glutamic acid residues. Thus, the positions of the asparagine and glutamine residues in the polypeptides analysed so far by CC-MS had to be determined by conventional methods [4-61. Since the assignment of these residues is also often difficult by the Edman