CN107266536A - 一种用于质谱检测的蛋白封闭剂及其应用 - Google Patents
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Abstract
本发明涉及质谱检测领域，特别涉及一种用于质谱检测的蛋白封闭剂及其应用。本发明提供的用于质谱检测的封闭蛋白，包括所述封闭蛋白的氨基酸如SEQ ID.No1所示，所述SEQ ID.No1中X为自然界存在的氨基酸，且不属于精氨酸和赖氨酸。本发明所提供的一种用于质谱检测的封闭蛋白在封闭质谱检测环境中的非特异性的蛋白质结合位点的应用。本发明还提供了一种用于质谱检测的封闭蛋白的制备方法。本发明提供用于质谱检测的封闭蛋白和用于质谱检测的蛋白封闭剂，不仅能有效封闭质谱检测环境中的非特异性的蛋白质结合位点，而且还能不被蛋白水解酶水解，大大避免了质谱检测的污染。
Description
技术领域
本发明涉及质谱检测领域，特别涉及一种用于质谱检测的蛋白封闭剂及其应用。
背景技术
蛋白质谱技术就是一种将质谱仪用于研究蛋白质的技术。它的基本原理是蛋白质经过蛋白酶的酶切消化后成肽段混合物，在质谱仪中肽段混合物电离形成带电离子，质谱分析器的电场、磁场将具有特定质量与电荷比值(即质荷比，M/Z)的肽段离子分离开来，经过检测器收集分离的离子，确定每个离子的M/Z值。经过质量分析器可分析出每个肽段的M/Z，得到蛋白质所有肽段的M/Z图谱，即蛋白质的一级质谱峰图。离子选择装置自动选取强度较大肽段离子进行二级质谱分析，输出选取肽段的二级质谱峰图，通过和理论上蛋白质经过胰蛋白酶消化后产生的一级质谱峰图和二级质谱峰图进行比对而鉴定蛋白质。
然而，由于质谱操作中的容器和移液器吸头等对蛋白质具有非特异吸附作用，会把质谱的目的蛋白吸附在容器和移液器吸头的表面，从生物体中提取微量蛋白样本，往往会有很大的损失。为了尽可能减少蛋白提取的损失，常将容器，吸管，移液器吸头等进行封闭的处理。目前常用的封闭剂为天然易得的蛋白质，这类蛋白能很好的封闭掉非特异吸附位点，从而大大减少目的蛋白的损失。但是，由于这类蛋白封闭剂始终添加在样本提取纯化过程中，假如提取的蛋白用于后续的质谱检测，那么会造成很大的污染。
发明内容
有鉴于此，本发明提供一种用于质谱检测的蛋白封闭剂，不仅能有效封闭质谱检测环境中的非特异性的蛋白质结合位点，而且还能不被蛋白水解酶水解，大大避免了质谱检测的污染。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种用于质谱检测的封闭蛋白，所述封闭蛋白的氨基酸如SEQID.No1所示，所述SEQ ID.No1中X为自然界存在的氨基酸，且不属于精氨酸和赖氨酸。
作为优选，所述SEQ ID.No1中X为组氨酸。
本发明所提供的一种用于质谱检测的封闭蛋白在封闭质谱检测环境中的非特异性的蛋白质结合位点的应用。
作为优选，所述的封闭蛋白不被蛋白水解酶水解。
作为优选，所述的蛋白水解酶为胰酶。
本发明还提供了一种用于质谱检测的封闭蛋白的制备方法，包括：
1)构建原核表达载体，所述原核表达载体包括SEQ ID.No1，得到重组封闭蛋白原核表达载体，使得重组封闭蛋白原核表达载体表达所述封闭蛋白；
2)将所述重组封闭蛋白原核表达载体整合到原核生物中，使得所述原核生物表达所述封闭蛋白，收集获得所述的封闭蛋白。
作为优选，所述原核表达载体含有组氨酸标签。
本发明还提供了一种用于质谱检测的蛋白封闭剂，包括用于质谱检测的封闭蛋白和缓冲液。
作为优选，所述缓冲液为PBS溶液或Nacl溶液。
本发明还提供了一种用于质谱检测的试剂盒，包括：用于质谱检测的蛋白封闭剂和质谱检测试剂。
有鉴于此，本发明所要解决的技术问题在于提供了一种封闭蛋白，该封闭蛋白能把质谱检测环境中的非特异性的蛋白质结合位点封闭起来，使得检测的目的蛋白不会吸附在容器、吸管、移液器吸头的表面，减少了目的蛋白的损失；而且，该封闭蛋白不含精氨酸和赖氨酸，质谱检测的蛋白水解酶不会水解该封闭蛋白，使得质谱检测时蛋白水解酶不会水解封闭蛋白，不会产生目的蛋白以外的多肽片段，显著增大了质谱检测的准确性。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示本发明的封闭蛋白与牛血清白蛋白酶解前后的SDS-PAGE图谱；
其中，A为牛血清白蛋白溶液的蛋白条带；B为胰酶水解后的牛血清白蛋白溶液的蛋白条带；C为封闭蛋白溶液的蛋白条带；D为胰酶水解后的封闭蛋白的蛋白条带。
具体实施方式
本发明公开了本发明提供一种用于质谱检测的蛋白封闭剂，不仅能有效封闭质谱检测环境中的非特异性的蛋白质结合位点，而且还能不被蛋白水解酶水解，大大避免了质谱检测的污染，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的一种用于质谱检测的蛋白封闭剂，其中所用原料及试剂均可由市场购得。
下面结合实施例，进一步阐述本发明：
实施例1
用于质谱检测的封闭蛋白的制备，具体方法如下：
根据SEQ ID No.1的蛋白质序列人工合成对应的封闭蛋白DNA序列。将封闭蛋白DNA序列重组到商业的原核表达载体(pET系列载体)中，得到重组封闭蛋白原核表达载体(pET-BSAmut-His)，pET-BSAmut-His载体含有6个His蛋白标签用于纯化，将pET-BSAmut-His载体整合在大肠杆菌中大量表达封闭蛋白。表达出来BSAmut-His蛋白后，用Ni离子柱子纯化。得到高纯度的封闭蛋白(BSAmut-His蛋白)，该封闭蛋白不含赖氨酸及精氨酸。
将纯化后的封闭蛋白与PBS混合，获得用于质谱检测的蛋白封闭剂。
实施例2
将实施例1获得的用于质谱检测的封闭蛋白进行蛋白酶酶解验证，具体操作如下：
试验组为实施例1的用于质谱检测的封闭蛋白；对照组为牛血清白蛋白。用胰酶水解试验组与对照组的蛋白，胰酶水解的条件按常规条件进行。水解前后分别进行SDS-PAGE验证。
从图1可知，A为牛血清白蛋白溶液的蛋白条带；B为胰酶水解后的牛血清白蛋白溶液的蛋白条带；C为封闭蛋白溶液的蛋白条带；D为胰酶水解后的封闭蛋白的蛋白条带，C和D的蛋白条带说明封闭蛋白无法被胰酶水解，因此，实施例的封闭蛋白在对质谱环境进行封闭后，蛋白水解酶不会作用封闭蛋白，不会对质谱数据造成污染。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 广州辉骏生物科技有限公司
<120> 一种用于质谱检测的蛋白封闭剂及其应用
<130> MP1708653
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 607
<212> PRT
<213> 人工序列
<222> (2)..(2)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (19)..(19)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (23)..(24)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (28)..(28)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (34)..(34)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (36)..(36)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (44)..(44)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (75)..(75)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (88)..(88)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (100)..(100)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (117)..(117)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (122)..(122)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (130)..(130)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (568)..(568)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
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<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<222> (597)..(597)
<223> Xaa can be any naturally occurring amino acid except arginine andLysine
<400> 1
Met Xaa Trp Val Thr Phe Ile Ser Leu Leu Leu Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser Xaa Gly Val Phe Xaa Xaa Asp Thr His Xaa Ser Glu Ile Ala
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His Xaa Phe Xaa Asp Leu Gly Glu Glu His Phe Xaa Gly Leu Val Leu
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Ile Ala Phe Ser Gln Tyr Leu Gln Gln Cys Pro Phe Asp Glu His Val
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Xaa Leu Val Asn Glu Leu Thr Glu Phe Ala Xaa Thr Cys Val Ala Asp
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Glu Ser His Ala Gly Cys Glu Xaa Ser Leu His Thr Leu Phe Gly Asp
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Glu Leu Cys Xaa Val Ala Ser Leu Xaa Glu Thr Tyr Gly Asp Met Ala
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Asp Cys Cys Glu Xaa Gln Glu Pro Glu Xaa Asn Glu Cys Phe Leu Ser
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His Xaa Asp Asp Ser Pro Asp Leu Pro Xaa Leu Xaa Pro Asp Pro Asn
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Thr Leu Cys Asp Glu Phe Xaa Ala Asp Glu Xaa Xaa Phe Trp Gly Xaa
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Tyr Leu Tyr Glu Ile Ala Xaa Xaa His Pro Tyr Phe Tyr Ala Pro Glu
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Leu Leu Tyr Tyr Ala Asn Xaa Tyr Asn Gly Val Phe Gln Glu Cys Cys
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Gln Ala Glu Asp Xaa Gly Ala Cys Leu Leu Pro Xaa Ile Glu Thr Met
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Xaa Glu Xaa Val Leu Ala Ser Ser Ala Xaa Gln Xaa Leu Xaa Cys Ala
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Ser Ile Gln Xaa Phe Gly Glu Xaa Ala Leu Xaa Ala Trp Ser Val Ala
225 230 235 240
Xaa Leu Ser Gln Xaa Phe Pro Xaa Ala Glu Phe Val Glu Val Thr Xaa
245 250 255
Leu Val Thr Asp Leu Thr Xaa Val His Xaa Glu Cys Cys His Gly Asp
260 265 270
Leu Leu Glu Cys Ala Asp Asp Xaa Ala Asp Leu Ala Xaa Tyr Ile Cys
275 280 285
Asp Asn Gln Asp Thr Ile Ser Ser Xaa Leu Xaa Glu Cys Cys Asp Xaa
290 295 300
Pro Leu Leu Glu Xaa Ser His Cys Ile Ala Glu Val Glu Xaa Asp Ala
305 310 315 320
Ile Pro Glu Asn Leu Pro Pro Leu Thr Ala Asp Phe Ala Glu Asp Xaa
325 330 335
Asp Val Cys Xaa Asn Tyr Gln Glu Ala Xaa Asp Ala Phe Leu Gly Ser
340 345 350
Phe Leu Tyr Glu Tyr Ser Xaa Xaa His Pro Glu Tyr Ala Val Ser Val
355 360 365
Leu Leu Xaa Leu Ala Xaa Glu Tyr Glu Ala Thr Leu Glu Glu Cys Cys
370 375 380
Ala Xaa Asp Asp Pro His Ala Cys Tyr Ser Thr Val Phe Asp Xaa Leu
385 390 395 400
Xaa His Leu Val Asp Glu Pro Gln Asn Leu Ile Xaa Gln Asn Cys Asp
405 410 415
Gln Phe Glu Xaa Leu Gly Glu Tyr Gly Phe Gln Asn Ala Leu Ile Val
420 425 430
Xaa Tyr Thr Xaa Xaa Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu
435 440 445
Val Ser Xaa Ser Leu Gly Xaa Val Gly Thr Xaa Cys Cys Thr Xaa Pro
450 455 460
Glu Ser Glu Xaa Met Pro Cys Thr Glu Asp Tyr Leu Ser Leu Ile Leu
465 470 475 480
Asn Xaa Leu Cys Val Leu His Glu Xaa Thr Pro Val Ser Glu Xaa Val
485 490 495
Thr Xaa Cys Cys Thr Glu Ser Leu Val Asn Xaa Xaa Pro Cys Phe Ser
500 505 510
Ala Leu Thr Pro Asp Glu Thr Tyr Val Pro Xaa Ala Phe Asp Glu Xaa
515 520 525
Leu Phe Thr Phe His Ala Asp Ile Cys Thr Leu Pro Asp Thr Glu Xaa
530 535 540
Gln Ile Xaa Xaa Gln Thr Ala Leu Val Glu Leu Leu Xaa His Xaa Pro
545 550 555 560
Xaa Ala Thr Glu Glu Gln Leu Xaa Thr Val Met Glu Asn Phe Val Ala
565 570 575
Phe Val Asp Xaa Cys Cys Ala Ala Asp Asp Xaa Glu Ala Cys Phe Ala
580 585 590
Val Glu Gly Pro Xaa Leu Val Val Ser Thr Gln Thr Ala Leu Ala
595 600 605
Claims (10)
1.一种用于质谱检测的封闭蛋白，其特征在于，所述封闭蛋白的氨基酸如SEQ ID.No1所示，所述SEQ ID.No1中X为自然界存在的氨基酸，且不属于精氨酸和赖氨酸。
2.根据根据权利要求1所述的用于质谱检测的封闭蛋白，其特征在于，所述SEQ ID.No1中X为组氨酸。
3.一种用于质谱检测的封闭蛋白在封闭质谱检测环境中的非特异性的蛋白质结合位点的应用。
4.根据根据权利要求3所述的应用，其特征在于，所述的封闭蛋白不被蛋白水解酶水解。
5.根据根据权利要求4所述的应用，其特征在于，所述的蛋白水解酶为胰酶。
6.一种用于质谱检测的封闭蛋白的制备方法，其特征在于，包括：
1)构建原核表达载体，所述原核表达载体包括SEQ ID.No1，得到重组封闭蛋白原核表达载体，使得重组封闭蛋白原核表达载体表达所述封闭蛋白；
2)将所述重组封闭蛋白原核表达载体整合到原核生物中，使得所述原核生物表达所述封闭蛋白，收集获得所述的封闭蛋白。
7.根据根据权利要求6所述的用于质谱检测的封闭蛋白的制备方法，其特征在于，所述原核表达载体含有组氨酸标签。
8.一种用于质谱检测的蛋白封闭剂，其特征在于，包括用于质谱检测的封闭蛋白和缓冲液。
9.根据权利要求8所述的用于质谱检测的蛋白封闭剂，其特征在于，所述缓冲液为PBS溶液或Nacl溶液。
10.一种用于质谱检测的试剂盒，其特征在于，包括：用于质谱检测的蛋白封闭剂和质谱检测试剂。
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Address after: 510663 Guangzhou City International Business Incubator D 504-505, science and Technology City, Guangzhou, Guangdong, 3 Applicant after: Guangzhou Huijun Biotechnology Co., Ltd. Address before: High tech Industrial Development Zone, Guangzhou City, Guangdong province 510000 Science City skim Springs Road No. 3, Guangzhou international business incubator D504-505 Applicant before: Guangzhou Hui Jun Biotechnology Co., Ltd. |
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Application publication date: 20171020 |