Study of peptides containing modified lysine residues by tandem mass spectrometry: precursor ion scanning of hexanal‐modified peptides

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Abstract
Upon hexanal‐modification in the presence of NaCNBH3, the oxidized B chain of insulin becomes mono‐ and further dialkylated on both the N‐terminal and Lys29 residues. A pseudo‐MS3 study was performed with a triple‐quadrupole mass spectrometer on the different modified lysine‐containing species to gain further insights into the characteristic fragmentation pattern. These fragmentations, in good agreement with true MS3 measurements obtained using an ion trap mass spectrometer, highlighted characteristic monoalkylated lysine (immonium—NH3) and protonated modified caprolactam ions at m/z 168 and 213, respectively. In contrast, no fragment ion derived from a modified lysine residue (immonium or caprolactam) was observed when dialkylation occurs on Lys29. However, a fragment ion corresponding to a protonated dihexylamine was observed at m/z 186. This loss, characteristic of dialkylated lysine fragmentation, was also observed upon dialkylation of Nα‐acetyllysine with either hexanal or pentanal. On the other hand, acetylation and malondialdehyde‐modification of the Nα‐acetyllysine side chain led mainly to the corresponding modified (immonium—NH3) fragment ions at m/z 126 and 138, respectively. Finally, it was demonstrated that precursor ion scanning for both m/z 168 and 213 ions led to specific and sensitive identification of peptides containing hexanal‐modified lysine residues within an unfractionated tryptic digest of hexanal‐modified apomyoglobin. Thus, Lys42, Lys45, Lys62, Lys63, Lys77, Lys87, Lys96, Lys98, Lys145 and Lys147 were found to be modified upon reaction with hexanal. Copyright © 2003 John Wiley & Sons, Ltd.