生物质谱技术和方法在蛋白类药物序列鉴定中的应用研究
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摘要
在生物仿制药出现以后,为保证它与原研产品功能的一致性,保证它的质量、安全和效能,首先需要对它的结构进行表征,包括完整蛋白分子量测定,氨基酸序列鉴定,各类修饰,如二硫键、糖链的鉴定等。本论文的研究主要集中在使用生物质谱技术对两种蛋白类药物分子,重组人生长激素和重组卵泡刺激素进行表征,对完整蛋白分子量,氨基酸序列,各类修饰,特别是糖链序列进行测定和解析。
本论文第二章的工作是对蛋白药PEG(聚乙二醇)修饰的重组人生长激素进行氨基酸的序列鉴定。还原烷基化实验后,使用UPLC-MS/MS仪器在正离子模式下进行液质联用在线分析,使用Uniprot数据库进行匹配搜索,鉴定到198条肽段,我们对原始数据和搜库结果进行了深入分析和总结,经过人工再次评估,结合一级质谱谱图,鉴定到的肽段达到204条,序列覆盖率几乎达到百分百,还发现了除胰蛋白酶酶解肽段外非正常胰蛋白酶酶解肽段的氨基酸序列,鉴定到诸如氨基酸替代,氧化、脱水、脱氨基等大量重要的化学修饰信息。这些信息对于蛋白类药物分子的氨基酸序列鉴定非常有意义,这对于鉴定肽段信息,准确鉴定蛋白分子结构,对鉴别其结构与功能方面的关系等提供了重要的信息。
论文第三章涉及的是对N-糖蛋白完整分子量的测定以及对从该蛋白PNGase F酶解分离得到的N-糖链的序列解析。对N-糖蛋白完整分子量的测定,得到了两个亚基的分子量,并且得知其中含有唾液酸和六元糖的糖单元。由于唾液酸的存在,在对N-糖糖链进行液质联用在线解析时,采用负离子模式,结合总离子流图(TIC)和提取离子流图(XIC)及MS和MS/MS谱图,在Analyst软件上进行数据分析,找到了糖链碎裂的分子离子峰,推断出可能的糖链序列组成。
此外,我们试图建立一种标准化的流程方法来解析糖蛋白中的糖链序列,由于还原端的衍生化可以简化谱图,特别是碎片离子的质量谱图,提高离子化效率,我们尝试用2-AB(2-氨基苯甲酰胺)这种荧光试剂进行糖链的衍生化。在前期对标记方法的尝试和摸索阶段,先使用六个葡萄单糖聚合的简单结构的麦芽六糖(Maltohexaose),标记反应完成后分离纯化除去过量的标记试剂,浓缩后进行质谱分析。首先使用MALDI-TOFTOF高分辨质谱,找到分子量增加120.06 Da的标记分子的加钠离子峰,然后使用UPLC-ESI-MS/MS液质联用在线检测,糖链标记前后1：1进样,观测到标记后麦芽六糖的质子峰信号强度明显增强,说明对于中性糖的标记是很有效果的,结合串级质谱谱图,进一步确认了这个糖链结构,然后把该方法应用到实际N-糖蛋白的糖链上,也取得了一定的效果。
应用质谱技术对蛋白类药物的表征,包括完整蛋白分子量分析,对氨基酸的序列分析,对各类修饰,特别是N-糖糖链的解析,有助于对蛋白类药物的结构鉴定,帮助蛋白仿制药和原研产品进行比较性研究,能更真实地反应出两者之间的相似与不同,对蛋白仿制药物分子的鉴定也更具体,有针对性,更真实可靠。
After the biological medicine "Biosimilars", their molecular structures need to be characterized in order to make sure the similarity with the original products, and ensure the similar quality, accuracy and efficacy. It is necessary to determine the molecular mass of intact protein, to identify the sequence of amino acids and modifications such as disulfide bond, glycosylation, etc. The research work of this master thesis mainly focuses on the characterization of two kinds of biological medicine with biomass spectrometry, mainly for the determination of intact protein molecular mass and the determination of amino acid sequence as well as its modifications especially with N-glycan chains.
The protein used in the second chapter is Recombinant Human Growth Hormone(rhGH) with PEGylation. After the reduction and alkylation response, the sample was analyzed by UPLC-ESI-MS-MS at positive ion mode, which yielded an identification of total 198 peptides from Uniprot database. Then the majority of original raw data from the search results are analyzed and summarized, through such manual evaluation together with the MS spectra, the peptides identified are up to 204, and the sequence coverage is close to 100%. The peptides, abnormally digested by trypsin, are discovered except the normal. Also, the substitution of amino acid and few chemical modifications such as oxidation, deamidated and dehydrated, are observed. Such information plays a very important pole in the characterization of the amino acid sequence of biological drugs. Moreover, it helps to characterize the peptide, the structure of protein, even the related function.
The work in the third chapter mainly focuses on the determination of intact N-glycoprotein molecular mass and the analysis of the sequence of N-glycan from the N-glycoprotein. The N-glycoprotein here is Recombinantfollicle stimulating hormone (FSH). The mass values of the two subunits are determined, and the sialic acid is also detected. The sample is unknown N-glycan chain form a drug company. Because of the existence of sialic acid, the UPLC-ESI-MS-MS experiment is done at the negative ion mode. Combined the TIC spectrum, XIC spectra, retention time, MS spectra and MS/MS spectra with the help of software "Analyst", the peaks of glycan fragments are identified. At last the glycanform are obtained successfully through the analysis and calculation on the data.
Later, the study was focused to establish a standard method to analyze the sequence of N-glycans, considering that the reducing end derivatization can help to decrease the complexity of mass spectra, especially the fragment ion mass spectra, the work adopt 2-AB(2-amino-benzamideg) as labeling. At the first step, maltohexaose polymerized by six glucoses are used in the experiment to verify the method. When the derivatization process is done, the labeled glycans need to be separated from too much labeling with the SPE cartridge. On the MALDI-TOF-TOF-MS, the increased mass 120.06 Da due to the derivatization can be easily found. Then the samples with 1:1 before and after labeling are subjected to UPLC-ESI-MS-MS, the signal of the peak with H+ can be detected with an obvious improvement, proving the effecitiveness of the method, through the structural confirmation with MS/MS spectra. Then at the second step, we apply the method to the complex N-glcans mixture.
The characterization of the biological medicine, including the determination of molecular mass of intact protein, and the analysis of the sequence of amino acids as well as modifications, especially with N-linked glycans, provides a possibility to identify the structure of biological drugs, especially when compared to the reference, with the precision and reliability of the results.
本论文第二章的工作是对蛋白药PEG(聚乙二醇)修饰的重组人生长激素进行氨基酸的序列鉴定。还原烷基化实验后,使用UPLC-MS/MS仪器在正离子模式下进行液质联用在线分析,使用Uniprot数据库进行匹配搜索,鉴定到198条肽段,我们对原始数据和搜库结果进行了深入分析和总结,经过人工再次评估,结合一级质谱谱图,鉴定到的肽段达到204条,序列覆盖率几乎达到百分百,还发现了除胰蛋白酶酶解肽段外非正常胰蛋白酶酶解肽段的氨基酸序列,鉴定到诸如氨基酸替代,氧化、脱水、脱氨基等大量重要的化学修饰信息。这些信息对于蛋白类药物分子的氨基酸序列鉴定非常有意义,这对于鉴定肽段信息,准确鉴定蛋白分子结构,对鉴别其结构与功能方面的关系等提供了重要的信息。
论文第三章涉及的是对N-糖蛋白完整分子量的测定以及对从该蛋白PNGase F酶解分离得到的N-糖链的序列解析。对N-糖蛋白完整分子量的测定,得到了两个亚基的分子量,并且得知其中含有唾液酸和六元糖的糖单元。由于唾液酸的存在,在对N-糖糖链进行液质联用在线解析时,采用负离子模式,结合总离子流图(TIC)和提取离子流图(XIC)及MS和MS/MS谱图,在Analyst软件上进行数据分析,找到了糖链碎裂的分子离子峰,推断出可能的糖链序列组成。
此外,我们试图建立一种标准化的流程方法来解析糖蛋白中的糖链序列,由于还原端的衍生化可以简化谱图,特别是碎片离子的质量谱图,提高离子化效率,我们尝试用2-AB(2-氨基苯甲酰胺)这种荧光试剂进行糖链的衍生化。在前期对标记方法的尝试和摸索阶段,先使用六个葡萄单糖聚合的简单结构的麦芽六糖(Maltohexaose),标记反应完成后分离纯化除去过量的标记试剂,浓缩后进行质谱分析。首先使用MALDI-TOFTOF高分辨质谱,找到分子量增加120.06 Da的标记分子的加钠离子峰,然后使用UPLC-ESI-MS/MS液质联用在线检测,糖链标记前后1：1进样,观测到标记后麦芽六糖的质子峰信号强度明显增强,说明对于中性糖的标记是很有效果的,结合串级质谱谱图,进一步确认了这个糖链结构,然后把该方法应用到实际N-糖蛋白的糖链上,也取得了一定的效果。
应用质谱技术对蛋白类药物的表征,包括完整蛋白分子量分析,对氨基酸的序列分析,对各类修饰,特别是N-糖糖链的解析,有助于对蛋白类药物的结构鉴定,帮助蛋白仿制药和原研产品进行比较性研究,能更真实地反应出两者之间的相似与不同,对蛋白仿制药物分子的鉴定也更具体,有针对性,更真实可靠。
After the biological medicine "Biosimilars", their molecular structures need to be characterized in order to make sure the similarity with the original products, and ensure the similar quality, accuracy and efficacy. It is necessary to determine the molecular mass of intact protein, to identify the sequence of amino acids and modifications such as disulfide bond, glycosylation, etc. The research work of this master thesis mainly focuses on the characterization of two kinds of biological medicine with biomass spectrometry, mainly for the determination of intact protein molecular mass and the determination of amino acid sequence as well as its modifications especially with N-glycan chains.
The protein used in the second chapter is Recombinant Human Growth Hormone(rhGH) with PEGylation. After the reduction and alkylation response, the sample was analyzed by UPLC-ESI-MS-MS at positive ion mode, which yielded an identification of total 198 peptides from Uniprot database. Then the majority of original raw data from the search results are analyzed and summarized, through such manual evaluation together with the MS spectra, the peptides identified are up to 204, and the sequence coverage is close to 100%. The peptides, abnormally digested by trypsin, are discovered except the normal. Also, the substitution of amino acid and few chemical modifications such as oxidation, deamidated and dehydrated, are observed. Such information plays a very important pole in the characterization of the amino acid sequence of biological drugs. Moreover, it helps to characterize the peptide, the structure of protein, even the related function.
The work in the third chapter mainly focuses on the determination of intact N-glycoprotein molecular mass and the analysis of the sequence of N-glycan from the N-glycoprotein. The N-glycoprotein here is Recombinantfollicle stimulating hormone (FSH). The mass values of the two subunits are determined, and the sialic acid is also detected. The sample is unknown N-glycan chain form a drug company. Because of the existence of sialic acid, the UPLC-ESI-MS-MS experiment is done at the negative ion mode. Combined the TIC spectrum, XIC spectra, retention time, MS spectra and MS/MS spectra with the help of software "Analyst", the peaks of glycan fragments are identified. At last the glycanform are obtained successfully through the analysis and calculation on the data.
Later, the study was focused to establish a standard method to analyze the sequence of N-glycans, considering that the reducing end derivatization can help to decrease the complexity of mass spectra, especially the fragment ion mass spectra, the work adopt 2-AB(2-amino-benzamideg) as labeling. At the first step, maltohexaose polymerized by six glucoses are used in the experiment to verify the method. When the derivatization process is done, the labeled glycans need to be separated from too much labeling with the SPE cartridge. On the MALDI-TOF-TOF-MS, the increased mass 120.06 Da due to the derivatization can be easily found. Then the samples with 1:1 before and after labeling are subjected to UPLC-ESI-MS-MS, the signal of the peak with H+ can be detected with an obvious improvement, proving the effecitiveness of the method, through the structural confirmation with MS/MS spectra. Then at the second step, we apply the method to the complex N-glcans mixture.
The characterization of the biological medicine, including the determination of molecular mass of intact protein, and the analysis of the sequence of amino acids as well as modifications, especially with N-linked glycans, provides a possibility to identify the structure of biological drugs, especially when compared to the reference, with the precision and reliability of the results.
引文
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