A comprehensive LC–MS based study of factors influencing biotinylation of critical reagents

<b>Aim:</b> Critical reagents (CR) are applied in ligand binding assays (LBA) and biotinylation is a widely conjugation method used for critical reagents. However, insufficient characterization and inconsistent biotinylation can lead to LBA failures and necessitate extensive troubleshooting. This publication developed the detection of biotinylated CR and evaluates efficiency of biotinylation conditions to ensure the reliability of reagents and accuracy when implemented in LBA. <b>Materials &amp; methods:</b> Intact mass analysis was applied to characterize a CR with complex glycosylation and biotinylation patterns. Peptide mapping was developed to identify the biotinylation sites. <b>Results</b>: Biotinylation degrees and sites were clearly illustrated. <b>Conclusion:</b> A CR and its biotinylation were successfully characterized. The relationship between biotinylation efficiency and labeling conditions was clearly illustrated. A critical reagent (NVS-protein) and the corresponding biotinylated products were characterized by intact mass spectrometry (MS) analysis with the use of an optimized deglycosylation and desialidation process. Biotin degrees of labeling on the critical reagent were successfully estimated by intact MS. The specific biotinylation sites were accurately identified by peptide mapping analysis. The factors that influence biotinylation of critical reagents were successfully evaluated with the combination of intact MS and peptide mapping analysis, such as challenge ratio, incubation time and light conditions.