Application of stable isotope dimethyl labeling for MRM based absolute antigen quantification of influenza vaccine

Determining the precursor/product ion pair and optimal collision energy are the critical steps for developing a multiple reaction monitoring (MRM) assay using triple quadruple mass spectrometer for protein quantitation. In this study, a platform consisting of stable isotope dimethyl labeling coupled with triple-quadruple mass spectrometer was used to quantify the protein components of the influenza vaccines. Dimethyl labeling of both the peptide N-termini and the ϵ-amino group of lysine residues was achieved by reductive amination using formaldehyde and sodium cyanoborohydrate. Dimethylated peptides are known to exhibit dominant a1 ions under gas phase fragmentation in a mass spectrometer. These a1 ions can be predicted from the peptide N-terminal amino acids, and their signals do not vary significantly across a wide range of collision energies, which facilitates the determination of MRM transition settings for multiple protein targets. The intrinsic a1 ions provide sensitivity for acquiring MRM peaks that is superior to that of the typical b/y ions used for native peptides, and they also provided good linearity (R² ≥ 0.99) at the detected concentration range for each peptide. These features allow for the simultaneous quantification of hemagglutinin and neuraminidase in vaccines derived from either embryo eggs or cell cultivation. Moreover, the low abundant ovalbumin residue originated from the manufacturing process can also be determined. The results demonstrate that the stable isotope dimethyl labeling coupled with MRM Mass spectrometry screening of a1 ions (i.e., SIDa-MS) can be used as a high-throughput platform for multiple protein quantification of vaccine products.