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Article(id=1201177208212644673, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201177206518145841, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2023-0284, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1678291200000, receivedDateStr=2023-03-09, revisedDate=1690732800000, revisedDateStr=2023-07-31, acceptedDate=null, acceptedDateStr=null, onlineDate=1764312563230, onlineDateStr=2025-11-28, pubDate=1704988800000, pubDateStr=2024-01-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1764312563230, onlineIssueDateStr=2025-11-28, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1764312563230, creator=13701087609, updateTime=1764312563230, updator=13701087609, issue=Issue{id=1201177206518145841, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='1', pageStart='1', pageEnd='268', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1764312562826, creator=13701087609, updateTime=1764312760268, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1201178034725417827, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201177206518145841, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1201178034725417828, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1201177206518145841, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=188, endPage=197, ext={EN=ArticleExt(id=1201177208657240911, articleId=1201177208212644673, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

The correct pairing of disulfide bonds maintains the correct folding mode and high-level structure formation of peptides and protein drugs, which is crucial for the quality control of products. In order to ensure that the disulfide bonds are correctly paired, disulfide bond analysis is an essential part of peptides and protein drug characterization. Mass spectrometry can be used to analyze disulfide bonds. However, insulin and its analogues have two pairs of disulfide bonds without restriction enzyme cutting site. Conventional collision-induced dissociation (CID) and high-energy induced cleavage (HCD) cannot accurately locate the complex disulfide bond. In our study, three methods were used to localize the complex disulfide, including enzyme digestion combined with key peptide fragment in source decay (ISD) fragmentation method, enzyme digestion combined with partial reduction alkylation method, intact protein source ISD and electron transfer dissociation (ETD) cleavage method, The applicability of insulin aspart, insulin lispro and insulin glargine were also investigated. This study provides a new way for the quality control of disulfide bonding mode of insulin and its analogues, and also provides a reference for the disulfide bond localization of peptides or proteins containing this complex disulfide bond.

, correspAuthors=Jing LI, Cheng-gang LINAG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 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, authorCompany=null, fund=null, authors=null, authorsList=Xin-yue HU, Xiao-li DING, Yue SUN, Hui ZHANG, Jing LI, Cheng-gang LINAG), CN=ArticleExt(id=1201177210355934114, articleId=1201177208212644673, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=利用高分辨质谱技术综合解析胰岛素类制品复杂二硫键结构, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

二硫键的正确配对维持了多肽和蛋白类药物的正确折叠方式和高级结构的形成, 对产品的质量控制至关重要。为了确保二硫键正确配对, 二硫键分析是多肽和蛋白质药物表征中必不可少的重要部分。质谱分析技术可用于二硫键分析, 然而, 胰岛素及其类似物中存在两对没有酶切位点的二硫键, 常规的碰撞诱导解离(CID) 和高能诱导裂解(HCD) 无法实现该复杂二硫键的准确定位。本研究通过3种方法综合定位该复杂二硫键, 包括酶切加关键肽段源内碎裂(ISD) 法、酶切加部分还原烷基化法、完整蛋白源内碎裂和电子转移解离(ETD) 裂解法, 并且考察了门冬胰岛素、赖脯胰岛素和甘精胰岛素的适用性, 为胰岛素及其类似物二硫键连接方式的质量控制提供了新途径, 也为含该类复杂二硫键多肽或蛋白类生物制品的二硫键定位提供借鉴。

, correspAuthors=李晶, 梁成罡, authorNote=null, correspAuthorsNote=
*李晶, Tel: 86-10-53851465, E-mail: ;
梁成罡, Tel: 86-10-53851638, E-mail:
, copyrightStatement=版权所有©《药学学报》编辑部2024, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=Vqcj19okGd+1dkXrg7sHIw==, magXml=Ic0MkUfvO1odVQyQIIgTug==, pdfUrl=null, pdf=qyuY+P25f5beUS9hX1AGEQ==, pdfFileSize=4625849, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=eBbF6RqCMNxN/rzA80d7jw==, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=l0Fb4rG3wM9h7zgR6ag+ug==, mapNumber=null, authorCompany=null, fund=null, authors=

#共同第一作者

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Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins [J]. Anal Bioanal Chem, 2018, 410: 2467-2484., articleTitle=null, refAbstract=null), Reference(id=1201177216861299122, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, 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=Chang SG, Choi KD, Jang SH, et al. Role of disulfide bonds in the structure and activity of human insulin [J]. 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A: b4-2/b10 ions; B: b5-2/y8 ions; C: b5-2/yMax ions; D: b6-2/yMax ions; E: y11-2/a8 ions; F: y11-2/b11 ions; G: y11-2/y11 ions; H: y11-2/y11-H<sub>2</sub>O ions , figureFileSmall=Ps4gRJWxmFb4R08FSmgOZg==, figureFileBig=A9rdFA2DiX05YEorqwFbRA==, tableContent=null), ArticleFig(id=1201177214961279227, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=EN, label=null, caption=null, figureFileSmall=gvwhl4+CXn1g3ZqhBp6Bfg==, figureFileBig=yDMAE5COmxwzhgf2RLNDqA==, tableContent=null), ArticleFig(id=1201177215070331144, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=CN, label=Figure 3, caption= Flow diagram of GLU-C and chymotrypsin digestion combined with partial reduction alkylation for localization of complex disulfide bonds in human insulin , figureFileSmall=gvwhl4+CXn1g3ZqhBp6Bfg==, figureFileBig=yDMAE5COmxwzhgf2RLNDqA==, tableContent=null), ArticleFig(id=1201177215221326104, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=EN, label=null, caption=null, figureFileSmall=0rEao1X8m+sGu/MGuEWqJw==, figureFileBig=PtYsUBhgv2TCrIzapBuTXQ==, tableContent=null), ArticleFig(id=1201177215359738144, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=CN, label=Figure 4, caption= Secondary sequence coverage of major peptides of insulin by GLU-C and chymotrypsin digestion combined with partial reduction alkylation. A: QCCTSICSLYQLE; B: FVNQHLCGSHLVE; C: GIVEQCCTSICSLY; D: FVNQHLCGSHLVEALY , figureFileSmall=0rEao1X8m+sGu/MGuEWqJw==, figureFileBig=PtYsUBhgv2TCrIzapBuTXQ==, tableContent=null), ArticleFig(id=1201177215460401449, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=EN, label=null, caption=null, figureFileSmall=7UT+12YI3uX3rRBcZPiezw==, figureFileBig=btj74ytl19n8yQZq9hRiFg==, tableContent=null), ArticleFig(id=1201177215561064755, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=CN, label=Figure 5, caption= Mass spectra of key fragment ions of insulin of disulfide bond localization by ISD. A: y7-2/y18; B: y7-2/y13; C: y6-2/y12; D: b6-1/y13-2/y13; E: b-1/y13-2/y14; F: b6-1/y11-2/y23 , figureFileSmall=7UT+12YI3uX3rRBcZPiezw==, figureFileBig=btj74ytl19n8yQZq9hRiFg==, tableContent=null), ArticleFig(id=1201177215649145148, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=EN, label=null, caption=null, figureFileSmall=7bgzDstHlN9gEXYzImS0Kw==, figureFileBig=mF5Wki+zC4RbRLURvXtQPw==, tableContent=null), ArticleFig(id=1201177215741419845, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=CN, label=Figure 6, caption= Mass spectra of key fragment ions of insulin of disulfide bond localization by ETD. A: z′3-2/z′22+H; B: z′2-2/z′23; C: c′′6-1/z′11-2/z′16; D: c′′6-1/y11-2/z′21 , figureFileSmall=7bgzDstHlN9gEXYzImS0Kw==, figureFileBig=mF5Wki+zC4RbRLURvXtQPw==, tableContent=null), ArticleFig(id=1201177215871443277, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Peptide nameModification siteModified nameObserved mass/DaMass error /Da
QCCTSICSLYQLE(C6, C7, C11)(IAM carbamidomethylation, M-biotin, IAM carbamidomethylation)2 128.891 1-1.25×10-6
FVNQHLCGSHLVE(C7)(M biotin)2 006.928 6-2.82×10-6
GIVEQCCTSICSLY(C6, C7, C11)(IAM carbamidomethylation, M-biotin, IAM carbamidomethylation)2 156.920 2-2.26×10-6
FVNQHLCGSHLVEALY(C7)(M biotin)2 354.112 1-2.85×10-6
), ArticleFig(id=1201177215955329366, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=CN, label=Table 1, caption=

Information table of GLU-C and chymotrypsin digestion of human insulin peptides respectively

, figureFileSmall=null, figureFileBig=null, tableContent=
Peptide nameModification siteModified nameObserved mass/DaMass error /Da
QCCTSICSLYQLE(C6, C7, C11)(IAM carbamidomethylation, M-biotin, IAM carbamidomethylation)2 128.891 1-1.25×10-6
FVNQHLCGSHLVE(C7)(M biotin)2 006.928 6-2.82×10-6
GIVEQCCTSICSLY(C6, C7, C11)(IAM carbamidomethylation, M-biotin, IAM carbamidomethylation)2 156.920 2-2.26×10-6
FVNQHLCGSHLVEALY(C7)(M biotin)2 354.112 1-2.85×10-6
), ArticleFig(id=1201177216051798369, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Insulin nameFragment ionObserved mass/DaExpected mass/DaMass error/Da
Insulin aspartb6-1/y14-2/y214 736.1174 736.182-13.83×10-6
b6-1/y13-2/y234 779.1184 779.188-14.56×10-6
b6-1/y14-2/y234 880.1664 880.236-14.34×10-6
y7-2/y142 516.1362 516.1235.13×10-6
y7-2/y122 303.9712 303.9700.39×10-6
y6-2/y122 175.9222 175.9124.64×10-6
Insulin lisprob6-1/y13-2/y133 678.6333 678.638-1.28×10-6
b6-1/y13-2/y143 791.7273 791.7221.27×10-6
b6-1/y13-2/y153 954.8143 954.7857.43×10-6
y7-2/y142 498.1512 498.1481.00×10-6
y7-2/y122 285.9952 285.996-0.52×10-6
y6-2/y142 370.0962 370.0902.40×10-6
Insulin glarginey7-2/y142 541.1962 541.1774.03×10-6
y7-2/y152 640.2462 640.2453.87×10-6
y6-2/y142 413.1642 413.11819.19×10-6
), ArticleFig(id=1201177216156655979, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=CN, label=Table 2, caption=

Key fragment ions of disulfide bond localization by in source decay (ISD) for other insulin analogue

, figureFileSmall=null, figureFileBig=null, tableContent=
Insulin nameFragment ionObserved mass/DaExpected mass/DaMass error/Da
Insulin aspartb6-1/y14-2/y214 736.1174 736.182-13.83×10-6
b6-1/y13-2/y234 779.1184 779.188-14.56×10-6
b6-1/y14-2/y234 880.1664 880.236-14.34×10-6
y7-2/y142 516.1362 516.1235.13×10-6
y7-2/y122 303.9712 303.9700.39×10-6
y6-2/y122 175.9222 175.9124.64×10-6
Insulin lisprob6-1/y13-2/y133 678.6333 678.638-1.28×10-6
b6-1/y13-2/y143 791.7273 791.7221.27×10-6
b6-1/y13-2/y153 954.8143 954.7857.43×10-6
y7-2/y142 498.1512 498.1481.00×10-6
y7-2/y122 285.9952 285.996-0.52×10-6
y6-2/y142 370.0962 370.0902.40×10-6
Insulin glarginey7-2/y142 541.1962 541.1774.03×10-6
y7-2/y152 640.2462 640.2453.87×10-6
y6-2/y142 413.1642 413.11819.19×10-6
), ArticleFig(id=1201177216261513593, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
Insulin nameFragment ionObserved mass/DaExpected mass/DaMass error/Da
Insulin aspartz′3-2/z′222 912.4712 912.32849.14×10-6
z′3-2/z′23+H2 970.4462 970.35730.03×10-6
c′′6-1/z′14-2/z′234 865.0174 865.225-42.77×10-6
c′′6-1/y14-2/z′143 911.6433 911.751-27.69×10-6
Insulin lisproz′5-2/z′20+H2 914.4722 914.35639.80×10-6
z′3-2/z′22+H2 895.4792 895.36140.69×10-6
c′′6-1/z′14-2/y234 863.4604 863.26939.17×10-6
c′′6-1/z′11-2/z′163 852.7203 852.742-5.74×10-6
Insulin glarginez′5-2/z′213 055.5483 055.44543.23×10-6
z′8-2/z′21+H3 460.8083 460.65833.84×10-6
), ArticleFig(id=1201177216357982599, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1201177208212644673, language=CN, label=Table 3, caption=

Key fragment ions of disulfide bond localization by electron transfer dissociation (ETD) for other insulin analogue

, figureFileSmall=null, figureFileBig=null, tableContent=
Insulin nameFragment ionObserved mass/DaExpected mass/DaMass error/Da
Insulin aspartz′3-2/z′222 912.4712 912.32849.14×10-6
z′3-2/z′23+H2 970.4462 970.35730.03×10-6
c′′6-1/z′14-2/z′234 865.0174 865.225-42.77×10-6
c′′6-1/y14-2/z′143 911.6433 911.751-27.69×10-6
Insulin lisproz′5-2/z′20+H2 914.4722 914.35639.80×10-6
z′3-2/z′22+H2 895.4792 895.36140.69×10-6
c′′6-1/z′14-2/y234 863.4604 863.26939.17×10-6
c′′6-1/z′11-2/z′163 852.7203 852.742-5.74×10-6
Insulin glarginez′5-2/z′213 055.5483 055.44543.23×10-6
z′8-2/z′21+H3 460.8083 460.65833.84×10-6
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利用高分辨质谱技术综合解析胰岛素类制品复杂二硫键结构
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胡馨月 # , 丁晓丽 # , 孙悦 , 张慧 , 李晶 * , 梁成罡 *
药学学报 | 研究论文 2024,59(1): 188-197
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药学学报 | 研究论文 2024, 59(1): 188-197
利用高分辨质谱技术综合解析胰岛素类制品复杂二硫键结构
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胡馨月#, 丁晓丽#, 孙悦, 张慧, 李晶* , 梁成罡*
作者信息
  • 中国食品药品检定研究院激素室, 国家卫生健康委员会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化学药品质量研究与评价重点实验室, 北京 102629

通讯作者:

*李晶, Tel: 86-10-53851465, E-mail: ;
梁成罡, Tel: 86-10-53851638, E-mail:
Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum
Xin-yue HU, Xiao-li DING, Yue SUN, Hui ZHANG, Jing LI* , Cheng-gang LINAG*
Affiliations
  • Division of Hormone, National Institutes for Food and Drug Control, NHC Key Laboratory of Research on Quality and Standardization of Biotech Products, NMPA Key Laboratory for Quality Research and Evaluation of Chemical Drugs, Beijing 102629, China
出版时间: 2024-01-12 doi: 10.16438/j.0513-4870.2023-0284
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摘要
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二硫键的正确配对维持了多肽和蛋白类药物的正确折叠方式和高级结构的形成, 对产品的质量控制至关重要。为了确保二硫键正确配对, 二硫键分析是多肽和蛋白质药物表征中必不可少的重要部分。质谱分析技术可用于二硫键分析, 然而, 胰岛素及其类似物中存在两对没有酶切位点的二硫键, 常规的碰撞诱导解离(CID) 和高能诱导裂解(HCD) 无法实现该复杂二硫键的准确定位。本研究通过3种方法综合定位该复杂二硫键, 包括酶切加关键肽段源内碎裂(ISD) 法、酶切加部分还原烷基化法、完整蛋白源内碎裂和电子转移解离(ETD) 裂解法, 并且考察了门冬胰岛素、赖脯胰岛素和甘精胰岛素的适用性, 为胰岛素及其类似物二硫键连接方式的质量控制提供了新途径, 也为含该类复杂二硫键多肽或蛋白类生物制品的二硫键定位提供借鉴。

胰岛素及其类似物  /  二硫键定位  /  部分还原烷基化  /  源内碎裂  /  电子转移裂解

The correct pairing of disulfide bonds maintains the correct folding mode and high-level structure formation of peptides and protein drugs, which is crucial for the quality control of products. In order to ensure that the disulfide bonds are correctly paired, disulfide bond analysis is an essential part of peptides and protein drug characterization. Mass spectrometry can be used to analyze disulfide bonds. However, insulin and its analogues have two pairs of disulfide bonds without restriction enzyme cutting site. Conventional collision-induced dissociation (CID) and high-energy induced cleavage (HCD) cannot accurately locate the complex disulfide bond. In our study, three methods were used to localize the complex disulfide, including enzyme digestion combined with key peptide fragment in source decay (ISD) fragmentation method, enzyme digestion combined with partial reduction alkylation method, intact protein source ISD and electron transfer dissociation (ETD) cleavage method, The applicability of insulin aspart, insulin lispro and insulin glargine were also investigated. This study provides a new way for the quality control of disulfide bonding mode of insulin and its analogues, and also provides a reference for the disulfide bond localization of peptides or proteins containing this complex disulfide bond.

insulin  /  disulfide bond localization  /  partial reduction alkylation  /  in source decay  /  electron transfer dissociation
胡馨月, 丁晓丽, 孙悦, 张慧, 李晶, 梁成罡. 利用高分辨质谱技术综合解析胰岛素类制品复杂二硫键结构. 药学学报, 2024 , 59 (1) : 188 -197 . DOI: 10.16438/j.0513-4870.2023-0284
Xin-yue HU, Xiao-li DING, Yue SUN, Hui ZHANG, Jing LI, Cheng-gang LINAG. Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum[J]. Acta Pharmaceutica Sinica, 2024 , 59 (1) : 188 -197 . DOI: 10.16438/j.0513-4870.2023-0284
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多肽和蛋白质中的二硫键对其热力学、力学和化学稳定性至关重要, 同时也参与对蛋白酶的抗性和活性的调节[1]。胰岛素及其类似物共含有三条二硫键, A链中存在一个链内二硫键, 位于A链6位和11位的半胱氨酸之间; A和B链则通过另外两对二硫键连接, 第一条在A链7位和B链7位半胱氨酸之间, 第二条在A链20位和B链19位半胱氨酸之间[2], 三对二硫键的正确配对维持了胰岛素及其类似物的正确折叠方式和高级结构的形成, 对产品质量也有至关重要的影响。
早期二硫键的定位方法主要包括X射线衍射晶体法[3]、多维核磁共振波谱法[4]、Edman降解法等。X射线衍射晶体法需要培养高度有序的蛋白结晶, 且X射线衍射晶体法和多维核磁共振波谱2种方法对样品需求量很大, 对样品纯度要求也较高。Edman降解法无法直接对含有链内二硫键和N端被封闭的多肽和蛋白质进行测序。近年来, 随着生物质谱的发展, 质谱技术逐渐应用到二硫键的定位研究中, 完整的蛋白质或多肽酶解成含有不同定位信息的肽段(自下而上法, 即“Bottom-up”法)[5], 通过软件分析实现对二硫键配对方式的表征, 但当多对二硫键同时存在时, 酶切片段中可能同时存在多对二硫键, 质谱对这种复杂二硫键的分析有一定的挑战性。胰岛素及其类似物的A: C6-A: C11和A: C7-B: C7这2个二硫键之间没有酶切位点, A6和A7又是相邻的半胱氨酸, 使这2对二硫键所连接的肽段成为一个闭环的结构, 因为常规的碰撞诱导解离(collision induced dissociation, CID) 和高能诱导裂解(higher energy collision induced dissociation, HCD) 产生的碰撞能量相对较低, 仅能够裂解多肽骨架, 几乎不能断裂二硫键[6], 采用CID或HCD裂解方式对带有该复杂二硫键的肽段序列进行分析, 则不能将肽段-C6C7TSIC11-进行碎片化裂解, 所以无法判定上述2对复杂二硫键正确连接方式。虽然人们已对胰岛素的结构和功能进行了深入研究[7], 但是对胰岛素二硫键定位的方法的报道仍然较少。黄亚娟等[8]报道了酶切质谱法联合氰基化裂解法对新型重组人门冬胰岛素的二硫键进行定位, 成功解析了门冬胰岛素两对复杂的二硫键, 但是氰基化裂解法也存在着操作较为繁琐, 多步前处理过程, 大量数据的处理和分析等问题。因此, 探索高效、快速、准确的方法来定位胰岛素及其类似物复杂二硫键, 具有一定挑战和意义。
随着质谱技术的发展, 质谱解离技术也渐渐丰富起来, 如CID、HCD、源内碎裂(in source decay, ISD) 技术、电子转移解离(electron transfer dissociation, ETD) 等。CID和HCD使酰胺键发生断裂主要生成b/y类型的碎片离子, HCD理论上可产生比CID更丰富的离子; ETD主要使N－C α键断裂生成c/z类型的碎片离子; ISD技术是在ESI离子源产生离子, 允许所有价态的离子发生断裂, 主要也是生成b/y类型的碎片离子。以上多种质谱解离技术常用做蛋白或者多肽的质谱鉴定分析[9]。胰岛素及其类似物的品种很多, 但其二硫键位置一致, 以人胰岛素的二硫键定位为例, 本研究工作尝试并且探讨了以下3种二硫键定位的方法, 包括酶切加关键肽段ISD法、酶切加部分还原烷基化法、完整蛋白ISD和ETD裂解法。以上3种方法综合定位胰岛素的两对复杂二硫键, 并且考察了门冬胰岛素、赖脯胰岛素和甘精胰岛素的适用性, 为胰岛素及其类似物二硫键连接方式的质量控制提供了新途径, 也为含该复杂二硫键多肽或蛋白类生物制品的二硫键定位提供借鉴。
材料与方法
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材料与试剂  人胰岛素、门冬胰岛素、赖脯胰岛素、甘精胰岛素(中国食品药品检定研究院激素室留样, 批号: GHIA19003, 023C220210603, GLPA20009, GCB2021002); 乙腈(批号: 164788)、甲酸(批号: 195715)、马来酰亚胺-PEG2-生物素(M-biotin, 批号: A39261), 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)、二硫苏糖醇(DL-dithiothreitol, DTT, 批号: SLBV6582)、碘乙酰胺(Iodoacetamide, IAM, 批号: SLCC6164)、糜蛋白酶(批号: 11418467001), 西格玛奥德里奇(上海) 贸易有限公司; ETD试剂盒[批号: W28102021, 沃特世(美国) 公司]; GLU-C酶(Worthington公司, 美国, 批号: 57E17522A)。
仪器耗材  Waters UPLC I-Class/SYNAPT Q-TOF和Waters-UNIFI数据处理软件[沃特世(美国) 公司]; Orbitrap Exploris 480和Biopharma Finder数据处理软件[赛默飞世尔科技(美国) 公司]; 电子天平(瑞士梅特勒-托利多公司)
酶切法的样品前处理  取10 mg胰岛素溶于10 mL TE溶液(tris-EDTA, pH 7.5), 取上述溶液100 μL, 加入10 μL的Glu-C酶(1 mg溶于1 mL的水中, 10 μg), 37 ℃孵育4 h, TE稀释至1 mL停止反应, 进行液质分析。
酶切+部分还原烷基化法的样品前处理  取10 mg胰岛素溶于10 mL TE溶液(tris-EDTA, pH 6.5), 取上述溶液100 μL, 加入5 μL的糜蛋白酶(25 μg溶于25 μL水, 加入5 μg) 或10 μL Glu-C酶(1 mg溶于1 mL水中, 加入10 μg), 37 ℃孵育4 h, 上述溶液用TE稀释至1 mL, 加入1 μL的0.1 mol·L-1 TCEP·HCl和1 μL M-biotin (将2 mg M-biotin溶于40 μL水, 配成浓度约为0.1 μmol·L-1), 65 ℃孵育15 min, 室温孵育1 h。向反应体系中加入1 μL的1 mol·L-1 DTT, 37 ℃孵育30 min, 加入2 μL氨水, 调节反应体系至碱性, 加入3 μL的1 mol·L-1 IAM, 暗处, 室温孵育30 min, 直接进行液质分析。
完整蛋白ISD和ETD裂解法的溶液配制方法  称取样品约10 mg, 用0.1%甲酸/水溶液定容至5 mL, 配成浓度为2 mg.mL-1的样品溶液; 取上述胰岛素样品溶液1 mL置于10 mL量瓶中, 用50 mmol·L-1碳酸氢铵水溶液定容至刻度, 混匀, 作为供试品溶液。
HCD裂解A: C20-B: C19二硫键鉴别液质方法  液相条件: 色谱柱ACQUITY UPLC peptide BEH C18 (150 mm × 2.1 mm, 300 Å, 1.7 μm); 流动相A为0.1%甲酸/水溶液, 流动相B为0.1%甲酸/乙腈溶液, 梯度洗脱: 0~3 min, 3% B; 3~85 min, 3%~32% B; 85~90 min, 32%~90% B; 90~95 min, 90% B; 95.1~100 min, 3% B。流速0.2 mL·min-1, 柱温50 ℃, 进样体积3 μL。质谱条件: 液质联用仪器为Orbitrap Exploris 480; 离子源: 电喷雾离子源正离子模式; 采集模式: 数据依赖采集模式; 毛细管电压3.8 kv, 源温度350 ℃, 鞘气35 Arb, 辅助气10 Arb, 正离子喷雾电压3 800 V, 一级m/z 150~2 000, 一级分辨率60 000@m/z 200, 二级分辨率15 000@m/z 200。
酶切+关键肽段ISD法的液质方法  液相条件: 色谱柱ACQUITY UPLC peptide BEH C18 (150 mm × 2.1 mm, 300 Å, 1.7 μm); 流动相A为0.1%甲酸/水溶液, 流动相B为0.1%甲酸/乙腈溶液, 梯度洗脱: 0~3 min, 1% B; 3~13 min, 1%~50% B; 13~14.5 min, 50%~90% B; 14.5~15.5 min, 90% B; 15.5~16 min, 90%~1% B; 16~20 min, 1% B。流速0.2 mL·min-1, 柱温50 ℃, 进样体积3 μL。质谱条件: 液质联用仪器为Waters UPLC I-Class/SYNAPT Q-TOF; 实时校正: 亮氨酸-脑啡肽(leucine enkephalin, LE, 200 pg·μL-1); 离子源: 电喷雾离子源正离子模式; 采集模式: MS模式; 毛细管电压: 2.5 kV, 源温度120 ℃, 锥孔电压: 80~90 V; 扫描范围: m/z 50~2 000。
酶切加部分还原烷基化法的液质方法  液相条件: 色谱柱ACQUITY UPLC peptide BEH C18 (150 mm × 2.1 mm, 300 Å, 1.7 μm); 流动相A为0.1%甲酸/水溶液, 流动相B为0.1%甲酸/乙腈溶液, 梯度洗脱: 0~3 min, 3%~10% B; 3~16 min, 10%~60% B; 16~21.5 min, 60%~90% B; 21.5~24.5 min, 90% B; 25~30 min, 3% B。流速0.2 mL·min-1, 柱温50 ℃, 进样体积3 μL。质谱条件: 液质联用仪器为Orbitrap Exploris 480; 离子源: 电喷雾离子源正离子模式; 采集模式: 数据依赖采集模式; 毛细管电压3.8 kV, 源温度350 ℃, 鞘气35 Arb, 辅助气10 Arb, 正离子喷雾电压3 800 V, 一级m/z 150~2 000, 一级分辨率60 000@m/z 200, 二级分辨率15 000@m/z 200。
完整蛋白ISD的液质方法  同酶切加关键肽段源内碎裂(ISD) 法的液质方法。
完整蛋白ETD裂解法的质谱方法  质谱直接进样法, 质谱方法如下: 液质联用仪器为Waters UPLC I-Class/SYNAPT Q-TOF; 离子源: 电喷雾离子源正离子模式; 采集模式: 母离子m/z 969.0, MS/MS模式; 毛细管电压: 2.5 kV, 源温度120 ℃, 锥孔电压: 30 V; 试剂放电电压: 0.9 kV, 试剂光放电电流: 80.0 µA, 样品流速2.5 μL·min-1, ETD试剂: 1, 3-苯二腈(m/z 128), Trap池波高电压: 0.14 V, 扫描范围: m/z 50~2 000。
数据处理  利用Biopharma Finder数据处理软件和Waters-UNIFI数据处理软件, 输入胰岛素序列, 一级二级质量数偏差设置为50×10-6。方法涉及酶解时, 编辑对应的酶, 烷基化修饰设置可变修饰为M-biotin和半胱氨酸碘乙酰胺化(carbamidomethylation)。
结果
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1 A: C20-B: C19位点鉴别结果
人胰岛素主要产生2条与二硫键相关的肽段, 其中, NYCN/ALYLVCGE肽段保留时间为46.20 min, 单一同位素分子质量为1 376.572 5 Da, 包含一对二硫键。如图 1A、B所示, NYCN和ALYLVCGE的碎片离子丰富, 基本覆盖全部氨基酸, 充分证明了A和B两条链通过A: C20和B: C19 (按照胰岛素整体序列编号, 下同) 的半胱氨酸相连接。QCCTSICSLYQLE/FVNQHLCGSHLVE肽段保留时间为58.95 min, 单一同位素分子质量为2 967.315 7 Da, 该肽段包含2条二硫键, A: C6/C7/C11-B: C7, 如图 1C、D所示, FVNQHLCGSHLVE片段碎片离子较为丰富, 而QCCTSICSLYQLE碎片离子少, 不能将肽段-CCTSIC-之间的离子进行碎片化裂解, 因此无法推断出与B链FVNQHLCGSHLVE片段的C7连接位点。门冬胰岛素、赖脯胰岛素、甘精胰岛素与人胰岛素结果相似, 无法推断QCCTSICSLYQLE/FVNQHLCGSHLVE肽段中二硫键的连接方式。
2 酶切+关键肽段ISD法对QCCTSICSLYQLE/FVNQHLCGSHLVE肽段中的复杂二硫键间接定位结果
ISD裂解能较CID和HCD能量高, 无需选择前体母离子即可在源内产生碎片离子, 从而引起整个肽段骨架的碎裂, 包括链间和链内二硫键的裂解。通过碎片离子的分析, 结果并未找到能够直接证明A: C6-A: C11二硫键连接方式的碎片离子, 如b2-y7等。通过进一步分析, 可以找到间接证明A: C7-B: C7的碎片, 如图 2A~D所示的4个碎片离子, 可以推断出A和B链的连接有2种方式A: C7-B: C7或者A: C6-B: C7; 如图 2E~H所示的4个碎片离子, A和B链的连接A: C7-B: C7或者A: C11-B: C7。综上, 间接推断出连接QCCTSICSLYQLE/FVNQHLCGSHLVE肽段A和B链的二硫键为A: C7-B: C7。以上碎片离子也说明ISD裂解引起了A: C6-A: C11链内二硫键的断裂, 即A: C6-A: C11的定位也可推断出。对门冬胰岛素、赖脯胰岛素和甘精胰岛素同法采集和处理, 皆可得到间接证明该复杂二硫键连接方式碎片离子组, 门冬胰岛素碎片离子为a4-2/b12 (1 740.771 Da, 偏差-3.45×10-6), b6-2/b8 (1 504.631 Da, 偏差-12.63×10-6), b6-2/a12 (1 940.871 Da, 偏差-11.33×10-6), b6-2/yMax (2 115.939 Da, 偏差-1.28×10-6), y11-2/b10 (2 380.054 Da, 偏差0.50×10-6), y11-2/b11 (2 493.155 Da, 偏差7.62×10-6), y11-2/y11 (2 493.155 Da, 偏差13.72×10-6), y11-2/yMax (2 739.308 Da, 偏差18.23×10-6)。赖脯胰岛素碎片离子为a3-2/b11 (1 540.691 Da, 偏差19.47×10-6), a5-2/a10 (1 587.673 Da, 偏差7.24×10-6), b5-2/y8 (1 377.553 Da, 偏差-5.44×10-6), b5-2/yMax (2 002.853 Da, 偏差2.10×10-6), y11-2/a12 (2 564.169 Da, 偏差-15.83×10-6), y11-2/y11 (2 493.084 Da, 偏差-14.84×10-6), y11-2/y11-NH3 (2 476.052 Da, 偏差-17.08×10-6), y11-NH3-2/y11 (2 476.052 Da, 偏差-17.08×10-6)。甘精胰岛素碎片离子为b5-2/b9 (1 506.563 Da, 偏差-19.71×10-6), b5-2/yMax (2 002.891 Da, 偏差16.73×10-6), b6-2/b11 (1 869.824 Da, 偏差1.98×10-6), b6-2/yMax (2 115.921 Da, 偏差9.45×10-6), y11-2/a12 (2 564.208 Da, 偏差-0.90×10-6), y11-2/y7 (2 000.842 Da, 偏差-17.59×10-6), y11-2/yMax (2 739.296 Da, 偏差13.87×10-6), y11-NH3-2/yMax (2 722.222 Da, 偏差-3.38×10-6) (注: 如2/y或2/b表示B链的y或b离子)。以上结果说明, 间接定位的离子组的第一组碎片离子需包含A: C6, A: C7和B: C7, 第二组碎片离子名称必须有A链的y11离子, B链需包含B: C7位。
3 酶切+部分还原烷基化法定位胰岛素的复杂二硫键结果
用GLU-C酶(主要酶切位点为E) 和糜蛋白酶(主要酶切位点为Y) 2种蛋白酶分别处理人胰岛素, 酶切后的肽段经TCEP·HCl部分还原和M-biotin烷基化, 随后经DTT完全还原和IAM烷基化。高分辨质谱对烷基化肽段进行分析, 通过半胱氨酸上M-biotin和IAM修饰的类型达到确认二硫键的目的, 分析流程图如图 3所示。GLU-C酶酶切+部分还原烷基化结果如表 1所示, QCCTSICSLYQLE和FVNQHLCGSHLVE是有烷基化的肽段, FVNQHLCGSHLVE中B: C7和QCCTSICSLYQLE中A: C7为TCEP还原的链间二硫键(部分还原条件下链间二硫键比链内二硫键更容易打开), 修饰位点为M-biotin, 可定位A: C7-B: C7; QCCTSICSLYQLE中A: C6和A: C11为DTT全部还原的链内二硫键, 烷基化为IAM, 即可证明A: C6-A: C11。同理, 采用酶切位点较为复杂的糜蛋白酶处理人胰岛素, 主要关注Y酶切位点, 得到的两条肽段GIVEQCCTSICSLY和FVNQHLCGSHLVEALY, 其修饰位点与GLU-C酶切时一致, 进一步证明了该两对复杂二硫键位置。如图 4A~D, 无论是GLU-C酶还是糜蛋白酶酶切加部分还原烷基化处理, 4条肽段的碎片离子丰富, 基本覆盖所有氨基酸, 且分子量偏差均在5×10-6之内, 结果可靠、准确。另外, 对门冬胰岛素、赖脯胰岛素和甘精胰岛素分别用GLU-C酶和糜蛋白酶同法处理, 得到的肽段与修饰位点与人胰岛素一致, 表明该方法适用性良好。
4 ISD与ETD裂解胰岛素完整蛋白结果
鉴于以上2种方法均需要酶切处理后再进行质谱采集, 本研究进一步尝试了对胰岛素分子整体碎裂的方法, 首先, 采用ISD-MS的方法, 寻找A: C20-B: C19定位的关键碎片离子, 如图 5A~C所示, y2-2/y18, y7-2/y13和y6-2/y12离子, 皆可以直接确定A: C20-B: C19二硫键; 在已知该二硫键前提下, 如图 5D~F所示, b6-1/y13-2/y13, b6-1/y13-2/y14和b6-1/y11-2/y23离子可以证明A: C6-A: C11二硫键位置, 则A: C7-B: C7的链间二硫键位置也可确定。另外, 选择前体母离子m/z 969.0 (6H+), 进一步尝试ETD-MS/MS的碎裂方式, 虽然产生的碎片离子不如ISD裂解丰富, 但是仍可以按照以上思路, 获得至少4对碎片离子, 确定3对二硫键位置。如图 6A、B所示, z′3-2/z′22+H和z′2-2/z′23离子, 皆定位A: C20-B: C19二硫键; 在已知该对二硫键前提下, 如图 6C~D, c′′6-1/z′11-2/z′16和c′′6-1/y11-2/z′21离子可以证明A: C6-A: C11二硫键位置, A: C7-B: C7的链间二硫键位置即可确定。本研究继续对门冬胰岛素、赖脯胰岛素和甘精胰岛素进行完整蛋白ISD和ETD碎裂采集分析, 结果如表 2和表 3所示, 门冬胰岛素、赖脯胰岛素完整分子裂解均有定位3对二硫键的关键碎片离子, 甘精胰岛素仅有A: C20-B: C19定位的关键碎片离子, 另外两对复杂二硫键定位离子未找到。虽然完整蛋白直接裂解的方法对甘精胰岛素具有一定局限性, 但是仍然可以为复杂二硫键的定位提供一定的思路。寻找A: C20-B: C19定位的关键碎片离子, 该类离子必须仅包含A: C20和B: C19, 之后确定另外两对二硫键位置, 推断A: C6-A: C11二硫键的关键碎片离子必须包含A链的C6和C11氨基酸, 但是不能包含A链C7和B链C7氨基酸。
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本研究对复杂闭环二硫键的定位采用了3种方法, 其中包含了利用化学试剂还原反应烷基化修饰定位的方法, 即酶切加部分还原烷基化法。该方法依赖还原剂和不同的烷基化试剂, DTT的还原能力较强, 对闭环二硫键来说, 无差别地全部还原, 无法定位这两对二硫键。选择TCEP作为还原剂, 它能够在酸性环境下还原[10], 进而抑制反应过程中二硫键错配的发生。在低浓度的TCEP (0.1~0.5 mmol·L-1) 下链间二硫键比链内二硫键更容易发生断裂, 可以将二硫键选择性的部分还原。针对烷基化封端的问题, 常用的烷基化试剂有碘乙酸(IAA)、碘乙酰胺(IAM) 和N‐乙基马来酰亚胺(NEM)[11]等。部分还原的目的在于对2对复杂二硫键打开后的巯基分别标记不同的烷基化试剂, 通过肽段不同的烷基化修饰确定二硫键正确的连接位置。部分研究表明TCEP在酸性环境中进行还原时可能也有较少部分二硫键交换反应(错配) 的发生, 而烷基化试剂(M-biotin) 的存在可以更加有效地减少这种现象的发生[12], 另外, 巯基和马来酰亚胺的加成反应快速高效[13], 所以本研究在TCEP部分还原后选择带有马来酰亚胺接头的PEG亲水性烷基化试剂M-biotin封端, 确定该复杂二硫键中的链间二硫键; 最后经DTT全部还原, 溶液pH调至碱性后, IAM烷基化, 实现另外1对二硫键的定位, 该方法对人胰岛素、门冬胰岛素、赖脯胰岛素和甘精胰岛素二硫键定位均适用。本研究了尝试ISD和ETD 2种断裂方式, 相对CID和HCD裂解来说, ISD和ETD裂解会同时发生二硫键和多肽骨架的断裂, 通过寻找关键碎片离子的方式定位二硫键, 无需进一步的前处理过程。ETD裂解是由电子亲和力足够低的负离子作为电子供体, 通过将电子转移给多肽离子从而触发氢自由基的释放。释放的氢自由基可以结合到多肽骨架的羧基基团, 导致肽链N-C α键发生断裂, 主要产生c和z离子[14-16], ETD裂解在对糖肽分析方面中已被报道卓有成效[17, 18]。研究发现ISD比ETD裂解产生更多的碎片离子, 可能因为ETD裂解较依赖肽段的位置、大小和电荷等因素[19], 而ISD是在离子从源向质谱仪真空室转移的过程中, ESI离子源所产生的离子是在一个相对高的压力区域产生的, 无需前体离子, 可以允许所有价态的离子发生断裂[20]。QCCTSICSLYQLE/FVNQHLCGSHLVE肽段解析过程中, ISD裂解引起了链内二硫键的断裂, 产生间接定位闭环二硫键的离子, 该方法对人胰岛素、门冬胰岛素、赖脯胰岛素和甘精胰岛素二硫键定位均适用, 而ETD裂解对该肽段进行分析时, 产生的碎片较少, 无法分析, 故在本文中未给出详细解析。研究也尝试了如不经过酶切还原烷基化等复杂的前处理过程, 直接对胰岛素完整蛋白进行ISD或ETD裂解, 考察其能否达到定位该两对复杂二硫键的目的。判断结果是通过先定位A: C20-B: C19, 后定位A: C6-A: C11去实现, 该方法对人胰岛素、门冬胰岛素和赖脯胰岛素具有良好的适用性, 而甘精胰岛素没有可定位的A: C6-A: C11, 可能因为甘精胰岛素的A链和B链序列或结构与人胰岛素、门冬胰岛素和赖脯胰岛素有一定差异, 使整体分子离子化效率较低。但是该方法也为胰岛素类二硫键定位和含复杂二硫键的多肽或者蛋白定位提供一定的思路, 在不经复杂前处理的过程中, 直接运用质谱的不同裂解方式的解析, 省时省力。综上3种方法, 酶切加部分还原烷基化法较其他两种方法能够更加直观、直接地评价胰岛素及其类似物的二硫键的位置。
随着重组类多肽或蛋白药物的推陈出新, 对其二硫键结构及相关质量控制方法也会越来越深入, 开发准确、快速的分析方法是极为重要。本文探讨的胰岛素类二硫键的定位方法为胰岛素及其类似物二硫键连接方式的质量控制提供了新途径, 也为含复杂二硫键多肽或蛋白类生物制品的二硫键定位提供借鉴。
致谢: Waters工程师何娟和ThermoFisher工程师龙珍在质谱数据采集和数据处理方面给予了专业意见和指导。
作者贡献: 胡馨月负责总体实验设计、实验操作、数据整理及文章撰写; 丁晓丽负责实验设计、数据处理及文章修改; 孙悦负责部分试验数据采集和分析; 张慧负责提出研究思路和解决实验问题; 李晶负责指导论文写作并对论文进行了修改; 梁成罡负责把握研究思路、指导论文写作和对论文进行检查。
利益冲突: 所有作者均声明不存在利益冲突。
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  • 中国食品药品检定研究院中青年发展研究基金课题(2022A3)
  • 国家重点研发计划课题(2021YFF0600804)
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doi: 10.16438/j.0513-4870.2023-0284
  • 接收时间：2023-03-09
  • 首发时间：2025-11-28
  • 出版时间：2024-01-12
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  • 收稿日期：2023-03-09
  • 修回日期：2023-07-31
基金
中国食品药品检定研究院中青年发展研究基金课题(2022A3)
国家重点研发计划课题(2021YFF0600804)
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    中国食品药品检定研究院激素室, 国家卫生健康委员会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化学药品质量研究与评价重点实验室, 北京 102629

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2种不同金属材料的力学参数
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Number of
species		 占总种数比例
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total species (%)		 属
Genus		 种数
Number of
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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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