首页 期刊 论文 学科刊群 资讯 加盟 关于
EN
image
Article(id=1308842112565731561, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308841897058198067, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2025-1593, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1767110400000, receivedDateStr=2025-12-31, revisedDate=1779984000000, revisedDateStr=2026-05-29, acceptedDate=null, acceptedDateStr=null, onlineDate=1789981876745, onlineDateStr=2026-09-21, pubDate=1783785600000, pubDateStr=2026-07-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789981876745, onlineIssueDateStr=2026-09-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789981876745, creator=13701087609, updateTime=1789981876745, updator=13701087609, issue=Issue{id=1308841897058198067, tenantId=1146029695717560320, journalId=1189982191388893191, year='2026', volume='61', issue='7', pageStart='2377', pageEnd='2820', issueExtLink='null', onlineDate='null', pubDate='1783785600000', pubDateStr='2026-07-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1789981825365, creator='13701087609', updateTime=1789984154811, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1308851667525792021, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308841897058198067, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1308851667525792022, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308841897058198067, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=2654, endPage=2659, ext={EN=ArticleExt(id=1308842112767058154, articleId=1308842112565731561, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Development of a serotyping method for adeno-associated virus capsid proteins via SDS-PAGE in-gel digestion followed by LC-MS analysis, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

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.

, authors=Chun-le HAN, Jin ZHOU, Ke-xin WANG, Yi-dan CAO, Yao WANG, Yi-bing LIAN, Lin PANG*, Yong-zhao ZHAI*, Chun-ming RAO*, authorsList=Chun-le HAN, Jin ZHOU, Ke-xin WANG, Yi-dan CAO, Yao WANG, Yi-bing LIAN, Lin PANG, Yong-zhao ZHAI, Chun-ming RAO, authorCompany=null, correspAuthors=Lin PANG, Yong-zhao ZHAI, Chun-ming RAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2026 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1308842113530421489, articleId=1308842112565731561, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=腺相关病毒衣壳蛋白SDS-PAGE胶内酶解LC-MS血清型鉴定方法的建立, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

重组腺相关病毒(recombinant adeno-associated virus, rAAV) 作为基因治疗的核心载体, 其衣壳蛋白的正确鉴定是保证产品安全性、有效性与批次间一致性的关键。现有基于溶液内酶切的液质联用(liquid chromatograph mass spectrometer, LC-MS) 肽图分析方法, 虽能确认蛋白一级结构, 但因衣壳蛋白(capsid protein, VP) 1~3共享完全相同的C-末端序列(以AAV9为例, 共有序列超过400个氨基酸), 导致混合酶切后产生的共有肽段无法归属其蛋白来源, 尤其难以独立评估低丰度VP1和VP2的序列覆盖完整性及特异性翻译后修饰。针对这一固有局限, 本研究首次系统性地建立了基于“十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 分离-胶内酶解-LC-MS分析”的rAAV衣壳蛋白肽图鉴定方法。本方法的核心策略是通过电泳预先将VP1~3三种衣壳蛋白进行物理分离, 并对其各自条带进行独立的胶内胰蛋白酶/胰凝乳蛋白酶酶切, 从而实现对每种蛋白序列覆盖度的特异性评估。值得注意的是, 尽管VP1~3在衣壳中的天然化学计量比约为1∶1∶10 (即VP1和VP2分别仅占总蛋白约1/12), 且经胶内酶切多步回收后肽段量进一步损失, 本方法仍能对低丰度的VP1和VP2实现高达98%以上的综合序列覆盖率, 充分体现了其卓越的检测灵敏度与特异性。方法学验证表明, 本方法专属性强(空白基质零干扰)、重复性高(三次平行肽段总覆盖率≥ 98%, 关键肽段保留时间相对标准偏差RSD < 1%)、耐用性良好(关键参数变动下覆盖率保持稳定)。本方法有效解决了因VP1~3的C-末端序列相同而导致的溶液内酶切无法归属肽段的难题, 为rAAV产品的精确血清型鉴定、质量属性研究与质量控制提供了稳定、可靠且高分辨率的分析新工具。

, authors=韩春乐, 周金, 王可心, 曹译丹, 王瑶, 廉亦冰, 庞琳*, 翟永召*, 饶春明*, authorsList=韩春乐, 周金, 王可心, 曹译丹, 王瑶, 廉亦冰, 庞琳, 翟永召, 饶春明, authorCompany=null, correspAuthors=庞琳, 翟永召, 饶春明, authorNote=

#

共同第一作者.

作者贡献:

韩春乐负责实验方案设计、样品前处理、数据采集与分析及论文初稿撰写; 周金、廉亦冰负责数据审核; 王可心负责样品前处理及方法考察与优化; 曹译丹、王瑶负责质谱参数优化与数据分析; 庞琳、翟永召负责研究经费获取、技术、材料支持与论文修改; 饶春明负责项目指导、研究方案把关及论文定稿。

, correspAuthorsNote=
庞琳, E-mail:
翟永召, E-mail:
饶春明, Tel: 86-10-67869966, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2026, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=H2wO6d4FDg1iqp/d9ypgLg==, magXml=ezHfZ1Jv57fTYQKF9hnpMg==, pdfUrl=null, pdf=Yx7yQzi6DY7tu0XDZ3R30g==, pdfFileSize=966907, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=RUH6RVCS4j/eMXxoYNIs5A==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=cpcz+fo9bfjdNWPgs0mDSw==, mapNumber=null, fund=null)}, authors=[Author(id=1308842113840799990, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842113912103160, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842113840799990, language=EN, stringName=Chun-le HAN, firstName=Chun-le, middleName=null, lastName=HAN, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842115178782969, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842113840799990, language=CN, stringName=韩春乐, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842115254280443, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=1, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842115333972221, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115254280443, language=EN, stringName=Jin ZHOU, firstName=Jin, middleName=null, lastName=ZHOU, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842115401081086, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115254280443, language=CN, stringName=周金, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842115489161472, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=2, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842115573047554, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115489161472, language=EN, stringName=Ke-xin WANG, firstName=Ke-xin, middleName=null, lastName=WANG, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842115652739331, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115489161472, language=CN, stringName=王可心, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842115719848197, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=3, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842115795345671, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115719848197, language=EN, stringName=Yi-dan CAO, firstName=Yi-dan, middleName=null, lastName=CAO, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842115862454536, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115719848197, language=CN, stringName=曹译丹, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842115929563402, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=4, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842115996672268, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115929563402, language=EN, stringName=Yao WANG, firstName=Yao, middleName=null, lastName=WANG, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842116067975437, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115929563402, language=CN, stringName=王瑶, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842116135084303, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=5, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842116214776081, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116135084303, language=EN, stringName=Yi-bing LIAN, firstName=Yi-bing, middleName=null, lastName=LIAN, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842116281884946, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116135084303, language=CN, stringName=廉亦冰, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842116348993812, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=6, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=panglin@joinn-lab.com, emailSecond=null, emailThird=null, correspondingAuthor=1, authorType=1, ext={EN=AuthorExt(id=1308842116453851414, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116348993812, language=EN, stringName=Lin PANG, firstName=Lin, middleName=null, lastName=PANG, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842116537737495, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116348993812, language=CN, stringName=庞琳, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842116613234969, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=7, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=zhaiyongzhao@joinn-lab.com, emailSecond=null, emailThird=null, correspondingAuthor=1, authorType=1, ext={EN=AuthorExt(id=1308842116701315355, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116613234969, language=EN, stringName=Yong-zhao ZHAI, firstName=Yong-zhao, middleName=null, lastName=ZHAI, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842116776812828, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116613234969, language=CN, stringName=翟永召, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842116843921694, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=8, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=raochunming@joinn-lab.com, emailSecond=null, emailThird=null, correspondingAuthor=1, authorType=1, ext={EN=AuthorExt(id=1308842116944584992, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116843921694, language=EN, stringName=Chun-ming RAO, firstName=Chun-ming, middleName=null, lastName=RAO, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842117020082465, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116843921694, language=CN, stringName=饶春明, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])])], keywords=[Keyword(id=1308842117103968546, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, orderNo=1, keyword=adeno-associated virus), Keyword(id=1308842117162688803, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, orderNo=2, keyword=capsid protein), Keyword(id=1308842117221409060, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, orderNo=3, keyword=serotyping), Keyword(id=1308842117292712229, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, orderNo=4, keyword=in-gel digestion), Keyword(id=1308842117359821094, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, orderNo=5, keyword=LC-MS), Keyword(id=1308842117422735655, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, orderNo=1, keyword=腺相关病毒), Keyword(id=1308842117481455912, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, orderNo=2, keyword=衣壳蛋白), Keyword(id=1308842117544370473, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, orderNo=3, keyword=血清型鉴定), Keyword(id=1308842117619867946, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, orderNo=4, keyword=胶内酶解), Keyword(id=1308842117686976811, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, orderNo=5, keyword=LC-MS)], refs=[Reference(id=1308842118643278134, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=1, rfOrder=0, authorNames=null, journalName=null, refType=null, unstructuredReference=Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery [J]. Nat Rev Drug Discov, 2019, 18: 358-378., articleTitle=null, refAbstract=null), Reference(id=1308842118706192695, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=2, rfOrder=1, authorNames=null, journalName=null, refType=null, unstructuredReference=Kuzmin DA, Shutova MV, Johnston NR, et al. The clinical landscape for AAV gene therapies [J]. Nat Rev Drug Discov, 2021, 20: 173-174., articleTitle=null, refAbstract=null), Reference(id=1308842118769107256, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=3, rfOrder=2, authorNames=null, journalName=null, refType=null, unstructuredReference=Foust KD, Nurre E, Montgomery CL, et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes [J]. Nat Biotechnol, 2009, 27: 59-65., articleTitle=null, refAbstract=null), Reference(id=1308842118827827513, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=4, rfOrder=3, authorNames=null, journalName=null, refType=null, unstructuredReference=Büning H, Srivastava A. Capsid modifications for targeting and improving the efficacy of AAV vectors [J]. Mol Ther Methods Clin Dev, 2019, 12: 248-265., articleTitle=null, refAbstract=null), Reference(id=1308842118890742074, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=5, rfOrder=4, authorNames=null, journalName=null, refType=null, unstructuredReference=FDA. Human Gene Therapy for Rare Diseases - Guidance for Industry [S]. 2020. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/human-gene-therapy-rare-diseases., articleTitle=null, refAbstract=null), Reference(id=1308842118953656635, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=6, rfOrder=5, authorNames=null, journalName=null, refType=null, unstructuredReference=Vliet KV, Mohluddin Y, McClung S, et al. Adeno-associated virus capsid serotype identification: Analytical methods development and application [J]. J Virol Methods, 2009, 159: 167-177., articleTitle=null, refAbstract=null), Reference(id=1308842119016571196, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=7, rfOrder=6, authorNames=null, journalName=null, refType=null, unstructuredReference=Tseng YS, Vliet KV, Rao L, et al. Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies [J]. J Virol Methods, 2016, 236: 105-110., articleTitle=null, refAbstract=null), Reference(id=1308842119096262973, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=8, rfOrder=7, authorNames=null, journalName=null, refType=null, unstructuredReference=Jin X, Liu L, Nass S, et al. Direct liquid chromatography/mass spectrometry analysis for complete characterization of recombinant adeno-associated virus capsid proteins [J]. Hum Gene Ther Methods, 2017, 28: 255-267., articleTitle=null, refAbstract=null), Reference(id=1308842119150788926, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=9, rfOrder=8, authorNames=null, journalName=null, refType=null, unstructuredReference=Lam AK, Zhang J, Frabutt D, et al. Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS [J]. Mol Ther Methods Clin Dev, 2022, 27: 186-194., articleTitle=null, refAbstract=null), Reference(id=1308842119217897791, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=10, rfOrder=9, authorNames=null, journalName=null, refType=null, unstructuredReference=Han CL, Wang KX, Wang SR, et al. Gene therapy adeno-associated virus (AAV) vector capsid protein serotype identification [J]. Chin J Pharm Anal (药物分析杂志), 2025, 45: 543-553., articleTitle=null, refAbstract=null), Reference(id=1308842119285006656, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=11, rfOrder=10, authorNames=null, journalName=null, refType=null, unstructuredReference=Zhang X, Jin X, Liu L, et al. Optimized reversed-phase liquid chromatography/mass spectrometry methods for intact protein analysis and peptide mapping of adeno-associated virus proteins [J]. Hum Gene Ther, 2021, 32: 1501-1511., articleTitle=null, refAbstract=null), Reference(id=1308842119335338305, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=12, rfOrder=11, authorNames=null, journalName=null, refType=null, unstructuredReference=Becerra SP, Rose JA, Hardy M, et al. Direct mapping of adeno-associated virus capsid proteins B and C: A possible ACG initiation codon [J]. Proc Natl Acad Sci U S A, 1985, 82: 7919-7923., articleTitle=null, refAbstract=null), Reference(id=1308842119389864258, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=13, rfOrder=12, authorNames=null, journalName=null, refType=null, unstructuredReference=Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (中华人民共和国药典) [S]. 2025 ed. Vol Ⅲ. Beijing: China Medical Science Press, 2025: 775-781., articleTitle=null, refAbstract=null), Reference(id=1308842119444390211, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=14, rfOrder=13, authorNames=null, journalName=null, refType=null, unstructuredReference=Snijder J, van de Waterbeemd M, Damoc E, et al. Defining the stoichiometry and cargo load of viral and bacterial nanoparticles by Orbitrap mass spectrometry [J]. J Am Chem Soc, 2014, 136: 7295-7299., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])], figs=[ArticleFig(id=1308842117808611628, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, label=null, caption=null, figureFileSmall=9Yw4USd9rQAOjBN0KMQ0sA==, figureFileBig=RUH6RVCS4j/eMXxoYNIs5A==, tableContent=null), ArticleFig(id=1308842117863137581, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, label=Fig.1, caption=Workflow of the SDS-PAGE/in-gel digestion/LC-MS method for rAAV capsid protein analysis. AAV: Adeno-associated virus; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; VP: Capsid protein, figureFileSmall=9Yw4USd9rQAOjBN0KMQ0sA==, figureFileBig=RUH6RVCS4j/eMXxoYNIs5A==, tableContent=null), ArticleFig(id=1308842118030909742, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, label=null, caption=null, figureFileSmall=yGB8yvJY7WdU967tDraRXQ==, figureFileBig=YxZctuYESECy/TiqEAUR7Q==, tableContent=null), ArticleFig(id=1308842118106407215, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, label=Fig.2, caption=rAAV9 SDS-PAGE electrophoresis image, figureFileSmall=yGB8yvJY7WdU967tDraRXQ==, figureFileBig=YxZctuYESECy/TiqEAUR7Q==, tableContent=null), ArticleFig(id=1308842118186098992, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, label=null, caption=null, figureFileSmall=ADn5IrsyPhD8h+pqMcwDng==, figureFileBig=UA0IcMf3OrOXpQKrjDO1VA==, tableContent=null), ArticleFig(id=1308842118253207857, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, label=Fig.3, caption=The TIC chromatogram of a trypsin-digested VP3 by LC-MS. A: Blank; B: rAAV9; TIC: Total ion chromatogram, figureFileSmall=ADn5IrsyPhD8h+pqMcwDng==, figureFileBig=UA0IcMf3OrOXpQKrjDO1VA==, tableContent=null), ArticleFig(id=1308842118328705330, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Parameter investigatedVariation rangeSum of coverage/%
VP1VP2VP3
Digestion time (trypsin/chymotrypsin)Standard condition16 h / 3 h99.86100.100
Reduced time15 h / 2 h99.86100.100
Extended time17 h / 4 h99.86100.100
Formic acid concentration in mobile phaseStandard concentration0.1%99.86100.100
Low concentration0.08%99.46100.100
High concentration0.12%98.5299.50100
Column modelPremier BEH C18-99.86100.100
Premier CSH C18-99.6099.17100
), ArticleFig(id=1308842118425174323, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, label=Tab.1, caption=

Robustness verification: Total peptide coverage of capsid proteins under variations in key operational parameters

, figureFileSmall=null, figureFileBig=null, tableContent=
Parameter investigatedVariation rangeSum of coverage/%
VP1VP2VP3
Digestion time (trypsin/chymotrypsin)Standard condition16 h / 3 h99.86100.100
Reduced time15 h / 2 h99.86100.100
Extended time17 h / 4 h99.86100.100
Formic acid concentration in mobile phaseStandard concentration0.1%99.86100.100
Low concentration0.08%99.46100.100
High concentration0.12%98.5299.50100
Column modelPremier BEH C18-99.86100.100
Premier CSH C18-99.6099.17100
), ArticleFig(id=1308842118483894580, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Batch No.Capsid proteinSum of coverage/%
Batch 1VP198.65
VP298.51
VP3100.
Batch 2VP199.86
VP299.34
VP3100.
), ArticleFig(id=1308842118551003445, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, language=CN, label=Tab.2, caption=

Analysis results of two independent rAAV9 batches

, figureFileSmall=null, figureFileBig=null, tableContent=
Batch No.Capsid proteinSum of coverage/%
Batch 1VP198.65
VP298.51
VP3100.
Batch 2VP199.86
VP299.34
VP3100.
)], attaches=null, journal=Journal(id=1189982048455397383, delFlag=0, nameCn=药学学报, nameEn=Acta Pharmaceutica Sinica, nameHistory1=null, nameHistory2=null, issn=0513-4870, eissn=null, cn=11-2163/R, coden=null, periodic=0, language=CN, oaType=null, ccby=null, superviseOffice=null, ownerOffice=null, pubOffice=null, editorOffice=null, officeType=null, aims=null, clcCode=null, officeProv=null, officeCity=null, officeAddr=null, officeZip=null, officeEmail=null, officePhone=null, editDirector=null, officeDirector=null, officeDirectorPhone=null, officeStaffNum=null, officeEmpNum=null, coverPicUrl=BTxjudbJDVO4PqdBR6On6Q==, journalPrice=null, startedYear=null, abbrevIsoEn=null, journalRemark=null, publicationField=null, createdTime=1761643429151, updatedTime=1788948913902, createdBy=18614031015, updatedBy=13041195026, firstLetterCn=Y, firstLetterEn=Y, subjectCode=Medical and Pharmaceutical Sciences, subjectName=null, subjectCodeEn=Medical and Pharmaceutical Sciences, subjectNameEn=null, picCn=BTxjudbJDVO4PqdBR6On6Q==, picEn=c4l1ckL55nWbhl1KrFdWIA==, jcr=null, cjcr=null, exts=[JournalExt(id=1304509553505227679, language=CN, name=药学学报, nameHistory1=null, nameHistory2=null, managedBy=中国科学技术协会, sponsoredBy=中国药学会、中国医学科学院药物研究所, publishedBy=《药学学报》编辑委员会编辑出版, editorOffice=, officeProv=null, officeCity=null, officeAddr=, officeZip=, editDirector=, officeDirector=null, officePhone=null, coverPicUrl=null, journalRemark=, submitArticleUrl=null, websiteUrl=, createdTime=1788948914171, updatedTime=1788948914171, createdBy=13041195026, updatedBy=13041195026, submissionGuidelinesUrl=, submissionAuthorUrl=https://www.yxxb.com.cn/journalx_yxxb/authorLogOn.action, submissionEditorUrl=https://www.yxxb.com.cn/journalx_yxxb/editorLogOn.action, submissionReviewUrl=https://www.yxxb.com.cn/journalx_yxxb/expertLogOn.action, submissionCeEditorUrl=, submissionAeEditorUrl=, option={"copyright":""}), JournalExt(id=1304509553559753632, language=EN, name=Acta Pharmaceutica Sinica, nameHistory1=null, nameHistory2=null, managedBy=, sponsoredBy=, publishedBy=, editorOffice=, officeProv=null, officeCity=null, officeAddr=, officeZip=, editDirector=, officeDirector=null, officePhone=null, coverPicUrl=null, journalRemark=, submitArticleUrl=null, websiteUrl=, createdTime=1788948914184, updatedTime=1788948914184, createdBy=13041195026, updatedBy=13041195026, submissionGuidelinesUrl=, submissionAuthorUrl=https://www.yxxb.com.cn/journalx_yxxb/authorLogOn.action, submissionEditorUrl=https://www.yxxb.com.cn/journalx_yxxb/editorLogOn.action, submissionReviewUrl=https://www.yxxb.com.cn/journalx_yxxb/expertLogOn.action, submissionCeEditorUrl=, submissionAeEditorUrl=, option={"copyright":""})], databaseList=null, tenantJournalId=1189982191388893191, websiteList=[Website(id=1189982271588340489, webName=null, webTitle=null, webDomain=null, webCopyrigh=null, webIpcNo=null, seoTitle=null, seoKeywords=null, seoDescription=null, tenantJournalId=null, journalId=1189982191388893191, journalNameCn=null, journalNameEn=null, grayFlag=null, tenantId=1146029695717560320, platformId=null, journalGroupId=null, journalGroupNameCn=null, journalGroupNameEn=null, type=1, domain=https://castjournals.cast.org.cn/joweb/yxxb/CN, language=CN, createTime=1761643482348, createBy=18614031015, updateTime=1761643498101, updateBy=18614031015, name=药学学报-中文, tplId=1146099689490845704, title=药学学报, delFlag=0, indexPage=/home, props=[WebsiteProps(id=1189982873114448678, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=articleTextType, value=kx, createTime=1761643625763, updateTime=1761643625763, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873093477155, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=banner, value=null, createTime=1761643625758, updateTime=1761643625758, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873135420201, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=grayFlag, value=0, createTime=1761643625768, updateTime=1761643625768, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873085088546, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=logo, value=https://castjournals.cast.org.cn/joweb/yxxb/CN/file/pic?fileId=w+t2v8bJnX5lh3+hRRJcDA==, createTime=1761643625756, updateTime=1761643625756, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873152197419, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=minRunFlag, value=0, createTime=1761643625772, updateTime=1761643625772, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873110254373, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=picServerUrl, value=https://castjournals.cast.org.cn/joweb/yxxb/CN/file/pic, createTime=1761643625762, updateTime=1761643625762, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873143808810, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=silenceFlag, value=0, createTime=1761643625770, updateTime=1761643625770, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873101865764, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=staticResourcePath, value=https://castjournals.cast.org.cn/joweb/cast_kjdb_cn_619/, createTime=1761643625760, updateTime=1761643625760, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873122837287, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=themeColor, value=null, createTime=1761643625765, updateTime=1761643625765, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982873127031592, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271588340489, code=themeStyle, value=null, createTime=1761643625766, updateTime=1761643625766, creator=18614031015, updator=18614031015)]), Website(id=1189982271655449355, webName=null, webTitle=null, webDomain=null, webCopyrigh=null, webIpcNo=null, seoTitle=null, seoKeywords=null, seoDescription=null, tenantJournalId=null, journalId=1189982191388893191, journalNameCn=null, journalNameEn=null, grayFlag=null, tenantId=1146029695717560320, platformId=null, journalGroupId=null, journalGroupNameCn=null, journalGroupNameEn=null, type=1, domain=https://castjournals.cast.org.cn/joweb/yxxb/EN, language=EN, createTime=1761643482364, createBy=18614031015, updateTime=1761643514085, updateBy=18614031015, name=药学学报-英文, tplId=1146101810881728533, title=Acta Pharmaceutica Sinica, delFlag=0, indexPage=/home, props=[WebsiteProps(id=1189982903015633534, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=articleTextType, value=kx, createTime=1761643632892, updateTime=1761643632892, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982902990467707, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=banner, value=null, createTime=1761643632886, updateTime=1761643632886, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982903036605057, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=grayFlag, value=0, createTime=1761643632897, updateTime=1761643632897, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982902982079098, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=logo, value=https://castjournals.cast.org.cn/joweb/yxxb/EN/file/pic?fileId=w+t2v8bJnX5lh3+hRRJcDA==, createTime=1761643632884, updateTime=1761643632884, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982903053382275, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=minRunFlag, value=0, createTime=1761643632901, updateTime=1761643632901, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982903007244925, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=picServerUrl, value=https://castjournals.cast.org.cn/joweb/yxxb/EN/file/pic, createTime=1761643632890, updateTime=1761643632890, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982903044993666, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=silenceFlag, value=0, createTime=1761643632899, updateTime=1761643632899, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982902998856316, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=staticResourcePath, value=https://castjournals.cast.org.cn/joweb/cast_kjdb_en_623/, createTime=1761643632888, updateTime=1761643632888, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982903019827839, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=themeColor, value=null, createTime=1761643632893, updateTime=1761643632893, creator=18614031015, updator=18614031015), WebsiteProps(id=1189982903028216448, tenantId=1146029695717560320, journalId=null, journalGroupId=null, siteId=1189982271655449355, code=themeStyle, value=null, createTime=1761643632895, updateTime=1761643632895, creator=18614031015, updator=18614031015)])], journalTitle=药学学报, weixinUrl=null, journalUrl=https://www.yxxb.com.cn/aps, iacademicId=null, status=1, seqNo=null, journalTitleEn=Acta Pharmaceutica Sinica, journalPhotoCn=BTxjudbJDVO4PqdBR6On6Q==, journalPhotoEn=c4l1ckL55nWbhl1KrFdWIA==, journalFirstLetter=Y, journalRecommend=null, journalNew=null, journalCollection=null, jcrJf=null, cjcrJf=null, jcrJfStr=null, cjcrJfStr=null, submissionFirstDecision=null, sciSubjectClassification=null, casSubjectClassification=null, citeScore=null, totalCitationFrequency=null, icpCode=null, psCode=null, advertisingLicenseCode=null, copyrightInformation=null, country=null, option=, provinceCode=null, provinceName=null, collectFlag=false, interPubPlatform=, interPubPlatformUrl=null), detailUrlCn=https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2025-1593, detailUrlEn=https://castjournals.cast.org.cn/joweb/yxxb/EN/10.16438/j.0513-4870.2025-1593, pdfUrlCn=https://castjournals.cast.org.cn/joweb/yxxb/CN/PDF/10.16438/j.0513-4870.2025-1593, pdfUrlEn=https://castjournals.cast.org.cn/joweb/yxxb/EN/PDF/10.16438/j.0513-4870.2025-1593, aliStartDate=0, aliEndDate=0, collectionFlag=false, citedCount=null, citedUrl=null, previewStatus=0, delFlag=0, hasFullText=1, orderTime=1783785600000, fullTextJson=null, articleText=null, reference=null)
收藏切换
腺相关病毒衣壳蛋白SDS-PAGE胶内酶解LC-MS血清型鉴定方法的建立
收藏切换
PDF下载
韩春乐 , 周金 , 王可心 , 曹译丹 , 王瑶 , 廉亦冰 , 庞琳 * , 翟永召 * , 饶春明 *
药学学报 | 研究论文 2026,61(7): 2654-2659
收起
收藏切换
药学学报 |研究论文 2026 , 61 (7) : 2654 -2659
腺相关病毒衣壳蛋白SDS-PAGE胶内酶解LC-MS血清型鉴定方法的建立
全屏
[Author(id=1308842113840799990, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=0, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842113912103160, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842113840799990, language=EN, stringName=Chun-le HAN, firstName=Chun-le, middleName=null, lastName=HAN, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842115178782969, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842113840799990, language=CN, stringName=韩春乐, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842115254280443, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=1, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842115333972221, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115254280443, language=EN, stringName=Jin ZHOU, firstName=Jin, middleName=null, lastName=ZHOU, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842115401081086, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115254280443, language=CN, stringName=周金, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842115489161472, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=2, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842115573047554, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115489161472, language=EN, stringName=Ke-xin WANG, firstName=Ke-xin, middleName=null, lastName=WANG, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842115652739331, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115489161472, language=CN, stringName=王可心, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842115719848197, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=3, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842115795345671, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115719848197, language=EN, stringName=Yi-dan CAO, firstName=Yi-dan, middleName=null, lastName=CAO, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842115862454536, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115719848197, language=CN, stringName=曹译丹, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842115929563402, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=4, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842115996672268, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115929563402, language=EN, stringName=Yao WANG, firstName=Yao, middleName=null, lastName=WANG, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842116067975437, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842115929563402, language=CN, stringName=王瑶, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842116135084303, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=5, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=null, emailSecond=null, emailThird=null, correspondingAuthor=0, authorType=1, ext={EN=AuthorExt(id=1308842116214776081, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116135084303, language=EN, stringName=Yi-bing LIAN, firstName=Yi-bing, middleName=null, lastName=LIAN, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842116281884946, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116135084303, language=CN, stringName=廉亦冰, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=null, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842116348993812, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=6, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=panglin@joinn-lab.com, emailSecond=null, emailThird=null, correspondingAuthor=1, authorType=1, ext={EN=AuthorExt(id=1308842116453851414, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116348993812, language=EN, stringName=Lin PANG, firstName=Lin, middleName=null, lastName=PANG, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842116537737495, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116348993812, language=CN, stringName=庞琳, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842116613234969, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=7, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=zhaiyongzhao@joinn-lab.com, emailSecond=null, emailThird=null, correspondingAuthor=1, authorType=1, ext={EN=AuthorExt(id=1308842116701315355, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116613234969, language=EN, stringName=Yong-zhao ZHAI, firstName=Yong-zhao, middleName=null, lastName=ZHAI, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842116776812828, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116613234969, language=CN, stringName=翟永召, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])]), Author(id=1308842116843921694, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, orderNo=8, firstName=null, middleName=null, lastName=null, nameCn=null, orcid=null, stid=null, country=null, authorPic=null, dead=0, email=raochunming@joinn-lab.com, emailSecond=null, emailThird=null, correspondingAuthor=1, authorType=1, ext={EN=AuthorExt(id=1308842116944584992, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116843921694, language=EN, stringName=Chun-ming RAO, firstName=Chun-ming, middleName=null, lastName=RAO, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null), CN=AuthorExt(id=1308842117020082465, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, authorId=1308842116843921694, language=CN, stringName=饶春明, firstName=null, middleName=null, lastName=null, prefix=null, suffix=null, authorComment=null, nameInitials=null, affiliation=null, department=null, xref=*, address=北京昭衍药物检定研究有限公司, 北京 102605, bio=null, bioImg=null, bioContent=null, aboutCorrespAuthor=null)}, companyList=[AuthorCompany(id=1308842113756913906, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, xref=null, ext=[AuthorCompanyExt(id=1308842113765302515, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China), AuthorCompanyExt(id=1308842113769496820, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308842112565731561, companyId=1308842113756913906, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=北京昭衍药物检定研究有限公司, 北京 102605)])])]
韩春乐, 周金, 王可心, 曹译丹, 王瑶, 廉亦冰, 庞琳* , 翟永召* , 饶春明*
作者信息
  • 北京昭衍药物检定研究有限公司, 北京 102605
通讯作者:
庞琳, E-mail:
翟永召, E-mail:
饶春明, Tel: 86-10-67869966, E-mail:
作者简介:

#

共同第一作者.

作者贡献:

韩春乐负责实验方案设计、样品前处理、数据采集与分析及论文初稿撰写; 周金、廉亦冰负责数据审核; 王可心负责样品前处理及方法考察与优化; 曹译丹、王瑶负责质谱参数优化与数据分析; 庞琳、翟永召负责研究经费获取、技术、材料支持与论文修改; 饶春明负责项目指导、研究方案把关及论文定稿。

Development of a serotyping method for adeno-associated virus capsid proteins via SDS-PAGE in-gel digestion followed by LC-MS analysis
Chun-le HAN, Jin ZHOU, Ke-xin WANG, Yi-dan CAO, Yao WANG, Yi-bing LIAN, Lin PANG* , Yong-zhao ZHAI* , Chun-ming RAO*
Affiliations
  • JOINN Pharmaceutical Quality Research and Testing (Beijing) Co., Ltd., Beijing 102605, China
出版时间: 2026-07-12 doi: 10.16438/j.0513-4870.2025-1593
文章导航
收藏切换
摘要
收起

重组腺相关病毒(recombinant adeno-associated virus, rAAV) 作为基因治疗的核心载体, 其衣壳蛋白的正确鉴定是保证产品安全性、有效性与批次间一致性的关键。现有基于溶液内酶切的液质联用(liquid chromatograph mass spectrometer, LC-MS) 肽图分析方法, 虽能确认蛋白一级结构, 但因衣壳蛋白(capsid protein, VP) 1~3共享完全相同的C-末端序列(以AAV9为例, 共有序列超过400个氨基酸), 导致混合酶切后产生的共有肽段无法归属其蛋白来源, 尤其难以独立评估低丰度VP1和VP2的序列覆盖完整性及特异性翻译后修饰。针对这一固有局限, 本研究首次系统性地建立了基于“十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) 分离-胶内酶解-LC-MS分析”的rAAV衣壳蛋白肽图鉴定方法。本方法的核心策略是通过电泳预先将VP1~3三种衣壳蛋白进行物理分离, 并对其各自条带进行独立的胶内胰蛋白酶/胰凝乳蛋白酶酶切, 从而实现对每种蛋白序列覆盖度的特异性评估。值得注意的是, 尽管VP1~3在衣壳中的天然化学计量比约为1∶1∶10 (即VP1和VP2分别仅占总蛋白约1/12), 且经胶内酶切多步回收后肽段量进一步损失, 本方法仍能对低丰度的VP1和VP2实现高达98%以上的综合序列覆盖率, 充分体现了其卓越的检测灵敏度与特异性。方法学验证表明, 本方法专属性强(空白基质零干扰)、重复性高(三次平行肽段总覆盖率≥ 98%, 关键肽段保留时间相对标准偏差RSD < 1%)、耐用性良好(关键参数变动下覆盖率保持稳定)。本方法有效解决了因VP1~3的C-末端序列相同而导致的溶液内酶切无法归属肽段的难题, 为rAAV产品的精确血清型鉴定、质量属性研究与质量控制提供了稳定、可靠且高分辨率的分析新工具。

腺相关病毒  /  衣壳蛋白  /  血清型鉴定  /  胶内酶解  /  LC-MS

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.

adeno-associated virus  /  capsid protein  /  serotyping  /  in-gel digestion  /  LC-MS
韩春乐, 周金, 王可心, 曹译丹, 王瑶, 廉亦冰, 庞琳, 翟永召, 饶春明. 腺相关病毒衣壳蛋白SDS-PAGE胶内酶解LC-MS血清型鉴定方法的建立. 药学学报, 2026 , 61 (7) : 2654 -2659 . DOI: 10.16438/j.0513-4870.2025-1593
Chun-le HAN, Jin ZHOU, Ke-xin WANG, Yi-dan CAO, Yao WANG, Yi-bing LIAN, Lin PANG, Yong-zhao ZHAI, Chun-ming RAO. Development of a serotyping method for adeno-associated virus capsid proteins via SDS-PAGE in-gel digestion followed by LC-MS analysis[J]. Acta Pharmaceutica Sinica, 2026 , 61 (7) : 2654 -2659 . DOI: 10.16438/j.0513-4870.2025-1593
正文
收起
腺相关病毒(adeno-associated virus, AAV) 因其非致病性、低免疫原性及可感染非分裂细胞等特性, 已成为基因治疗领域最成功的递送载体之一[1]。截至目前, 已有多种基于AAV的基因治疗药物获批上市, 超过百项临床试验正在进行中[2]。不同血清型的AAV具有独特的组织趋向性, 例如, AAV9因其能够穿越血脑屏障的特性, 在神经系统疾病的基因治疗中展现出巨大潜力[3]。AAV的生物学特性与其衣壳蛋白(capsid protein, VP) 密切相关, 不同血清型间衣壳蛋白的氨基酸序列差异直接决定了病毒的组织趋向性、转导效率及免疫逃逸能力[4]。因此, 在生产过程中对rAAV衣壳蛋白进行准确的血清型鉴定和一级结构确证, 是确保产品身份、纯度、效价及批次一致性的基石, 也是监管机构的明确要求[5]。
传统的AAV血清型鉴定方法主要依赖于十二烷基硫酸钠–聚丙烯酰胺凝胶电泳(SDS-PAGE)、蛋白质印迹(Western blot) 或血清型特异性酶联免疫吸附分析(ELISA)[6]。然而, SDS-PAGE仅能提供表观分子量信息, 对序列高度同源(如AAV1与AAV6) 或分子量相近的不同血清型区分能力有限; Western blot及ELISA则严重依赖于高质量、高特异性抗体的可获得性, 且对于新出现的工程化改造衣壳往往缺乏对应的检测试剂[7]。随着质谱技术的发展, 液相色谱-质谱联用(liquid chromatograph mass spectrometer, LC-MS) 技术凭借其高分辨率、高灵敏度及提供直接质量信息的优势, 已成为生物大分子深度表征的金标准。已有研究利用LC-MS直接测定完整衣壳蛋白的分子量以区分不同血清型[8-10], 或通过溶液内酶切后进行肽图分析来确认蛋白序列[11]。然而, 常规的溶液内酶切策略存在一个固有局限: 由于VP1~3是通过可变剪接和不同起始密码子翻译而来, 它们共享一段很长的、完全相同的C-末端序列[12]。以AAV9为例, 其VP1、VP2与VP3的C-末端有超过400个氨基酸的序列是完全一致的。若对三者混合物直接进行胰蛋白酶酶切, 针对这段共有序列, 理论上会产生一系列完全相同的肽段。这些肽段在质谱检测中无法被区分其来源自VP1、VP2或VP3, 从而导致无法精确、独立地评估其中任何一种蛋白(尤其是比例较低的VP1和VP2) 在该区域的序列覆盖完整性, 也难以考察各蛋白在共有序列区域可能存在的特异性翻译后修饰。
为解决这一难题, 本研究创新性地提出并建立了“先分离, 后鉴定”的分析策略。本方法首先利用SDS-PAGE的高分辨率将VP1~3三者完全分离, 随后对切取的目标蛋白条带分别进行胶内酶解, 最后通过高分辨LC-MS对酶切肽段进行分析(图1)。这种策略从源头上实现了对3种衣壳蛋白的物理隔离与独立分析, 彻底避免了共有肽段的归属不确定性。本文系统阐述了本方法的建立过程, 并参照中国药典(2025年版三部通则9101“分析方法验证指导原则”)[13], 对其专属性、重复性及耐用性进行了全面、严格的方法学验证, 旨在为rAAV产品的研发与质控提供一种精确、稳定且具有普适性的衣壳蛋白鉴定新方法。
材料与方法
收起
供试品及试剂    供试品rAAV9 (蛋白浓度: 400.17 μg·mL-1) 为北京昭衍药物检定研究有限公司留存。胰蛋白酶(V5117)、胰凝乳蛋白酶(V1062) 购自美国Promega公司; 碳酸氢铵(NH4HCO3, 09830)、二硫苏糖醇(DTT, D0632)、碘乙酰胺(IAA, I1149) 购自美国Sigma公司; 乙腈(A998-4) 购自美国Fisher Chemical公司; 甲酸(FA, 50144) 购自DIKMA公司; 4%~20%预制蛋白胶(M00655) 购自GenScript公司。
仪器    Waters XEVO G3液相色谱-高分辨质谱联用仪(美国Waters公司); ChemiScope 6100凝胶成像系统(上海勤翔科学仪器有限公司)。C18色谱柱(Acquity Premier Peptide BEH和CSH, 130 Å, 150 mm × 2.1 mm, 1.7 µm) 购自美国Waters公司。
实验条件的优化
电泳  取rAAV9供试品80 μL, 加入5×蛋白上样缓冲液, 100 ℃变性5 min。使用预制胶进行SDS-PAGE电泳(200 V, 80~120 min)。电泳结束后, 考马斯亮蓝染色, 依据Marker指示切取VP1 (约82 kDa)、VP2 (约67 kDa)、VP3 (约60 kDa) 目标条带。
切胶  切取条带时应尽量靠近条带边缘, 减少多余空白胶的引入, 以降低后续脱色和萃取步骤的背景干扰, 使用洁净刀片, 避免条带间交叉污染。
脱色  胶块经脱色、脱水后, 进行还原(10 mmol·L-1, 56 ℃, 50 min) 与烷基化(55 mmol·L-1 IAA, 室温避光30 min), 脱色需充分至胶块完全透明, 残留的考马斯亮蓝染料会抑制胰蛋白酶活性并干扰质谱检测; 可多次更换脱色液(50%乙腈/50 mmol·L-1 NH4HCO3) 并震荡脱色, 直至胶块无色; 脱水不充分会导致后续酶液无法有效渗入胶内, 影响酶切效率, 使用100%乙腈脱水至胶块完全收缩变白, 并真空干燥或自然风干彻底去除乙腈。
酶切  分别加入胰蛋白酶或胰凝乳蛋白酶工作液(0.02 μg·μL-1), 加入胰蛋白酶的样品在37 ℃过夜酶切, 加入胰凝乳蛋白酶的样品于25 ℃温育3 h。酶液体积应足以完全覆盖胶块, 通常VP1和VP2因蛋白量少, 胶块体积小, 加入酶量为8~10 μL, VP3胶块加入酶量20~25 μL (根据胶块体积调整), 确保胶块充分吸胀; 酶切过程中需保持密闭, 防止蒸发。
萃取  酶切后肽段用萃取液(含5%甲酸, 67%乙腈) 提取, 提取肽段时可采用多次萃取(如2或3次) 并使用超声辅助(每次5~10 min), 以提高疏水性肽段的回收率, 氮吹浓缩至干。
色谱方法   色谱柱柱温: 40 ℃。流动相A: 水(含0.1%甲酸); 流动相B: 乙腈(含0.1%甲酸)。梯度洗脱: 0~2 min, 2% B; 2~5 min, 2%~5% B; 5~90 min, 5%~32% B; 90~95 min, 32%~60% B; 95~100 min, 60%~95% B; 100~105 min, 95% B; 105~106 min, 95%~2% B; 106~120 min, 2% B; 流速: 0.2 mL·min-1; 上样体积20 μL。
质谱条件   高分辨质谱采用ESI正离子模式, MSE采集, 扫描范围50~2 000 Da。毛细管电压+2.5 kV, 锥孔电压40 V, 低能碰撞能6 V, 高能碰撞能25~42 V, 离子源温度140 ℃, 脱溶剂气温度550 ℃, 脱溶剂气流速800 L·h-1, 锥孔气流速50 L·h-1。
数据分析   原始数据采用UNIFI软件(3.13.0.682) 处理。数据库为rAAV9衣壳蛋白理论序列。搜索参数: 前体离子质量容差10×10-6, 碎片离子容差20×10-6; 允许最多2个漏切位点; 至少匹配3个b或y离子。翻译后修饰包括: 天冬酰胺脱酰胺及甲硫氨酸氧化、VP1与VP3的N-端乙酰化。
方法学验证
专属性  取空白基质(不含rAAV9的相同缓冲液), 与供试品同步进行SDS-PAGE电泳, 在对应于VP1、VP2、VP3的分子量位置切取凝胶条带, 随后进行相同的胶内还原、烷基化、酶切(胰蛋白酶/胰凝乳蛋白酶)、肽段萃取及LC-MS分析。将空白样品的检测结果与rAAV9样品比较, 评估空白基质是否存在干扰性肽段信号。
重复性  取同一批次rAAV9样品, 由同一操作员按照完整流程独立制备并分析三份平行样品。每份样品均分别进行SDS-PAGE分离、切取VP1/VP2/VP3条带、独立胶内酶切(胰蛋白酶和胰凝乳蛋白酶分别处理)、LC-MS数据采集。计算每种衣壳蛋白的总覆盖率(两种酶切肽段合并后的序列覆盖率), 并统计所有关键肽段的液相色谱保留时间的相对标准偏差(RSD)。
耐用性  考察关键操作参数在合理范围内微小变动时对测定结果的影响。具体包括: 胰蛋白酶酶切时间(15、16、17 h) 及胰凝乳蛋白酶酶切时间(2、3、4 h); 流动相中甲酸浓度(0.08%、0.1%、0.12%); 色谱柱型号(Waters Premier BEH C18与Premier CSH C18)。其他条件保持不变, 分别计算每种衣壳蛋白的总覆盖率, 并与标准条件(胰蛋白酶16 h/胰凝乳蛋白酶3 h、0.1%甲酸、BEH C18柱) 下的结果进行比较。
结果
收起
1　衣壳蛋白的SDS-PAGE分离
SDS-PAGE电泳结果显示, rAAV9样品的3种主要衣壳蛋白得到了良好的分离(图2), 条带清晰, 分子量与预期相符, 为后续的胶内切取与分别酶切奠定了基础。
2　方法专属性验证
结果表明, 在所有空白样品的检测中, 均未发现任何可归属于目标rAAV9衣壳蛋白的特征肽段信号, 其序列覆盖率为0%。与此形成鲜明对比的是, 平行处理的rAAV9样品中, VP1、VP2、VP3的特征肽段均被清晰、明确地鉴定。以胰蛋白酶酶切VP3为例, 空白样品和rAAV9的液质肽图见图3。这一结果充分证明, 本方法所采用的样品前处理流程及LC-MS检测条件能够有效排除复杂基质可能带来的干扰, 具备优异的专属性, 可确保鉴定结果的准确无误。
3　方法重复性验证
本研究中, 单一酶切覆盖率是指分别使用胰蛋白酶或胰凝乳蛋白酶对某一衣壳蛋白条带进行胶内酶解后, 经LC-MS鉴定到的所有肽段所覆盖的氨基酸残基数占该蛋白总氨基酸残基数的百分比。总覆盖率(表1) 是指将同一种衣壳蛋白经两种酶分别酶切后鉴定到的所有肽段进行合并, 去除重复覆盖的氨基酸区域后, 计算覆盖的氨基酸残基总数占该蛋白总氨基酸残基数的百分比。
3份平行样品的测定结果显示, VP1的总覆盖率稳定在98.25%至98.92%之间, VP2和VP3的总覆盖率均达到100%, 全部满足预设的≥ 98%的严苛标准。这一数据表明, 本方法能够稳定、可靠地实现对衣壳蛋白近乎完全序列覆盖的鉴定。进一步, 对鉴定出的所有关键肽段(代表蛋白不同区域) 的液相色谱保留时间进行统计分析, 3次重复分析中共有53条关键肽段的保留时间RSD均小于1%, 其中43条肽段低于0.2%。这从另一个维度证明了本方法在整个分析流程, 包括色谱分离环节, 具有出色的精密度。
4　方法耐用性验证
结果显示(表1), 在酶切时间(胰蛋白酶15~17 h, 胰凝乳蛋白酶2~4 h)、流动相甲酸浓度(0.08%~0.12%) 及色谱柱型号(BEH C18与CSH C18) 等参数发生有意图的变化时, 所有测试条件下的衣壳蛋白肽段总覆盖率与标准条件相比, 差异均≤ 5%。这充分说明, 本方法对前处理及仪器分析过程中可能出现的微小波动不敏感, 具有良好的耐受性和可靠性, 具备成为标准质检方法的潜力。
5　样品测定
为评估本方法在实际样品分析中的效果, 采用已建立的胶内酶解LC-MS方法对两批独立的rAAV9供试品进行了衣壳蛋白血清型鉴定及序列覆盖率分析。结果表明, 本方法对不同批次的rAAV样品均能实现高置信度的序列覆盖, 具有良好应用价值, 可满足rAAV产品常规质控中衣壳蛋白鉴定的需求(表2)。
讨论
收起
AAV衣壳蛋白的精确表征对于基因治疗产品的质量把控至关重要。尽管基于LC-MS的完整蛋白分子量测定能够快速区分主要血清型[8, 14], 且溶液内酶切肽图分析已被用于确认蛋白一级结构[11], 但这些策略均无法实现对3种衣壳蛋白组分的独立、精细解析。本研究建立的方法可将经典的胶内酶解创造性地应用于解决rAAV衣壳蛋白特异性鉴定的实际难题。
本方法的根本优势源于其“物理分离为先”的设计理念。通过SDS-PAGE将VP1~3三者预先分离, 相当于在酶切前为每种蛋白建立了独立的“反应舱”。随后的还原、烷基化、酶切及肽段提取步骤均在各自胶块内独立完成。这种策略规避了溶液内酶切的最大痛点: 3种蛋白C-末端超过400个氨基酸序列完全相同, 混合酶切后产生的共有肽段(尤其是来源于高丰度VP3的肽段) 会严重干扰对低丰度VP1和VP2的鉴定, 导致其序列覆盖率评估不准或特有修饰无法被检出。而本研究通过对3种蛋白的独立处理, 确保了最终进入质谱分析的肽段混合物具有明确的蛋白来源归属, 从而能够分别计算出每种衣壳蛋白独立、真实的序列覆盖率。方法重复性验证结果显示, 即使对于相对比例较低的VP1和VP2, 本方法仍能实现高达98%以上的综合覆盖率, 这充分证明了该策略的有效性。
本方法的另一个突出优势是检测灵敏度高, 这得益于高分辨质谱仪的高效离子化与检测能力。需要强调的是, 整个分析流程始于SDS-PAGE, 而最终的质谱检测对象是来源于凝胶条带的酶切肽段。在电泳上样时, 总蛋白上样量约为20 μg, 但考虑到VP1~3在衣壳中的天然化学计量比约为1∶1∶10, 因此实际加载到凝胶上的VP1和VP2蛋白质量仅为约2 μg。更重要的是, 随后的胶内酶切流程, 包括脱色、脱水、酶解及肽段提取等步骤, 会不可避免地造成目标肽段的进一步损失, 最终进入LC-MS分析的肽段实际量远低于初始上样量, 但是本方法仍能对VP1和VP2实现高达98%以上的综合序列覆盖率。这强有力地证明了所采用的LC-MS具备极高的灵敏度和检测下限, 能够对极微量(纳摩尔甚至皮摩尔级别) 的酶切肽段进行有效鉴定。这一特性对于分析来源有限、珍贵的样品或需要高效利用物料的过程研究样品尤为重要。
从方法性能来看, 全面的验证结果证实了本方法的可靠性、精确性与稳定性。专属性实验确保了方法在复杂生物基质背景下的“纯净”检测能力。优异的重复性(高覆盖率与低保留时间偏差) 表明, 从电泳分离、胶内操作到LC-MS检测的整个多步骤流程均得到了良好控制, 具备成为常规分析方法的条件。出色的耐用性则赋予了该方法强大的适应能力, 使其能够承受不可避免的实验条件微小波动, 保证了在不同场景下结果的可比性与可信度。
与文献[8, 9, 11]报道的其他LC-MS策略相比, 本方法在鉴定深度和特异性上实现了显著提升。例如, Lam等[9]报道了快速的完整蛋白分析和高通量肽图分析, 但其肽图分析仍基于混合酶切, 未解决蛋白特异性归属问题。Jin等[8, 11]的工作虽详细研究了多种血清型完整蛋白质量及AAV2的肽图, 但其肽图覆盖的确认依赖于多种酶的溶液内酶切, 流程复杂且仍未实现3种蛋白的完全区分。
此外, 在鉴定衣壳蛋白的翻译后修饰方面, 本方法充分发挥了二级质谱(MS/MS) 解析的优势。当目标肽段发生如N-端乙酰化、甲硫氨酸氧化或天冬酰胺脱酰胺等修饰时, 其准确分子量会发生特定的质量偏移。通过获取这些肽段的MS/MS谱图, 分析其产生的b系列和y系列碎片离子, 不仅可以确认修饰的存在, 更能依据特征碎片离子的质量数, 将修饰类型精确归属到特定的氨基酸残基上。这种基于二级质谱碎片信息的解析, 提供了远高于一级质谱质量数匹配的确证能力, 确保了修饰鉴定结果的高置信度, 进一步体现了本方法在深度表征方面的技术优势。
需要说明的是, 由于涉及具体产品的知识产权与序列保密要求, 本文在结果与讨论中未列出具体的衣壳蛋白氨基酸序列或特征肽段序列, 但这并不影响对本方法有效性及性能的评估。在应用本方法时, 用户可通过将实验获得的肽段质量数与理论序列数据库进行比对, 从而实现准确的血清型鉴定与序列覆盖度计算, 这一过程完全不依赖于公开序列信息, 非常适用于需要保密的在研产品或工程化衣壳的分析。
当然, 本方法也存在一定的局限性。胶内酶切操作步骤相对繁琐, 对操作人员的技能要求较高, 主要体现在切胶精度、脱色终点判断、胶块脱水程度及肽段提取效率等关键环节, 任何细微的操作偏差都可能影响肽段回收率和鉴定结果的稳定性。在实际产业化应用中, 这种依赖人工操作的多步骤流程可能成为通量提升和标准化检测的瓶颈。然而, 本研究通过标准化的操作流程和严格的方法学验证(专属性、重复性、耐用性), 已将操作者间差异控制在可接受范围内。未来可探索与自动化切胶、酶解工作站联用, 以减少人为误差, 提升方法的通量和操作一致性, 从而更好地适应产业化的高通量质控需求。

文献
收起
参考文献 引证文献
排序方式：
1
Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery [J]. Nat Rev Drug Discov, 2019, 18: 358-378.
2
Kuzmin DA, Shutova MV, Johnston NR, et al. The clinical landscape for AAV gene therapies [J]. Nat Rev Drug Discov, 2021, 20: 173-174.
3
Foust KD, Nurre E, Montgomery CL, et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes [J]. Nat Biotechnol, 2009, 27: 59-65.
4
Büning H, Srivastava A. Capsid modifications for targeting and improving the efficacy of AAV vectors [J]. Mol Ther Methods Clin Dev, 2019, 12: 248-265.
5
FDA. Human Gene Therapy for Rare Diseases - Guidance for Industry [S]. 2020. https://www.fda.gov/regulatory-information/search-fda-guidance-documents/human-gene-therapy-rare-diseases.
6
Vliet KV, Mohluddin Y, McClung S, et al. Adeno-associated virus capsid serotype identification: Analytical methods development and application [J]. J Virol Methods, 2009, 159: 167-177.
7
Tseng YS, Vliet KV, Rao L, et al. Generation and characterization of anti-Adeno-associated virus serotype 8 (AAV8) and anti-AAV9 monoclonal antibodies [J]. J Virol Methods, 2016, 236: 105-110.
8
Jin X, Liu L, Nass S, et al. Direct liquid chromatography/mass spectrometry analysis for complete characterization of recombinant adeno-associated virus capsid proteins [J]. Hum Gene Ther Methods, 2017, 28: 255-267.
9
Lam AK, Zhang J, Frabutt D, et al. Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS [J]. Mol Ther Methods Clin Dev, 2022, 27: 186-194.
10
Han CL, Wang KX, Wang SR, et al. Gene therapy adeno-associated virus (AAV) vector capsid protein serotype identification [J]. Chin J Pharm Anal (药物分析杂志), 2025, 45: 543-553.
11
Zhang X, Jin X, Liu L, et al. Optimized reversed-phase liquid chromatography/mass spectrometry methods for intact protein analysis and peptide mapping of adeno-associated virus proteins [J]. Hum Gene Ther, 2021, 32: 1501-1511.
12
Becerra SP, Rose JA, Hardy M, et al. Direct mapping of adeno-associated virus capsid proteins B and C: A possible ACG initiation codon [J]. Proc Natl Acad Sci U S A, 1985, 82: 7919-7923.
13
Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China (中华人民共和国药典) [S]. 2025 ed. Vol Ⅲ. Beijing: China Medical Science Press, 2025: 775-781.
14
Snijder J, van de Waterbeemd M, Damoc E, et al. Defining the stoichiometry and cargo load of viral and bacterial nanoparticles by Orbitrap mass spectrometry [J]. J Am Chem Soc, 2014, 136: 7295-7299.
2026年第61卷第7期
PDF下载
199
95
引用本文
BibTeX
文章信息
doi: 10.16438/j.0513-4870.2025-1593
  • 接收时间：2025-12-31
  • 首发时间：2026-09-21
  • 出版时间：2026-07-12
补充材料
相关文章
文章信息
作者
出版历史
  • 收稿日期：2025-12-31
  • 修回日期：2026-05-29
基金
作者信息
    北京昭衍药物检定研究有限公司, 北京 102605

通讯作者:

庞琳, E-mail:
翟永召, E-mail:
饶春明, Tel: 86-10-67869966, E-mail:
参考文献
分享链接
https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2025-1593
分享至
全文二维码

扫描看全文

引用本文
BibTeX
本文的引用情况
2种不同金属材料的力学参数
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
关闭全屏
相关链接
论文
最新文章
热读文章
热点专题
内参
蓝皮书
产业发展报告
传播力报告
期刊分级目录
资讯
学术新闻
行业新闻
学术会议
行业会议
关于
关于我们
联系我们
北京市海淀区学院南路86号
100081
010-62662699（运营及技术支持）
qkjq@cast.org.cn

中国科学技术协会版权所有 京ICP 备10216604号-4 海淀分局备案 1101084647
科技导报社主办 中国科协信息中心技术支持