Full
Outline
Recombinant adeno-associated virus (rAAV) is a pivotal vector for gene therapy, and accurate identification of its capsid proteins is crucial for ensuring product safety, efficacy, and batch-to-batch consistency. Although existing LC-MS peptide mapping methods based on in-solution digestion can confirm the primary structure of proteins, they suffer from an inherent limitation: VP1, VP2, and VP3 share an identical C-terminal sequence (over 400 amino acids in the case of AAV9). Consequently, common peptides generated from mixed digestion cannot be assigned to their parental protein origin, making it particularly difficult to independently assess the sequence coverage integrity and specific post-translational modifications of low-abundance VP1 and VP2. To address this inherent limitation, this study establishes, for the first time, a systematic peptide mapping method for rAAV capsid proteins based on "SDS-PAGE separation, in-gel digestion, and LC-MS analysis". The core strategy of this method involves the initial physical separation of the three capsid proteins (VP1, VP2, and VP3) by electrophoresis, followed by independent in-gel trypsin/chymotrypsin digestion of each individual protein band, thereby enabling specific evaluation of the sequence coverage for each protein. It is noteworthy that despite the natural stoichiometry of VP1, VP2, and VP3 in the capsid being approximately 1:1:10 (i.e., VP1 and VP2 each constitute only about 1/12 of the total protein), and despite further loss of peptide quantities during the multi-step recovery process of in-gel digestion, this method achieves a combined sequence coverage of over 98% for the low-abundance VP1 and VP2, fully demonstrating its exceptional detection sensitivity and specificity. Method validation demonstrated that the approach is highly specific (no interference from blank matrix), reproducible (total peptide coverage ≥ 98% in triplicate runs, retention time RSD of key peptides < 1%), and robust (stable coverage under variations of key parameters). This method effectively resolves the challenge of ambiguous peptide assignment caused by identical C-terminal sequences of VP1/VP2/VP3 in solution-based digestion, providing a reliable, stable, and high-resolution analytical tool for accurate serotyping, quality attribute investigation, and quality control of rAAV products.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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