CN105137088A - Whole-body-protein quantitative analysis method - Google Patents
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Abstract
本发明涉及一种整体蛋白质定量分析方法，首先将两组不同生理或病理条件的整体蛋白质分别作为控制组与疾病组，并分别对可能存在的二硫键进行还原，同时对巯基进行烷基化保护；然后对两组烷基化的蛋白质的氨基酸特定官能团进行全同重化学共价标记；同重标记后两组蛋白质按等比例混合进行高分辨质谱和串级质谱分析得到一个数据组；最后对数据组进行定性、定量数据库搜索，得到蛋白质的ID及疾病组每一个蛋白质相对于控制组的相对比例，即疾病条件下所有蛋白的上调或下调情况，上调或下调倍数最大的蛋白即为与该疾病发生、发展相关的蛋白。与现有技术相比，本发明方法标记效率高，准确度高，适用于整体蛋白质基于高分辨串级质谱的定量分析。
The invention relates to a method for quantitative analysis of whole protein. Firstly, two groups of whole protein with different physiological or pathological conditions are respectively used as control group and disease group, and the possible disulfide bonds are respectively reduced, and the sulfhydryl groups are alkylated at the same time. protection; then the amino acid specific functional groups of the two groups of alkylated proteins were chemically covalently labeled with the same weight; after the same weight labeling, the two groups of proteins were mixed in equal proportions and analyzed by high-resolution mass spectrometry and tandem mass spectrometry to obtain a data set; finally, the The data group is searched qualitatively and quantitatively to obtain the ID of the protein and the relative proportion of each protein in the disease group relative to the control group, that is, the up-regulation or down-regulation of all proteins under the disease condition. Proteins related to disease occurrence and development. Compared with the prior art, the method of the invention has high labeling efficiency and high accuracy, and is suitable for quantitative analysis of whole proteins based on high-resolution tandem mass spectrometry.
Description
技术领域technical field
本发明涉及一种蛋白质分子的分析方法，尤其是涉及一种整体蛋白质定量分析方法，主要涉及与生物质谱相关的系统生物学、蛋白质组学等技术领域。The invention relates to a method for analyzing protein molecules, in particular to a method for quantitatively analyzing whole proteins, and mainly relates to the technical fields of systems biology and proteomics related to biological mass spectrometry.
背景技术Background technique
近两三年来，商业化的高质量分辨、高质量测量精度质谱仪有了飞跃式发展，也就是轨道阱质谱仪的出现。轨道阱质谱仪跟傅里叶变换离子回旋共振质谱仪分辨率和精度相当；但价格相对便宜，质谱采集速度更快、解离效率更高。该质谱仪的相对普及为分子量相对较大的整体蛋白的质谱及串级质谱分析提供了坚实的基础。在整体蛋白定量标记方面，基于SILAC的体内标记和基于TMT的体外标记，由于靶向氨基酸的非完全标记(如重的赖氨酸或精氨酸在SILAC中的非完全替代)和非靶向氨基酸的非选择性标记(如苏氨酸的TMT标记)，其应用范围受到了比较大的限制。二甲基(同位素，isotopic或同重,isobaric)标记由于其试剂易得，标记效率高，在多肽定量方面得到了广泛的研究和应用；我国科学家在这个定量技术方面也做出了巨大的贡献，发展了多种不同形式的二甲基标记；其中中国科学院大连化学物理研究所张玉奎院士和张丽华研究员课题组发展的N端同重二甲基标记和复旦大学杨芃原教授和陆豪杰教授课题组发展的赖氨酸ε-氨基的胍化保护组合起来可直接应用于蛋白的定量标记，有较好的前景。In the past two or three years, commercial mass spectrometers with high-quality resolution and high-quality measurement accuracy have developed by leaps and bounds, that is, the emergence of orbitrap mass spectrometers. Orbitrap mass spectrometers have the same resolution and accuracy as Fourier transform ion cyclotron resonance mass spectrometers; but they are relatively cheap, with faster mass spectrometry acquisition and higher dissociation efficiency. The relative popularity of this mass spectrometer provides a solid foundation for the mass spectrometry and tandem mass spectrometry analysis of the overall protein with a relatively large molecular weight. In terms of overall protein quantitative labeling, SILAC-based in vivo labeling and TMT-based in vitro labeling, due to incomplete labeling of targeted amino acids (such as incomplete substitution of heavy lysine or arginine in SILAC) and non-targeted Non-selective labeling of amino acids (such as TMT labeling of threonine) has relatively limited application range. Dimethyl (isotopic or isobaric) labeling has been widely studied and applied in peptide quantification due to its easy availability of reagents and high labeling efficiency; Chinese scientists have also made great contributions to this quantitative technology , developed a variety of different forms of dimethyl marks; among them, the N-terminal isobaric dimethyl marks developed by the research group of Academician Zhang Yukui and Researcher Zhang Lihua of Dalian Institute of Chemical Physics, Chinese Academy of Sciences and the research group of Professor Yang Pengyuan and Professor Lu Haojie of Fudan University The combination of the guanidine protection of the ε-amino group of lysine can be directly applied to the quantitative labeling of proteins, which has a good prospect.
在整体蛋白质数据库新算法和引擎发展方面，中国专利CN103389335A、CN104359967A、CN104765984A公布了同位素轮廓指纹比对算法(isotopicenvelopefingerprinting,iEF)，直接在原始数据上进行匹配离子的搜索和蛋白质的鉴定。该算法无需对实验数据进行“去同位素”预处理，可以节省相应的时间；根据各个离子中是否有同位素峰缺失和同位素峰相对强度的偏差对理想及非理想实验数据进行很好的区分，保证蛋白鉴定的可信度；根据已知重叠离子的同位素峰相对强度关系对重叠实验数据进行有效的解析。基于该算法申请人成功地开发了整体蛋白数据库搜索引擎ProteinGoggle，在单个标准蛋白(泛素、肌红蛋白)及整体蛋白质组混合物(组蛋白H4家族翻译后修饰异构体、大肠杆菌)的定性鉴定中，表现出较高的可信度和较好的应用前景。在定量分析方面，ProteinGoggle有着它内在的和独特的潜在优势；一方面基于同位素轮廓及其中每个同位素的指纹比对可以快速精确地搜索同位素或同重标记的定量离子对；另一方面，搜索过程中每个离子中每个同位素峰的实验强度都被记录和输出，可以用来直接计算相对定量比。In terms of the new algorithm and engine development of the overall protein database, Chinese patents CN103389335A, CN104359967A, and CN104765984A have announced the isotopic contour fingerprinting algorithm (isotopic envelope fingerprinting, iEF), which directly searches for matching ions and identifies proteins on the original data. This algorithm does not need to perform "de-isotope" preprocessing on the experimental data, which can save corresponding time; according to whether there is a lack of isotope peak in each ion and the deviation of the relative intensity of the isotope peak, the ideal and non-ideal experimental data can be well distinguished to ensure The reliability of protein identification; effective analysis of overlapping experimental data based on the relative intensity relationship of isotopic peaks of known overlapping ions. Based on this algorithm, the applicant successfully developed the overall protein database search engine ProteinGoggle, in the qualitative analysis of single standard proteins (ubiquitin, myoglobin) and overall proteome mixtures (post-translational modification isoforms of histone H4 family, Escherichia coli) In identification, it shows high reliability and good application prospect. In terms of quantitative analysis, ProteinGoggle has its inherent and unique potential advantages; on the one hand, based on the isotope profile and the fingerprint comparison of each isotope, it can quickly and accurately search for isotopic or isobaric-labeled quantitative transitions; on the other hand, the search The experimental intensity of each isotope peak in each ion is recorded and output during the process, which can be used to directly calculate the relative quantitative ratio.
发明内容Contents of the invention
本发明的目的就是为了克服上述现有技术存在的缺陷而提供一种标记效率高、无需预先对部分官能团进行保护、准确度高的整体蛋白质定量方法。The purpose of the present invention is to overcome the defects in the prior art and provide an overall protein quantification method with high labeling efficiency, no need to protect some functional groups in advance, and high accuracy.
本发明的目的可以通过以下技术方案来实现：The purpose of the present invention can be achieved through the following technical solutions:
一种整体蛋白质定量分析方法，包括以下步骤：A method for overall protein quantitative analysis, comprising the following steps:
(1)将两组不同生理或病理条件的整体蛋白质分别作为控制组与疾病组，并分别对可能存在的二硫键进行还原，同时对巯基进行烷基化保护；(1) Two groups of whole proteins with different physiological or pathological conditions were used as the control group and the disease group respectively, and the possible disulfide bonds were respectively reduced, and the sulfhydryl group was protected by alkylation;
(2)对两组烷基化的蛋白质的氨基酸特定官能团进行全同重化学共价标记；(2) Carry out identical heavy chemical covalent labeling to the amino acid specific functional groups of two groups of alkylated proteins;
(3)同重标记后两组蛋白质按等比例混合进行高分辨质谱和串级质谱分析得到一个数据组；(3) After isobaric labeling, the two groups of proteins are mixed in equal proportions for high-resolution mass spectrometry and tandem mass spectrometry analysis to obtain a data set;
(4)对数据组进行定性、定量数据库搜索，得到蛋白质的ID及疾病组每一个蛋白质相对于控制组的相对比例，即疾病条件下所有蛋白的上调或下调情况，上调或下调倍数最大的蛋白即为与该疾病发生、发展相关的蛋白。(4) Perform a qualitative and quantitative database search on the data set to obtain the ID of the protein and the relative proportion of each protein in the disease group relative to the control group, that is, the up-regulation or down-regulation of all proteins under disease conditions, and the protein with the largest up- or down-regulation fold It is the protein related to the occurrence and development of the disease.
优选地，步骤(1)中用二硫苏糖醇对可能存在的二硫键进行还原，用碘乙酰胺对所有巯基进行烷基化保护。Preferably, dithiothreitol is used in step (1) to reduce possible disulfide bonds, and iodoacetamide is used to protect all sulfhydryl groups by alkylation.
优选地，步骤(2)中氨基酸特定官能团包括赖氨酸ε-氨基及N端氨基，同时该方法同样适用于蛋白质序列中赖氨酸ε-氨基及N端氨基除外的其他特定官能团的全同重化学共价标记。Preferably, the amino acid specific functional group in step (2) includes lysine ε-amino group and N-terminal amino group, and this method is also applicable to the identity of other specific functional groups except lysine ε-amino group and N-terminal amino group in the protein sequence. Heavy chemical covalent labeling.
优选地，步骤(2)中，一组蛋白质中的氨基酸特定官能团标记为-N(CH2D)2，另一组蛋白质中的氨基酸特定官能团标记为-N(13CH3)2，单个标记后氨基的质量差异为0.00584Da。同样的，本发明方法同样适用于蛋白质序列中赖氨酸ε-氨基及N端氨基其他全同重化学共价标记。Preferably, in step (2), the amino acid specific functional group in one group of proteins is marked as -N(CH 2 D) 2 , the amino acid specific functional group in another group of proteins is marked as -N( 13 CH 3 ) 2 , and the single label The mass difference of the post amino group is 0.00584Da. Similarly, the method of the present invention is also applicable to other identical heavy chemical covalent labeling of lysine ε-amino group and N-terminal amino group in the protein sequence.
对于标记为-N(CH2D)2和-N(13CH3)2的方法而言，一组蛋白质用甲醛和氘代氰基硼氢化钠标记，另一组蛋白质用13C标记甲醛和氰基硼氢化钠标记；反应过程和条件如下：烷基化的蛋白质重新溶解在乙酸钠缓冲溶液后，加入4％(v/v)甲醛溶液；在搅拌条件下加入600mM的新配制的氰基硼氢化钠溶液，室温反应1小时后，用4％(v/v)的氨水淬灭。其中，所述的乙酸钠缓冲溶液为100mmol/L，pH为5-6。For the methods labeled -N(CH 2 D) 2 and -N( 13 CH 3 ) 2 , one set of proteins was labeled with formaldehyde and sodium deuterocyanoborohydride, and another set of proteins was labeled with 13 C formaldehyde and Labeling with sodium cyanoborohydride; the reaction procedure and conditions are as follows: after the alkylated protein was redissolved in sodium acetate buffer solution, 4% (v/v) formaldehyde solution was added; 600 mM freshly prepared cyano Sodium borohydride solution, after reacting at room temperature for 1 hour, quenched with 4% (v/v) ammonia water. Wherein, the sodium acetate buffer solution is 100mmol/L, and the pH is 5-6.
步骤(3)所述的等比例混合指：按照控制组与疾病组的重量、体积或摩尔量以1:1比例进行混合。The equal proportion mixing described in step (3) refers to mixing in a ratio of 1:1 according to the weight, volume or molar weight of the control group and the disease group.
步骤(4)中对数据组进行定性、定量数据库搜索，得到蛋白质的ID及疾病组每一个蛋白质相对于控制组的相对比例为本领域的常规技术，优选使用背景技术中介绍的整体蛋白数据库搜索引擎ProteinGoggle，同时也可以使用其他数据库。In step (4), it is a routine technique in the art to perform qualitative and quantitative database searches on the data set to obtain the ID of the protein and the relative ratio of each protein in the disease group relative to the control group, preferably using the overall protein database search introduced in the background technology Engine ProteinGoggle, but other databases can also be used.
与现有技术相比，本发明具有以下优点及有益效果：Compared with the prior art, the present invention has the following advantages and beneficial effects:
1、本发明的解析方法基于所述质谱的原始二级质谱，通过计算含标记基团碎片离子的平均相对强度从而计算标记蛋白在不同生理或病理条件下的相对强度。本发明的定量方法对整体蛋白质序列中赖氨酸ε-氨基及N端氨基的同重标记和其高分辨串级质谱标记碎片离子同位素轮廓指纹比对原位数据解析，标记效率高，准确度高，适用于整体蛋白质基于高分辨串级质谱的定量分析。1. The analysis method of the present invention is based on the original MS/MS spectrum of the mass spectrum, and calculates the relative intensity of the marker protein under different physiological or pathological conditions by calculating the average relative intensity of the fragment ions containing the marker group. The quantitative method of the present invention analyzes the isobaric labeling of lysine ε-amino group and N-terminal amino group in the overall protein sequence and its high-resolution tandem mass spectrometry labeling fragment ion isotope profile fingerprint comparison in situ data analysis, with high labeling efficiency and accuracy High, suitable for quantitative analysis of whole protein based on high-resolution tandem mass spectrometry.
2、本发明方法仅需对蛋白质进行二硫键还原，和对巯基进行烷基化保护，然后进行全同重化学共价标记即可，不需要对蛋白质序列中赖氨酸残基上的ε-氨基进行胍化保护，方法简单快捷，同时标记效率高、副反应少、准确度高。2. The method of the present invention only needs to reduce the disulfide bond of the protein, carry out alkylation protection on the sulfhydryl group, and then carry out isotropic heavy chemical covalent labeling, and does not need to carry out the ε- The amino group is protected by guanidinization, the method is simple and fast, and at the same time, it has high labeling efficiency, less side reactions and high accuracy.
附图说明Description of drawings
图1为基于蛋白质赖氨酸ε-氨基及N端氨基同重二甲基标记的定量流程图。Figure 1 is a quantitative flow chart based on isobaric dimethyl labeling of protein lysine ε-amino group and N-terminal amino group.
图2为蛋白质分子赖氨酸ε-氨基和N端氨基全同重二甲基标记示意图。Fig. 2 is a schematic diagram of isobaric dimethyl labeling of lysine ε-amino group and N-terminal amino group of protein molecules.
具体实施方式Detailed ways
下面结合附图和具体实施例对本发明进行详细说明。The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments.
实施例Example
一种整体蛋白质定量分析方法，采用如图1所示的整体流程，包括以下步骤：A method for quantitative analysis of overall protein, using the overall process shown in Figure 1, comprising the following steps:
(1)两种不同生理或病理条件的整体蛋白(以控制组与疾病组为例)先统一用二硫苏糖醇对可能存在的二硫键进行还原；然后用碘乙酰胺对所有巯基进行烷基化保护；(1) Two whole proteins with different physiological or pathological conditions (take the control group and the disease group as an example) first use dithiothreitol to reduce the possible disulfide bonds; then use iodoacetamide to reduce all sulfhydryl groups. Alkylation protection;
(2)烷基化后的两组蛋白质分别用甲醛和氘代氰基硼氢化钠、13C标记甲醛和氰基硼氢化钠对蛋白分子中的赖氨酸ε-氨基及N端氨基进行全部同重标记(如图2所示)；也就是分别标记为-N(CH2D)2和-N(13CH3)2，其单个标记后氨基的质量差异为0.00584Da。反应过程和条件如下：烷基化后的蛋白重新溶解在乙酸钠缓冲溶液(100mM，pH5-6)后，加入刚刚配制的4％甲醛溶液；在搅拌条件下加入600mM的新配制的氰基硼氢化钠溶液。室温反应1小时后，反应用4％的氨水淬灭；(2) After alkylation, the two groups of proteins were labeled with formaldehyde and deuterated sodium cyanoborohydride, and 13 C-labeled formaldehyde and sodium cyanoborohydride, respectively, to completely remove the lysine ε-amino group and the N-terminal amino group in the protein molecule. Isobaric labeling (as shown in Figure 2); that is, labeling as -N(CH 2 D) 2 and -N( 13 CH 3 ) 2 respectively, the mass difference of the amino group after a single labeling is 0.00584Da. The reaction process and conditions are as follows: after the alkylated protein is redissolved in sodium acetate buffer solution (100mM, pH5-6), add the freshly prepared 4% formaldehyde solution; add 600mM newly prepared cyanoboron under stirring condition Sodium hydride solution. After reacting at room temperature for 1 hour, the reaction was quenched with 4% ammonia;
(3)同重标记后两组蛋白按等比例混合(按照控制组与疾病组的重量、体积或摩尔量以1:1比例进行混合)进行高分辨质谱和串级质谱分析得到一个数据组；(3) After isobaric labeling, the two groups of proteins are mixed in equal proportions (mixed according to the weight, volume or molar ratio of the control group and the disease group at a ratio of 1:1) for high-resolution mass spectrometry and tandem mass spectrometry analysis to obtain a data set;
(4)ProteinGoggle对数据组进行定性、定量数据库搜索，得到蛋白质的ID及疾病组每一个蛋白质相对于控制组的相对比例，即疾病条件下所有蛋白的上调或下调情况。上调或下调倍数最大的蛋白即与该疾病发生、发展相关的蛋白。(4) ProteinGoggle conducts qualitative and quantitative database searches on the data set to obtain the ID of the protein and the relative proportion of each protein in the disease group relative to the control group, that is, the up-regulation or down-regulation of all proteins under disease conditions. The protein with the largest up-regulation or down-regulation fold is the protein related to the occurrence and development of the disease.
上述解析方法基于所述质谱的原始二级质谱，通过计算含标记基团碎片离子的平均相对强度从而计算标记蛋白在不同生理或病理条件下的相对强度。该定量方法对整体蛋白质序列中赖氨酸ε-氨基及N端氨基的同重标记和其高分辨串级质谱标记碎片离子同位素轮廓指纹比对原位数据解析，标记效率高，准确度高，适用于整体蛋白质基于高分辨串级质谱的定量分析。The above analysis method is based on the original MS/MS spectrum of the mass spectrum, and calculates the relative intensity of the marker protein under different physiological or pathological conditions by calculating the average relative intensity of the fragment ions containing the marker group. This quantitative method analyzes the isobaric labeling of lysine ε-amino group and N-terminal amino group in the overall protein sequence and its high-resolution tandem mass spectrometry-labeled fragment ion isotope profile fingerprint comparison in situ data analysis, with high labeling efficiency and high accuracy. It is suitable for quantitative analysis of whole protein based on high-resolution tandem mass spectrometry.
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改，并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此，本发明不限于上述实施例，本领域技术人员根据本发明的揭示，不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。The above descriptions of the embodiments are for those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments, and apply the general principles described here to other embodiments without creative efforts. Therefore, the present invention is not limited to the above-mentioned embodiments. Improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.
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