Differentiation of Norvaline and Valine in Peptides by Hot Electron Capture Dissociation

During the production of recombinant proteins, misincorporation of Nva (norvaline) is common and causes heterogeneity or even toxicity. To characterize Nva and differentiate it from Val (Valine), a systematic study was conducted using hot electron capture dissociation (HECD) and Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. The thorough investigation of the fragmentation behaviors of a set of model peptides led us to reveal the characteristic/diagnostic fragment ions, w ions, which can be used to differentiate Val and Nva. However, when both Nva and Val were present in one peptide, the observation of interfering ions may mislead the interpretation. Interestingly, HECD also produced v ions, which have the same nominal mass as the M+1 isotope of the w ion and can only be determined by MS with ultrahigh mass resolution and high mass accuracy. The energy-dependent study of the v ion provided an unambiguous identification of Nva and Val since the v ion was generated only when Val was present, not Nva within the electron energy range we studied. In addition, an electron energy-dependent curve provided an overall picture on how w ions and v ions, as well as interfering ions, behaved as the electron energy increased from 1.5 to 14 eV. The results suggest that careful selection of electron energy during a HECD experiment is crucial for the unambiguous differentiation of Val and Nva.