Primary structure determination of peptides and proteins by mass spectrometry
A pressing problem in modern organic chemistry is the determination of the structure of polypeptides and proteins, for it is here that the needs and interests of biochemistry have far surpassed present chemical possibilities, while elucidation of many a biological problem on the molecular level requires a clear understanding of the relation between structure and biological function. I should like to survey here the studies we have carried out in recent years that have led to the proposal of a new, general method for determining the amino acid sequence in peptides and proteins by means of mass spectrometry. As is well known the primary structure of proteins determines the architecture of the molecule as a whole, including its conformational specificities and in the long run its biological properties. Therefore, determination of the amino acid sequence is one of the basic requisites for further progress in physicochemical and biological investigations of proteins. What means do we actually possess for making such a determination? At present the selective chemical or enzymatic degradation of the protein molecule into polypeptide fragments and their separation presents no basic difficulties. However, determination of the amino acid sequence in these polypeptide fragments rapidly and with unequivocal results is not always an easy task, particularly in view of the fact that in the majority of cases one has to deal with microquantities of substance. All methods used for this purpose are based on the stepwise chemical or enzymatic cleavage of the amino acid residues, beginning with the Nor C-terminus. Usually thereby the positions of not more than 1—3 residues can reliably be established. The only exception is Edman's phenylthiohydantoin method which allows determination of 4—6 N-terminal amino acid residues. Possibly, using Edman's automatic "sequenator" the method will permit determination directly in the protein chain of much larger fragments1. However, the sequenator has not left the experimental stage. At the same time the problem of the primary structure elucidation of proteins is so important and the aims are so varied that it is necessary to have available several fundamentally differing methods, not only for the sake of mutual control, but also for the solution of the particular problems. For instance, the sequenator cannot be used to determine the primary structure