A-C-B human proinsulin: A novel insulin agonist and intermediate in the synthesis of human insulin

A Novel Protein Cross-Linking Reaction in Stressed Neutral Protamine Hagedorn Formulations of Insulin

“…The results of endo-Glu-C digestion show that the protamine is linked to a residue somewhere within the first 13 amino acid residues of the insulin B chain. Tryptic digestion shows that the linkage can be made between B [1][2][3][4][5][6][7][8][9][10][11][12][13] and any arginine residue in protamine.…”

“…Inspection of the amino acid residues in the insulin B chain peptide B [1][2][3][4][5][6][7][8][9][10][11][12][13] can rule out a number of the residues for reaction with protamine. The sequence 16 is as follows:…”

“…The most likely cross-linking reaction involves the imidazole group of histidine and the guanidino group of arginine, based on the combination of the known geometric association of insulin histidine and protamine arginine residues and the process of eliminating all of the other residues in the B [1][2][3][4][5][6][7][8][9][10][11][12][13] peptide. The mass lost during the reaction strongly suggests that two bonds are formed: a loss of four hydrogens leads to ∆ ) -4, which is very close to the measured experimental value.…”

“…The peaks in the mass range 4000-4400 Da correspond to unmodified protamine. The peaks in the mass range 5500-5900 Da correspond to protamine attached to the proteolytic fragment of insulin B chain, B [1][2][3][4][5][6][7][8][9][10][11][12][13]. These peaks also have ∆ ≈ -5 Da.…”

“…Using the hypothetical reaction deduced in the section below, an MS/MS experiment was performed on CIPP material that had been subjected to a trypsin digestion, followed by a disulfide bond reduction and an endo-Glu-C digestion. The quadruply charged ion that would correspond to the B [1][2][3][4][5][6][7][8][9][10][11][12][13] cross-linked to the dipeptide RR less 5 Da (m/z ) 453.1) was selected and MS/MS sequencing performed on that ion. The MS/MS data showed that the RR peptide was attached in the interval B [4][5]: the ion signals excluded all but these two residues as possible attachment sites.…”