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Article(id=1199703586465743259, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1199703581889753882, articleNumber=1001-2494(2025)01-0086-08, orderNo=null, doi=10.11669/cpj.2025.01.011, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1722268800000, receivedDateStr=2024-07-30, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1763961224428, onlineDateStr=2025-11-24, pubDate=1736265600000, pubDateStr=2025-01-08, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763961224428, onlineIssueDateStr=2025-11-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763961224428, creator=13701087609, updateTime=1763961224428, updator=13701087609, issue=Issue{id=1199703581889753882, tenantId=1146029695717560320, journalId=1190317699101192196, year='2025', volume='60', issue='1', pageStart='1', pageEnd='104', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=0, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763961223337, creator=13701087609, updateTime=1763967062652, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1199728073798157161, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1199703581889753882, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1199728073798157162, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1199703581889753882, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=86, endPage=93, ext={EN=ArticleExt(id=1199703587866640813, articleId=1199703586465743259, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Characterization of Recombinant Human Follicle-Stimulating Hormone Disulfide Bonds by High Resolution Mass Spectrometry, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To establish a high resolution mass spectrometry method for characterization of recombinant human follicle-stimulating hormone disulfide bonds. METHODS For pre-treatment, follicle-stimulating hormone(FSH) was diluted to 0.5 mg·mL-1 with diluent, subtilin, trypsin, and PNGase F were added into the stock solution respectively, and 1 μL 10% formic acid was finally added to stop the reaction. For LC-MS analysis, ACQUITY UPLC Peptide BEH C18(2.1 mm×100 mm, 1.7 μm) column was used, the mobile phase was 0.1% formic acid/aqueous solution(A)-0.1% formic acid/acetonitrile solution(B), and gradient elution was performed. The mass spectrum acquisition mode was ddMS2, the ion source was ESI+, and the scanning range was m/z 250-2 000. RESULTS The 6-p-disulfide bond of β-subunit was successfully identified, with the values β as followes: C3=β:C51. β:C17=β:C66; β:C20=β:C104; β:C28=β:C82; β:C32=β:C84; β:C87=β:C94. The 5-p-disulfide bond of α-subunit was successfully identified, α:C7=α:C31; α:C10=α:C60; α:C28=α:C82; α:C32=α:C84; α:C59=α:C87. CONCLUSION The localization method of disulfide bond of recombinant human follicle stimulating hormone(hFSH) is established, which provides a new idea for the quality control of disulfide bond connection.

, correspAuthors=Chenggang LIANG, Jing LI, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, 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, authorCompany=null, fund=null, authors=null, authorsList=Xinyue HU, Yuxing XIANG, Yue SUN, Lüyin WANG, Yi LI, Xiaoming ZHANG, Ping LÜ, Chenggang LIANG, Jing LI), CN=ArticleExt(id=1199703589959598629, articleId=1199703586465743259, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=利用高分辨质谱法表征重组人促卵泡激素二硫键的方法研究, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 建立高分辨质谱法表征重组人促卵泡激素二硫键的方法。方法 前处理方法是将卵泡刺激素(follicle-stimulating hormone,FSH)原液用稀释液稀释至0.5 mg·mL-1,分别加入枯草杆菌蛋白酶、胰蛋白酶、PNGase F酶解,最后加入1 μL 10%的甲酸,停止反应。液质方法:采用ACQUITY UPLC Peptide BEH C18(2.1 mm×100 mm,1.7 μm),以0.1%甲酸/水溶液(A)-0.1%甲酸/乙腈溶液(B)为流动相,梯度洗脱;质谱采集模式为ddMS2,离子源为ESI+,扫描范m/z 250~2 000。结果 成功鉴定出β亚基6对二硫键,分别为β:C3=β:C51; β:C17=β:C66; β:C20=β:C104; β:C28=β:C82; β:C32=β:C84; β:C87=β:C94;成功鉴定出α亚基5对二硫键,分别为α:C7=α:C31; α:C10=α:C60; α:C28=α:C82; α:C32=α:C84; α:C59=α:C87。结论 建立了重组人促卵泡激素二硫键的定位方法,为该类产品二硫键连接方式的质量控制提供了新思路。

, correspAuthors=梁成罡, 李晶, authorNote=null, correspAuthorsNote=
*梁成罡,男,博士,研究员 研究方向:激素类药物质量控制及标准研究 Tel:(010) 53851638;
李晶,女,博士,研究员 研究方向:激素类药物质量控制及标准研究 Tel:(010) 53851465
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胡馨月,女,博士,助理研究员 研究方向:激素类药物质量控制;

向禹兴,男,硕士研究生 研究方向:生物技术药物分析。胡馨月与向禹兴为共同第一作者

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胡馨月,女,博士,助理研究员 研究方向:激素类药物质量控制;

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向禹兴,男,硕士研究生 研究方向:生物技术药物分析。胡馨月与向禹兴为共同第一作者

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Chin J Pharm Anal(药物分析杂志), 2023, 43(1):12-19., articleTitle=Analysis and study on the primary structure of the lysozyme using UPLC-Q TOF MS, refAbstract=null), Reference(id=1199727466655875842, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, doi=null, pmid=null, pmcid=null, year=2023, volume=58, issue=1, pageStart=33, pageEnd=40, url=null, language=null, rfNumber=[7], rfOrder=6, authorNames=ZHAO X Y, LI M, WU G, journalName=Chin Pharm J(中国药学杂志), refType=null, unstructuredReference=ZHAO X Y, LI M, WU G, et al. Primary structure characterization of anti-CD79b antibody-vc-MMAE[J]. Chin Pharm J(中国药学杂志), 2023, 58(1):33-40., articleTitle=Primary structure characterization of anti-CD79b antibody-vc-MMAE, refAbstract=null), Reference(id=1199727466798482179, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, doi=null, pmid=null, pmcid=null, year=2024, volume=59, issue=1, pageStart=188, pageEnd=197, url=null, language=null, rfNumber=[8], rfOrder=7, authorNames=HU X Y, DINGX L, SUN Y, journalName=Acta Pharm Sin(药学学报), refType=null, unstructuredReference=HU X Y, DINGX L, SUN Y, et al. Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum[J]. Acta Pharm Sin(药学学报), 2024, 59(1):188-197., articleTitle=Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum, refAbstract=null), Reference(id=1199727466894951173, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, doi=null, pmid=null, pmcid=null, year=2001, volume=383, issue=6, pageStart=961, pageEnd=968, url=null, language=null, rfNumber=[9], rfOrder=8, authorNames=AMORESANO A, ORRÙ S, SICILIANO R A, journalName=Biol Chem, refType=null, unstructuredReference=AMORESANO A, ORRÙ S, SICILIANO R A, et al. Assignment of the complete disulphide bridge pattern in the human recombinant follitropin β-chain[J]. Biol Chem, 2001, 383(6):961-968., articleTitle=Assignment of the complete disulphide bridge pattern in the human recombinant follitropin β-chain, refAbstract=null)], funds=[Fund(id=1199727465494053604, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, awardId=2023SKLDRS0108, language=CN, fundingSource=药品监管科学全国重点实验室第一批课题项目资助(2023SKLDRS0108), fundOrder=null, country=null), Fund(id=1199727465586328294, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, awardId=2024SKLDRS0203, language=CN, fundingSource=药品监管科学全国重点实验室第二批课题项目资助(2024SKLDRS0203), fundOrder=null, country=null), Fund(id=1199727465674408681, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, awardId=2024HYZX23, language=CN, fundingSource=中国食品药品检定研究院化学药品检定所重点实验室项目资助(2024HYZX23), fundOrder=null, country=null)], companyList=[AuthorCompany(id=1199727456564380101, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, xref=1, ext=[AuthorCompanyExt(id=1199727456576963015, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, companyId=1199727456564380101, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 State Key Laboratory of Drug Regulatory Science, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs, National Institutes for Food and Drug Control, Beijing 102629, China), AuthorCompanyExt(id=1199727456589545927, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, companyId=1199727456564380101, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1 中国食品药品检定研究院, 药品监管科学全国重点实验室, 国家卫生健康委生物技术产品检定方法及其标准化重点实验室, 国家药品监督管理局生物制品质量研究与评价重点实验室, 国家药品监督管理局化学药品质量研究与评价重点实验室, 北京 102629)]), AuthorCompany(id=1199727456673432012, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, xref=2, ext=[AuthorCompanyExt(id=1199727456686014924, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, companyId=1199727456673432012, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 China Pharmaceutical University, Nanjing 210009, China), AuthorCompanyExt(id=1199727456698597838, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, companyId=1199727456673432012, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2 中国药科大学, 南京 210009)])], figs=[ArticleFig(id=1199727462243467933, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Fig.1, caption=The structure of human follicle-stimulating hormone(hFSH), figureFileSmall=2XJHu90JJs3on4c8PMdLuw==, figureFileBig=is/WuAh2kLJU5J4wxK9g0w==, tableContent=null), ArticleFig(id=1199727462360908451, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=图1, caption=重组人促卵泡激素(hFSH)结构图, figureFileSmall=2XJHu90JJs3on4c8PMdLuw==, figureFileBig=is/WuAh2kLJU5J4wxK9g0w==, tableContent=null), ArticleFig(id=1199727463552090794, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Fig.2, caption=Secondary mass spectra of β:C3=β:C51 and β:C17=β:C66 peptides in β-subunit of hFSH

A-β:C3=β:C51 NSCE terminal secondary mass spectrometry; B-β:C3=β:C51 KTCTF terminal secondary mass spectrometry; C-β:C17=β:C66 ECR terminal secondary mass spectrometry;D-β:C17=β:C66 PGCAH terminal secondary mass spectrometry.

, figureFileSmall=4nU0ialXetpbhd/AmHw2JA==, figureFileBig=0/Na8w7mFdvUUUr57nYxeg==, tableContent=null), ArticleFig(id=1199727463665337006, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=图2, caption=hFSH的β亚基β:C3=β:C51和β:C17=β:C66肽段二级质谱图

A-β:C3=β:C51中NSCE端二级质谱图;B-β:C3=β:C51中KTCTF端二级质谱图;C-β:C17=β:C66中ECR端二级质谱图;D-β:C17=β:C66中PGCAH端二级质谱图。

, figureFileSmall=4nU0ialXetpbhd/AmHw2JA==, figureFileBig=0/Na8w7mFdvUUUr57nYxeg==, tableContent=null), ArticleFig(id=1199727463786971828, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Fig.3, caption=Secondary mass spectra of β:C20=β:C104 and β:C28=β:C82 peptides in β-subunit of hFSH

A-β:C20=β:C104 FCIS terminal secondary mass spectrometry; B-β:C20=β:C104 YCSF terminal secondary mass spectrometry; C-β:C28=β:C82 WCA terminal secondary mass spectrometry; D-β:C28=β:C82 TQCH terminal secondary mass spectrometry.

, figureFileSmall=EAoA/udNvDsuPEQPj0Mo9w==, figureFileBig=cIgMN+EI43JbsR+wirdI3w==, tableContent=null), ArticleFig(id=1199727463908606647, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=图3, caption=hFSH的β亚基β:C20=β:C104和β:C28=β:C82肽段二级质谱图

A-β:C20=β:C104中FCIS端二级质谱图;B-β:C20=β:C104中YCSF端二级质谱图;C-β:C28=β:C82中WCA端二级质谱图;D-β:C28=β:C82中TQCH端二级质谱图。

, figureFileSmall=EAoA/udNvDsuPEQPj0Mo9w==, figureFileBig=cIgMN+EI43JbsR+wirdI3w==, tableContent=null), ArticleFig(id=1199727464047018679, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Fig.4, caption=Secondary mass spectra of β:C32=β:C84 peptide and β:C87=β:C94 peptide in β-subunit of hFSH

A-β:C32=β:C84 GYCY terminal secondary mass spectrometry; B-β:C32=β:C84 HCGK terminal secondary mass spectrum; C-β:C87=β:C94 GKCD terminal secondary mass spectrometry; D-β:C87=β:C94 DCTVR terminal secondary mass spectrometry.

, figureFileSmall=0RUtas2Ta2/xE5mXGmKv8A==, figureFileBig=21uV/6mqk/wNw2q2AyyM2A==, tableContent=null), ArticleFig(id=1199727464147681979, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=图4, caption=hFSH的β亚基β:C32=β:C84肽段和β:C87=β:C94肽段二级质谱图

A-β:C32=β:C84中GYCY端二级质谱图;B-β:C32=β:C84中HCGK端二级质谱图;C-β:C87=β:C94中GKCD端二级质谱图;D-β:C87=β:C94中DCTVR端二级质谱图。

, figureFileSmall=0RUtas2Ta2/xE5mXGmKv8A==, figureFileBig=21uV/6mqk/wNw2q2AyyM2A==, tableContent=null), ArticleFig(id=1199727464252539585, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Fig.5, caption=Secondary mass spectra of α:C7=α:C31 peptide and α:C10=α:C60 peptide in α-subunit of hFSH

A-α:C7=α:C31 CP terminal secondary mass spectrometry; B-α:C7=α:C31 GC terminal secondary mass spectrum; C-α:C10=α:C60 CTL terminal secondary mass spectrometry; D-α:C10=α:C60 CVAK terminal secondary mass spectrum.

, figureFileSmall=m6zdvRr1IgiQCGX/kjorwQ==, figureFileBig=+R/XUTHMccSmqHbi+Kjg2Q==, tableContent=null), ArticleFig(id=1199727464332231365, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=图5, caption=hFSH的α亚基α:C7=α:C31肽段和α:C10=α:C60肽段二级质谱图

A-α:C7=α:C31中CP 端二级质谱图; B-α:C7=α:C31中GC端二级质谱图;C-α:C10=α:C60中CTL端二级质谱图;D-α:C10=α:C60中CVAK端二级质谱图。

, figureFileSmall=m6zdvRr1IgiQCGX/kjorwQ==, figureFileBig=+R/XUTHMccSmqHbi+Kjg2Q==, tableContent=null), ArticleFig(id=1199727464458060486, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Fig.6, caption=Secondary mass spectra of α:C28=α:C82 peptide, α:C32=α:C84 peptide and α:C59=α:C87 peptide in α-subunit of hFSH

A-α:C28=α:C82 LQC terminal secondary mass spectrometry; B-α:C28=α:C82 AC terminal secondary mass spectrometry; C-α:C32=α:C84 CF terminal secondary mass spectrometry; D-α:C32=α:C84 HCS terminal secondary mass spectrometry; E-α:C59=α:C87 TC terminal secondary mass spectrometry; F-α:C59=α:C87 TCY terminal secondary mass spectrometry.

, figureFileSmall=Gr1FdF3MPQpBPDzIqacirA==, figureFileBig=sxuwgRd2MpeeE83IZKCspg==, tableContent=null), ArticleFig(id=1199727464588083915, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=图6, caption=hFSH的α亚基α:C28=α:C82肽段、α:C32=α:C84肽段和α:C59=α:C87肽段二级质谱图

A-α:C28=α:C82中LQC端二级质谱图; B-α:C28=α:C822中AC端二级质谱图; C-α:C32=α:C84中CF端二级质谱图; D-α:C32=α:C84中HCS端二级质谱图; E-α:C59=α:C87中TC端二级质谱图; F-α:C59=α:C87中TCY端二级质谱图。

, figureFileSmall=Gr1FdF3MPQpBPDzIqacirA==, figureFileBig=sxuwgRd2MpeeE83IZKCspg==, tableContent=null), ArticleFig(id=1199727464743273165, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Fig.7, caption=Extracted ion chromatogram of alpha subunit disulfide peptide

A-peptide CP=GC extraction ion flow diagram; B-peptide CTL=CVAK extraction ion flow diagram; C-peptide LQC=AC extraction ion flow diagram; D-peptide CF=HCS extraction ion flow diagram; E-peptide TC=TCY extraction ion flow diagram.

, figureFileSmall=NqZsJBiMBqowI/D90j7s3A==, figureFileBig=MMqfKUvuyHeS1k8SVqBrKg==, tableContent=null), ArticleFig(id=1199727464848130766, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=图7, caption=α亚基二硫键肽段提取离子流

A-肽段CP=GC提取离子流图;B-肽段CTL=CVAK提取离子流图;C-肽段LQC=AC提取离子流图;D-肽段CF=HCS提取离子流图;E-肽段TC=TCY提取离子流图。

, figureFileSmall=NqZsJBiMBqowI/D90j7s3A==, figureFileBig=MMqfKUvuyHeS1k8SVqBrKg==, tableContent=null), ArticleFig(id=1199727464948794066, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Tab.1, caption=

Information of β-subunit disulfide bond matching peptide of FSH

, figureFileSmall=null, figureFileBig=null, tableContent=
Connection type The peptide containing disulfide bonds MW(Theoretical) MW(Measured) Deviation/×10-6 ASR
β:C3=β:C51 NSCE=KTCTF 1 047.399 0 1 047.400 1 -1.0 1.0
NSCE=CTF 818.256 2 818.257 5 -1.6 1.0
NSCEL=KTCTF 1 160.480 2 1 160.484 2 -3.4 1.0
CE=CTF 617.181 8 617.182 5 -1.1 1.0
β:C17=β:C66 ECR=PGACH 887.334 9 887.337 8 -3.2 1.0
ECR=RVPGCA 1 005.445 9 1 005.448 4 -2.5 1.0
CRF=VPGCAH 1 004.430 5 1 004.432 0 -1.5 1.0
ECRF=PGCAH 1 034.403 4 1 034.406 2 -2.7 1.0
ECRF=CAH 880.330 0 880.332 0 -2.3 1.0
β:C20=β:C104 FCIS=YCSF 984.370 7 984.372 1 -1.4 1.0
FCI=GPSYCS 991.374 3 991.377 9 -3.6 1.0
FCI=SYCS 837.301 4 837.303 7 -2.7 1.0
β:C28=β:C82 WCA=TQCH 863.304 3 863.305 4 -1.3 1.0
WCA=CH 634.197 8 634.199 2 -2.2 1.0
β:C32=β:C84 GYCY=HCGK 945.344 9 945.347 3 -2.5 1.0
GYCYT=HCGK 1 046.393 1 1 046.395 0 -1.8 1.0
GYCY=CGK 808.286 4 808.288 4 -2.5 1.0
YCY=CG 623.170 7 623.172 0 -2.1 1.0
β:C87=β:C94 GKCD=DCTVR 1 011.407 8 1 011.411 4 -3.6 1.0
GKCD=STDCTVR 1 199.487 5 1 199.491 1 -3.0 1.0
GKCD=TDCTVR 1 112.456 1 1 112.459 0 -2.6 1.0
), ArticleFig(id=1199727465053651671, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=表1, caption=

hFSH的β亚基二硫键匹配肽段信息

, figureFileSmall=null, figureFileBig=null, tableContent=
Connection type The peptide containing disulfide bonds MW(Theoretical) MW(Measured) Deviation/×10-6 ASR
β:C3=β:C51 NSCE=KTCTF 1 047.399 0 1 047.400 1 -1.0 1.0
NSCE=CTF 818.256 2 818.257 5 -1.6 1.0
NSCEL=KTCTF 1 160.480 2 1 160.484 2 -3.4 1.0
CE=CTF 617.181 8 617.182 5 -1.1 1.0
β:C17=β:C66 ECR=PGACH 887.334 9 887.337 8 -3.2 1.0
ECR=RVPGCA 1 005.445 9 1 005.448 4 -2.5 1.0
CRF=VPGCAH 1 004.430 5 1 004.432 0 -1.5 1.0
ECRF=PGCAH 1 034.403 4 1 034.406 2 -2.7 1.0
ECRF=CAH 880.330 0 880.332 0 -2.3 1.0
β:C20=β:C104 FCIS=YCSF 984.370 7 984.372 1 -1.4 1.0
FCI=GPSYCS 991.374 3 991.377 9 -3.6 1.0
FCI=SYCS 837.301 4 837.303 7 -2.7 1.0
β:C28=β:C82 WCA=TQCH 863.304 3 863.305 4 -1.3 1.0
WCA=CH 634.197 8 634.199 2 -2.2 1.0
β:C32=β:C84 GYCY=HCGK 945.344 9 945.347 3 -2.5 1.0
GYCYT=HCGK 1 046.393 1 1 046.395 0 -1.8 1.0
GYCY=CGK 808.286 4 808.288 4 -2.5 1.0
YCY=CG 623.170 7 623.172 0 -2.1 1.0
β:C87=β:C94 GKCD=DCTVR 1 011.407 8 1 011.411 4 -3.6 1.0
GKCD=STDCTVR 1 199.487 5 1 199.491 1 -3.0 1.0
GKCD=TDCTVR 1 112.456 1 1 112.459 0 -2.6 1.0
), ArticleFig(id=1199727465154314970, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=EN, label=Tab.2, caption=

Information of α-subunit disulfide matching peptide of hFSH

, figureFileSmall=null, figureFileBig=null, tableContent=
Connection type The peptide containing disulfide bonds MW(Theoretical) MW(Measured) Deviation/×10-6 ASR
α:C7=α:C31 CP=GC 394.098 1 394.097 2 -2.2 1.0
DCP=C 452.102 8 452.103 6 -1.8 3.0
α:C10=α:C60 CTL=CVAK 752.353 9 752.356 1 -2.9 1.0
CTL=CV 553.222 7 553.224 0 -2.3 1.0
CTLQENP=CV 1 021.418 0 1 021.420 9 -2.8 1.0
α:C28=α:C82 LQC=AC 552.202 1 552.203 6 -2.7 1.0
ILQC=TAC 766.332 8 766.335 3 -3.3 1.3
α:C32=α:C84 CF=HCS 611.190 1 611.183 2 -1.1 1.5
CF=CST 575.169 2 575.172 0 -4.8 1.0
α:C59=α:C87 TC=TCY 605.178 6 605.182 5 -6.4 1.0
), ArticleFig(id=1199727465271755485, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1199703586465743259, language=CN, label=表2, caption=

hFSH 的α亚基二硫键匹配肽段信息

, figureFileSmall=null, figureFileBig=null, tableContent=
Connection type The peptide containing disulfide bonds MW(Theoretical) MW(Measured) Deviation/×10-6 ASR
α:C7=α:C31 CP=GC 394.098 1 394.097 2 -2.2 1.0
DCP=C 452.102 8 452.103 6 -1.8 3.0
α:C10=α:C60 CTL=CVAK 752.353 9 752.356 1 -2.9 1.0
CTL=CV 553.222 7 553.224 0 -2.3 1.0
CTLQENP=CV 1 021.418 0 1 021.420 9 -2.8 1.0
α:C28=α:C82 LQC=AC 552.202 1 552.203 6 -2.7 1.0
ILQC=TAC 766.332 8 766.335 3 -3.3 1.3
α:C32=α:C84 CF=HCS 611.190 1 611.183 2 -1.1 1.5
CF=CST 575.169 2 575.172 0 -4.8 1.0
α:C59=α:C87 TC=TCY 605.178 6 605.182 5 -6.4 1.0
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利用高分辨质谱法表征重组人促卵泡激素二硫键的方法研究
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胡馨月 1 , 向禹兴 2 , 孙悦 1 , 王绿音 1 , 李懿 1 , 张孝明 1 , 吕萍 1 , 梁成罡 1, * , 李晶 1, *
中国药学杂志 | 论著 2025,60(1): 86-93
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中国药学杂志 | 论著 2025, 60(1): 86-93
利用高分辨质谱法表征重组人促卵泡激素二硫键的方法研究
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胡馨月1, 向禹兴2, 孙悦1, 王绿音1, 李懿1, 张孝明1, 吕萍1, 梁成罡1, *, 李晶1, *
作者信息
  • 1 中国食品药品检定研究院, 药品监管科学全国重点实验室, 国家卫生健康委生物技术产品检定方法及其标准化重点实验室, 国家药品监督管理局生物制品质量研究与评价重点实验室, 国家药品监督管理局化学药品质量研究与评价重点实验室, 北京 102629
  • 2 中国药科大学, 南京 210009

    • 胡馨月,女,博士,助理研究员 研究方向:激素类药物质量控制;

    向禹兴,男,硕士研究生 研究方向:生物技术药物分析。胡馨月与向禹兴为共同第一作者

通讯作者:

*梁成罡,男,博士,研究员 研究方向:激素类药物质量控制及标准研究 Tel:(010) 53851638;
李晶,女,博士,研究员 研究方向:激素类药物质量控制及标准研究 Tel:(010) 53851465
Characterization of Recombinant Human Follicle-Stimulating Hormone Disulfide Bonds by High Resolution Mass Spectrometry
Xinyue HU1, Yuxing XIANG2, Yue SUN1, Lüyin WANG1, Yi LI1, Xiaoming ZHANG1, Ping LÜ1, Chenggang LIANG1, *, Jing LI1, *
Affiliations
  • 1 State Key Laboratory of Drug Regulatory Science, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Biological Products, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs, National Institutes for Food and Drug Control, Beijing 102629, China
  • 2 China Pharmaceutical University, Nanjing 210009, China
出版时间: 2025-01-08 doi: 10.11669/cpj.2025.01.011
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摘要
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目的 建立高分辨质谱法表征重组人促卵泡激素二硫键的方法。方法 前处理方法是将卵泡刺激素(follicle-stimulating hormone,FSH)原液用稀释液稀释至0.5 mg·mL-1,分别加入枯草杆菌蛋白酶、胰蛋白酶、PNGase F酶解,最后加入1 μL 10%的甲酸,停止反应。液质方法:采用ACQUITY UPLC Peptide BEH C18(2.1 mm×100 mm,1.7 μm),以0.1%甲酸/水溶液(A)-0.1%甲酸/乙腈溶液(B)为流动相,梯度洗脱;质谱采集模式为ddMS2,离子源为ESI+,扫描范m/z 250~2 000。结果 成功鉴定出β亚基6对二硫键,分别为β:C3=β:C51; β:C17=β:C66; β:C20=β:C104; β:C28=β:C82; β:C32=β:C84; β:C87=β:C94;成功鉴定出α亚基5对二硫键,分别为α:C7=α:C31; α:C10=α:C60; α:C28=α:C82; α:C32=α:C84; α:C59=α:C87。结论 建立了重组人促卵泡激素二硫键的定位方法,为该类产品二硫键连接方式的质量控制提供了新思路。

重组人促卵泡激素  /  二硫键定位  /  高分辨质谱  /  酶解法

OBJECTIVE To establish a high resolution mass spectrometry method for characterization of recombinant human follicle-stimulating hormone disulfide bonds. METHODS For pre-treatment, follicle-stimulating hormone(FSH) was diluted to 0.5 mg·mL-1 with diluent, subtilin, trypsin, and PNGase F were added into the stock solution respectively, and 1 μL 10% formic acid was finally added to stop the reaction. For LC-MS analysis, ACQUITY UPLC Peptide BEH C18(2.1 mm×100 mm, 1.7 μm) column was used, the mobile phase was 0.1% formic acid/aqueous solution(A)-0.1% formic acid/acetonitrile solution(B), and gradient elution was performed. The mass spectrum acquisition mode was ddMS2, the ion source was ESI+, and the scanning range was m/z 250-2 000. RESULTS The 6-p-disulfide bond of β-subunit was successfully identified, with the values β as followes: C3=β:C51. β:C17=β:C66; β:C20=β:C104; β:C28=β:C82; β:C32=β:C84; β:C87=β:C94. The 5-p-disulfide bond of α-subunit was successfully identified, α:C7=α:C31; α:C10=α:C60; α:C28=α:C82; α:C32=α:C84; α:C59=α:C87. CONCLUSION The localization method of disulfide bond of recombinant human follicle stimulating hormone(hFSH) is established, which provides a new idea for the quality control of disulfide bond connection.

recombinant human follicle-stimulating hormone  /  disulfide bond localization  /  high resolution mass spectrometry  /  enzymolysis
胡馨月, 向禹兴, 孙悦, 王绿音, 李懿, 张孝明, 吕萍, 梁成罡, 李晶. 利用高分辨质谱法表征重组人促卵泡激素二硫键的方法研究. 中国药学杂志, 2025 , 60 (1) : 86 -93 . DOI: 10.11669/cpj.2025.01.011
Xinyue HU, Yuxing XIANG, Yue SUN, Lüyin WANG, Yi LI, Xiaoming ZHANG, Ping LÜ, Chenggang LIANG, Jing LI. Characterization of Recombinant Human Follicle-Stimulating Hormone Disulfide Bonds by High Resolution Mass Spectrometry[J]. Chinese Pharmaceutical Journal, 2025 , 60 (1) : 86 -93 . DOI: 10.11669/cpj.2025.01.011
正文
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重组人促卵泡激素(human follicle-stimulating hormone, hFSH)是糖蛋白激素(Glycoprotein hormones,GPHs)家族的一员。GPHs还包括人促黄体生成素(human luteinizing hormone, hLH)、人促甲状腺激素(human thyroid-stimulating hormone, hTSH)和人绒毛膜促性腺激素(human chorionic gonadotropin, hCG)。GPHs家族都是由1个共同的α和1个激素特异性β亚基的非共价结合形成的异二聚体[1-2],两个亚基通过β半胱氨酸环相互作用连接。hFSH是相对分子质量为35.5×103的异源二聚体,α亚基由92个氨基酸组成,包含2个N糖基化位点(Asn-52和Asn-78),含有10个半胱氨酸残基,形成5对链内二硫桥;β亚基由111个氨基酸组成,分别包含2个N糖基化位点(Asn-7和Asn-24),包含12半胱氨酸残基,形成6对链内二硫键[3](图1)。在结构方面,两个亚基具有的二硫键共价连接蛋白两部分,形成蛋白的疏水核心。在功能方面,稳定蛋白结构,帮助蛋白折叠;利用氧化还原反应,调节蛋白功能。二硫键的正确配对是维持FSH正确折叠方式和高级结构形成的关键因素,对产品的质量控制至关重要。根据国际人用药品注册技术协调会(ICH) Q6B指导原则及《中国药典》2020年版三部人用重组DNA蛋白制品总论的通则要求,应对该类药物进行包括二硫键是否正确配对在内的结构特性分析。FSH两个亚基具有复杂而又丰富多样的二硫键结构[4],特别是α亚基中的第31和32位、59和60位,分别存在4个紧密相邻的半胱氨酸,并各自与其他位置的半胱氨酸形成二硫键,很难被特异性蛋白酶有效切割,从而给该类家族蛋白药物α亚基二硫键的准确表征带来较大困难。GPHs家族所有糖蛋白激素药物的α亚基二硫键位置均高度相似,α亚基在这些激素中起到一定的交叉反应作用,但并不决定它们的特异性功能,β亚基则是GPHs家族特有的,这两个亚基共同决定了GPHs家族的生物学特性和功能。审批审评中对该类家族创新药物2个亚基二硫键的特性表征要求,尤其对α亚基二硫键定位解析定位鲜有报道,技术上存在较大难点,研发企业很难提供信噪比较高的二级质谱图谱,一定程度上制约了该类药物的研发上市进程。
早期阶段,通过X射线衍射晶体法对FSH的二硫键进行定位。而X射线衍射晶体法需要培养高度有序的蛋白结晶,且对样品需求量很大,对样品纯度要求也较高。质谱分析技术可用于二硫键分析,经过蛋白前处理和LC-MS/MS分析,匹配正确的二硫键位置。现研究蛋白质二硫键的实验方法中,自上而下质谱法较为普遍,主要有两种手段[5]:非还原条件下分析完成二硫键肽段或者还原条件下断开二硫键分析半胱氨酸定位,获得的肽段结构信息,数据分析软件与手动解析相结合鉴定二硫键结构。然而,FSH二硫键定位的难点在于无特异性理想的水解酶可以将每对相邻二硫键所含肽段酶解,往往一条肽段包含两个以上半胱氨酸,无法确定其连接位点;使用还原或部分还原方法也不能很好地控制二硫键的选择性断裂。
本研究采用非特异性酶和特异性酶相结合的策略,非还原酶切和非还原酶切后还原处理相对比的手段,利用高分辨质谱仪器,最终通过分析软件成功解析出FSH的11对二硫键,方法可行,且准确度高;尤其是对α亚基二硫键的解析,可为GPHs家族复杂糖蛋白激素药物的审批审评与科学监管提供一定的技术储备。
1 材料与方法
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1.1 材料
1.1.1 仪器
Orbitrap Exploris 480(美国赛默飞世尔科技公司);Waters-UNIFI数据处理软件(美国沃特世公司);电子天平(瑞士梅特勒-托利多公司)。
1.1.2 试药
乙腈(批号:164788)、甲酸(批号:195715)[赛默飞世尔科技(中国)有限公司];三(2-羧乙基)膦盐酸盐(TCEP. HCl,批号:LSBZ2552)、枯草杆菌酶(批号:P5459,来源地衣芽孢杆菌,glycerol solution 50%)[西格玛奥德里奇(上海)贸易有限公司];胰蛋白酶(批号:V5111,每支25 μg,Promega北京); PNGase F(批号:P0704L,NEW ENGLAND BioLabs)。
1.2 方法
1.2.1 溶液配制
①原液稀释液:取0.193 g Na2HPO4·12H2O和0.067 g NaH2PO4·H2O,加入100 mL纯化水,混匀即得。②100 mmol·L-1的TCEP溶液:取28.6 mg的TCEP. HCl,加入1 mL纯化水混匀即得。
1.2.2 非还原酶切处理
将FSH原液用稀释液稀释至0.5 mg·mL-1,取该溶液100 μL加入枯草杆菌蛋白酶1 μL(取10 μL枯草杆菌酶加入840 μL超纯水,混匀),37 ℃酶解17 h;后加入1 μL胰蛋白酶(取每支25 μg的胰蛋白酶1支,加超纯水25 μL溶解,混匀),37 ℃酶解8 h;最后,加入1 μL PNGase F,37 ℃酶解17 h,加入1 μL体积分数10%的甲酸,停止反应。
1.2.3 非还原酶切后还原处理
将FSH原液用稀释液稀释至0.5 mg·mL-1,取该溶液100 μL加入枯草杆菌蛋白酶1 μL(取10 μL枯草杆菌酶加入840 μL超纯水,混匀),37 ℃酶解17 h;后加入1 μL胰蛋白酶(取规格为25 μg/支胰蛋白酶1支,加入超纯水25 μL,混匀),37 ℃酶解8 h;最后,加入1 μL PNGase F,37 ℃酶解17 h,加入1 μL 100 mmol·L-1的TCEP溶液,37 ℃孵育30 min,加入1 μL体积分数10%的甲酸,停止反应。
1.2.4 液质条件
液相条件:采用ACQUITY UPLC Peptide BEH C18(2.1 mm×100 mm,1.7 μm),以0.1%甲酸/水溶液(A)-0.1%甲酸/乙腈溶液(B)为流动相,梯度洗脱(0~60 min, 0.5% B→20% B;60~70 min,20% B→98% B;70~75 min,98%B;75~75.1 min,98% B→0.5% B;75.1~85 min, 0.5%B),流速为0.2 mL·min-1,柱温为60 ℃,进样量20 μL。
质谱条件:质谱采集模式为ddMS2,鞘气35,辅气10;离子源为正离子,离子源传输管温度320 ℃,扫描范围m/z 250~2 000,一级分辨率60 000@110 m/z;二级分辨率15 000@110 m/z。
1.2.5 处理方法
β亚基处理方法设置:输入β亚基理论序列,可变修饰为氧化、还原、半胱氨酸化,设置检测下限强度为1×105,偏差10×10-6,非特异性酶切(nonspecific);α亚基处理方法设置:输入α亚基理论序列,可变修饰为氧化、还原、半胱氨酸化,设置检测下限强度为1×104,偏差10×10-6,非特异性酶切(nonspecific)。
2 实验结果
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2.1 β亚基鉴定结果
对检索结果进行筛选,分别搜索β:C3=β:C51, β:C17=β:C66, β:C20=β:C104,β:C28=β:C82,β:C32=β:C84, β:C87=β:C94的6对二硫键具有MS2的肽段。如表1所示,6对二硫键均检出多对可定位的C=C连接肽段,一级偏差均在10×10-6之内,每条肽段的平均结构分辨率(average structural resolution,ASR)为1.0。选择β:C3=β:C51的NSCE=KTCTF和β:C17=β:C66的ECR=PGACH肽段(图2),选择β:C20=β:C104的FCIS=YCSF肽段和β:C28=β:C82的WCA=TQCH肽段(图3),选择β:C32=β:C84的GYCY=HCGK肽段和β:C87=β:C94的GKCD=DCTVR肽段(图4),进一步对以上6条C=C肽段进行二级图谱详细解析,通过分析互补的碎片离子,可确认β亚基6对二硫键的连接方式。其中,图2A中的b3、 y2、y3,图2B中的y3、y4和b3可确认NSCE=KTCTF的C3-C3连接方式;图2C中的b2、y2,以及图2D中的y3、y4和b3、b4,可确认ECR=PGACH的C2-C4连接方式;图3A中b2、b3、y3和图3B中的b2、b3、y3可确认FCIS=YCSF的C2-C2连接方式;图3C中b2、y2和3D中b3、y2、y3可确认WCA=TQCH的C2-C3的连接方式;图4A中b3、y2、y3和图4B中y3可确认GYCY=HCGK的C3-C2连接方式;图4C中b3、y2、y3和图4D中y4可确认GKCD=DCTVR的C3和C2连接方式。
2.2 α亚基鉴定结果
α亚基中存在2个相邻半胱氨酸分别与其他位置半胱氨酸成二硫键的情况,分别为C31、C32和C59、C60,则要求水解酶必须在以上2个相邻半胱氨酸之间进行酶解,设置检测限较β亚基更低,符合肽段长度可能更短。经过检索,分别搜索α:C7=α:C31, α:C10=α:C60, α:C28=α:C82, α:C32=α:C84, α:C59=α:C87的5对二硫键具有MS2的肽段(表2)。α:C10=α:C60, α:C28=α:C82, α:C32=α:C84二硫键均具有多对可定位的C=C连接肽段,一级偏差均在10×10-6之内,Average structural resolution均小于1.5。α:C59=α:C87检索到1个匹配肽段,一级偏差均在10×10-6之内。值得注意的是,α:C7=α:C31的证明肽段解析难度很大,可以直接定位的肽段需要仅包含29位M到31位C的3个氨基酸序列MGC或者仅有GC两个氨基酸序列的肽段,实际检出了1个目标肽段为CP=GC,ASR为1.0;另外一个为DCP=C,它的重复检出率很高,因DCP=C不能确定为哪个位置的C,判断时采用排除法,首先检索到了α:C7~α:C31/α:C32的肽段,虽然不确定C7与C31和C32的连接,通过α:C32=α:C84多条肽段的确证,排除C7与C32的连接,确定DCP=C为α:C7=α:C31的连接,虽然是间接判断,但是可以作为该对二硫键定位的补充。
对以下肽段进行二级碎片分析:选择α:C7=α:C31的CP=GC和α:C10=α:C60的CTL=CVAK肽段(图5),选择α:C28=α:C82的LQC=AC肽段和α:C32=α:C84的CF=HCS肽段,α:C59=α:C87的TC=TCY(图6)。图5A中的a1离子可证明CP=GC的C1-C2连接方式;图5C、5D中所有氨基酸均具有二级碎片,可证明CTL=CVAK的C1-C1连接方式;图6A、6B具有二级碎片,但无直接证明LQC=AC连接方式碎片离子;图6C、6D具有二级碎片,但无直接证明CF=HCS连接方式的碎片离子;图6E中y1、图6F中的y2离子可证明TC=TCY的C1-C2连接方式。虽然存在无直接证明C-C连接的碎片离子,以上5条肽段每条仅有两个半胱氨酸,两条肽段仅能通过两个C-C键相连接,所以可以定位α亚基5对二硫键。为增加判定准确性,对比非还原酶切和非还原酶切后还原处理的两组样品数据,将非还原酶切处理后样品数据的5对C=C肽段提取离子流(XIC)信号(图7),再对酶切后还原处理的数据手动提取以上5对C=C肽段提取离子流,根据保留时间进行判断。结果显示,非还原酶切后还原处理的样品数据并未提取到以上5对C=C肽段提取离子流信号,证明在非还原酶切后还原处理的样品中以上均二硫键打开。综上,可进一步确认α亚基5对二硫键的连接方式。
3 讨论
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《中国药典》2020年版三部人用重组制品总论一级结构测定中要求“一级结构,即包括二硫键连接方式的氨基酸序列(包含二硫键的完整性和正确性)”。二硫键不仅维持蛋白的功能与稳定性,而且其表征为结构的质量控制的重要组成部分,对产品的质量控制至关重要。Yin等[6]利用胰蛋白酶水解的方法解析出溶酶菌的4对二硫键,为cys6-cys127、cys30-cys115、cys64-cys80、cys76-cys94;Zhao等[7]使用NEM封闭游离巯基加胰蛋白酶水解的方法定位了CD79b为靶点的抗体偶联药物二硫键,以上两种均采用“自下而上”质谱分析策略,特异性的酶将蛋白分解为肽段,并且一条肽段中仅含有一对二硫键,如链间二硫键或链内二硫键。二硫键具有复杂多样性,其中也包含酶解肽段同时包含二对二硫键,如胰岛素类经特异性酶解后同时包含了链间和链内二硫键,我们在先前的研究中利用了部分还原酶解与质谱不同的裂解方式多种手段成功解析了胰岛素类的复杂二硫键[8]。然而对于FSH来说,二硫键类型更加复杂多样,酶切的肽段可能包含多个半胱氨酸残基,不局限于以上两种二硫键的组合方式,尤其是α亚基中还存在2对相邻半胱氨酸分别与其他位置半胱氨酸成二硫键的情况,分别为C31、C32和C59、C60,要求水解酶必须在这2个相邻半胱氨酸之间进行酶解才可能有效鉴别。其他GPHs家族也同样存在此类棘手问题,鉴于现实存在的技术难点,目前在新药申报审评中对该类产品的特性鉴定二硫键鉴别并未做出明确要求,如每个亚基的每对二硫键是否可以定位并具有一级二级质谱信息等。因此,给新药审评机构相关药学或质量控制指导原则的制定带来困扰,同时企业申报过程中对该类产品二硫键解析也没有明确的方向。
有关报道文献利用了非特异性酶和特异性酶联合酶解的方式定位了FSH的β亚基6对二硫键[9],但近年来α亚基的二硫键定位未见文献报道。本研究仍采用非特异性枯草杆菌酶和特异性胰蛋白酶联合水解FSH,其优势主要有几点:①枯草杆菌酶有利于释放亚基的紧密构象;②防止形成1个以上的S-S桥的团簇,分离出单个半胱氨酸残基的肽段;③提供有助于解析质谱数据的裂解肽段。采用该种策略可以产生足以分开二硫键配对位点的多肽片段,但也不可避免肽段长度过小,响应较低的问题。经多次重复实验表明,β亚基输入全序列解析,每对二硫键均有多对肽段相匹配,二级碎片质量良好,可明确定位。α亚基可识别二硫键的肽段较β亚基,长度更小,响应更低,并且保留时间靠前,还出现肽段保留时间相近或共流出的现象,α:C7=α:C31和α:C59=α:C87解析难度最大,建议鉴定α亚基二硫键时,可直接提取目标肽段的m/z或输入目标肽段序列进行二级解析,同时增加非还原酶切后还原处理的样品进行对比佐证。另外,在前处理过程中,不可避免地有少量半胱氨酸发生交换反应,在解析过程中也可发现少部分游离的半胱氨酸或二硫键交换的产物,应注意枯草杆菌酶的选择和浓度、缓冲体系及pH、仪器状态等,保证实验的重现性和相应目标肽段的重复检出。
本研究探讨了重组人促卵泡激素二硫键的定位方法,并成功鉴定出β亚基6对二硫键和α亚基5对二硫键,为FSH乃至长效FSH(如FSH-CTP, FSH-Fc等)二硫键连接方式的质量控制提供新思路,也为制定GPHs家族类生物制品的药学评价指导原则提供借鉴。
基金
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  • 药品监管科学全国重点实验室第一批课题项目资助(2023SKLDRS0108)
  • 药品监管科学全国重点实验室第二批课题项目资助(2024SKLDRS0203)
  • 中国食品药品检定研究院化学药品检定所重点实验室项目资助(2024HYZX23)
文献
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参考文献 引证文献
排序方式：
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CAHOREAU C, KLETT D, COMBARNOUS Y. Structure-function relationships of glycoprotein hormones and their subunits' ancestors.[J] .Front Endocrinol(Lausanne), 2015, 6:26.
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ULLOA-AGUIRRE A, ZARIÑÁN T, JARDÓN-VALADEZ E, et al. Structure-function relationships of the follicle-stimulating hormone receptor[J]. Front Endocrinol(Lausanne), 2018, 9:707.
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BOUSFIELD G R, HARVEY D J. Follicle-stimulating hormone glycobiology[J]. Endocrinology, 2019, 160(6):1515-1535.
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XU H, et al. Identification and characterization of disulfide bonds in proteins and peptides from tandem MS data by use of the Mass Matrix MS/MS search engine[J]. J Proteome Res, 2008, 7(1), 138-144.
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WANG X Y, YAN G Q, ZHOU X W, et al. Assignment of Polypeptide Disulfide Bonds by Chemical Cleavage and Bio-mass Spectrometry?[J]. Chem J Chin Univ(高等学校化学学报), 2020, 7(41):1505-1512.
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YIN H R, ZHANG Y, FANG X X, et al. Analysis and study on the primary structure of the lysozyme using UPLC-Q TOF MS[J]. Chin J Pharm Anal(药物分析杂志), 2023, 43(1):12-19.
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ZHAO X Y, LI M, WU G, et al. Primary structure characterization of anti-CD79b antibody-vc-MMAE[J]. Chin Pharm J(中国药学杂志), 2023, 58(1):33-40.
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HU X Y, DINGX L, SUN Y, et al. Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum[J]. Acta Pharm Sin(药学学报), 2024, 59(1):188-197.
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AMORESANO A, ORRÙ S, SICILIANO R A, et al. Assignment of the complete disulphide bridge pattern in the human recombinant follitropin β-chain[J]. Biol Chem, 2001, 383(6):961-968.
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文章信息
doi: 10.11669/cpj.2025.01.011
  • 接收时间：2024-07-30
  • 首发时间：2025-11-24
  • 出版时间：2025-01-08
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出版历史
  • 收稿日期：2024-07-30
基金
药品监管科学全国重点实验室第一批课题项目资助(2023SKLDRS0108)
药品监管科学全国重点实验室第二批课题项目资助(2024SKLDRS0203)
中国食品药品检定研究院化学药品检定所重点实验室项目资助(2024HYZX23)
作者信息
    1 中国食品药品检定研究院, 药品监管科学全国重点实验室, 国家卫生健康委生物技术产品检定方法及其标准化重点实验室, 国家药品监督管理局生物制品质量研究与评价重点实验室, 国家药品监督管理局化学药品质量研究与评价重点实验室, 北京 102629
    2 中国药科大学, 南京 210009

通讯作者:

*梁成罡,男,博士,研究员 研究方向:激素类药物质量控制及标准研究 Tel:(010) 53851638;
李晶,女,博士,研究员 研究方向:激素类药物质量控制及标准研究 Tel:(010) 53851465
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https://castjournals.cast.org.cn/joweb/zgyxzz/CN/10.11669/cpj.2025.01.011
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2种不同金属材料的力学参数
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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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