This study aimed to investigate the cause of an unidentified peak that appeared ahead of the VP3 subunit peak during the purity analysis of adeno-associated virus(AAV)capsid subunits by capillary electrophoresis sodium dodecyl sulfate(CE-SDS)technique.Initially,a point mutation was introduced to the second start codon within the base sequence of the recombinant adeno-associated virus(rAAV)capsid protein's VP3 subunit via PCR to generate a mutant rAAV.Subsequently,the relationship between this mutation and the unidentified signal peak was elucidated using CE-SDS technique.Additionally,the relative molecular weight and peptide analysis of the unknown peak was detected by LC-MS.The results indicated that if the VP3 subunit's base sequence contain two initiation codons(ATG),the translation might start from the second initiation codon.And the expression prod-uct was a variant of the VP3 subunit(VP3'),which aligned with the unidentified signal peak observed in CE-SDS analysis.Consequently,this unidentified peak should be regarded as a AAV capsid subunit peak during the purity assessment of AAV capsid proteins.
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