CN113552269B - 山羊绒蛋白标记方法及其差异表达蛋白的肽段序列 - Google Patents
山羊绒蛋白标记方法及其差异表达蛋白的肽段序列 Download PDFInfo
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Abstract
本发明公开了一种山羊绒蛋白标记方法，提取绒山羊的绒和毛的蛋白质，测定总蛋白浓度；总蛋白的酶解，记录酶解情况及其蛋白含量，采用高效液相色谱二级质谱方法分析肽段组成和含量；将SWATH质谱获取的肽段数据作为非目标数据，IDA模式的无标记定量方法获取的肽段数据作为目标数据；测定差异表达蛋白含量，利用差异表达蛋白含量进行绒和毛的鉴别。本发明具有高度敏感性、动态范围广、快速的扫描速度、较高覆盖率、成本相对较低、试验操作简便和不受限制。
Description
技术领域
本发明属于纤维鉴别技术领域，具体涉及一种山羊绒蛋白标记方法。
背景技术
蛋白质组学(Proteomics)是研究蛋白质的结构、功能、组成、表达及其活动规律的一门科学。包括蛋白的定性及定量、蛋白的结构与功能、蛋白翻译后修饰等内容。定量蛋白质组学(Quantitative Proteomics)是对一个基因组表达的全部蛋白质或一个复杂混合体系内所有蛋白质进行精确鉴定和定量的一门科学。它可用于筛选和寻找任何因素下所引起的样本之间的差异表达蛋白，定量蛋白质组学可分为标记定量组学和非标记定量组学两种。非标记定量即Label-free定量，通常用于分析大规模蛋白的鉴定和定量，不需要对比较样本做特定标记处理，只需要比较特定肽段/蛋白在不同样品间的色谱质谱响应信号就可得到样品间蛋白表达量的差异。常见方法有IDA(information dependent acquisition)和SWATH(sequential windowed acquisition of all theoretical fragment ions)。SWATH是较为新颖的一种非标记高通量蛋白定量技术。目前为止，SWATH技术允许完整和永久地记录生物样本中肽前体中所有可检测的碎片离子。在一次进样分析中可以同时得到一个较高质量的定量结果和蛋白质组学样品中每个肽段的数据。这种采集模式将以25Dalton为一个系列区间进行扫描，在扫描范围内通过高速度的扫描可以获得全部离子的所有碎片信息。具有高分辨采集模式的SWATH技术，可以对样品中所有的化合物进行追寻、根源茶渣以及系统分析等相应工作，通过一次实验就可以获得完整的定性定量的结果分析，不需要在进行方法的改善，并通过均匀扫描和采集检测范围内所有前体离子及二级产物离子信息，并依据二级产物离子峰面积进行蛋白质定量的工作。
实现绒毛鉴定的方案有：氨基酸分析法、蛋白质分析法、DNA的分析方法。氨基酸分析法是通过甲酸萃取蛋白质，分析羊绒和绵羊毛纤维中甘氨酸和酪氨酸含量的差异进行鉴定的，但是这种方法数量级差异较小，实验难度大，设备要求高。蛋白质分析法是利用动物纤维中的Ⅱ型角蛋白N-末端和C-末端结构域的物种特异性差异进行纤维鉴别，但是动物纤维中的蛋白质含量会受因同一种属不同个体蛋白质含量的离散性、动物的生长饲养状况、生长时期而产生较大差异，因此检测结果的准确性也会受到影响。DNA的分析方法利用物种特异探针进行DNA点杂交分析到PCR技术的应用以及DNA序列的测定。
目前对羊绒羊毛鉴别的方法主要有：形态特征观察鉴别、溶液浸润和染色鉴别、光谱分析、碱溶度差异分析、结晶结构和热力学学性能分析、摩擦和拉伸性能分析、生物芯片技术等。针对形态特征观察鉴别法，人工观察鳞片结构慢、受主观因素干扰大、导致结果误差大。溶液浸润和染色鉴别法对绒毛本身损伤大、判定精确度有限，鉴别效果并不明显，只适用于纤维宏观形态差异的区分，测试精度有限。光谱分析法，其可靠性在很大程度上取决于建立完善的模型和数据库；检测所得结果很大程度上依赖于所建模型数据库的完整性，数据库要有代表性，并且要有足够的数据积累，否则很容易对检测结果造成不良的影响。碱溶度差异法，需要在特定的试验条件下进行，且耗时长。生物芯片，该方法对纤维要求严格，仅适用于未经过热处理或化学处理的原状态纤维。结晶结构和热力学学性能分析，不同纤维的结晶度以及结晶构成是不同的，X-射线衍射法就是一种利用结晶度来鉴别羊绒和羊毛的一种方法。摩擦和拉伸性能分析，根据羊毛和羊绒纤维表面鳞片的不同特征，对纤维的顺、逆鳞片的动静摩擦系数进行测试，对技术要求较高。
发明内容
针对现有技术存在的问题，本发明的目的在于提供一种山羊绒蛋白标记方法，用于羊毛羊绒的鉴别，具有高度敏感性、动态范围广、快速的扫描速度、较高覆盖率、成本相对较低、试验操作简便和不受限制。
为达到上述目的，本发明使用的技术解决方案是：
山羊绒蛋白标记方法，包括：
提取绒山羊的绒和毛的蛋白质，测定总蛋白浓度；总蛋白的酶解，记录酶解情况及其蛋白含量，采用高效液相色谱二级质谱方法分析肽段组成和含量；
将SWATH质谱获取的肽段数据作为非目标数据，IDA模式的无标记定量方法获取的肽段数据作为目标数据；
测定差异表达蛋白含量，利用差异表达蛋白含量进行绒和毛的鉴别。
进一步，在液相色谱串联质谱技术系统上，采用IDA模式的无标记定量方法和SWATH所有碎片离子的顺序窗口采集；依据Transition峰面积计算出肽段含量，通过肽段含量得出绒和毛中蛋白质的含量。
进一步，以差异表达基因分析来筛选差异表达蛋白。
进一步，差异表达蛋白的肽段序列为：
MSRHFSSRSGYRCGGGGGGFSSGSAGVVSYQRRSTSSSVRRSGGGGGRFSGGVCGGGAGGGFGSRSLINLGGSKSISISVAGGGGRGGFGGGYGGGYGGGGFGGGGFGGGGFGGGGFGGGGIGGGFGGGGFGGGGFGGGGFPVCPGGIQEVTINQSLLQPLNVEIDPEIQKIKSQEREQIKSLNNQFASFIDKVRFLEQQNQVLQTKWELLQQINTSTRTYSLEPFFEAYISNLRRNVDQLKSDRSRLDSELKNMQDLVEDYRSKYEDEINKRTNAENEFVGIKKDVDAAYITKVDLQAKFDNLLQEIDFFKTLFQAELSQMQTHISDTNVILSMDNNRNLDLNSIIDEVKNQYEEIAQRSKAEAEALYQTKYEELQITAGQHGDSLKNTKMEISELNRVIQRLRSEIDSVKKQISSLQQAISDAEQRGENAIKDAQNKLNELEDALQKAKEDMARLLRDYQELMNTKLALDVEIATYRTLLEGEESRMSGECVPNVSVSVSTSHTSISGGGGRGGGGFSSGGGGYSSGGGGGYGSGGGGGYGSGGGSSYGSRGGGSYGSGGGSSGSRRGGSGGGGGGSGGSFSSSGGRASSTKTSGGSSSVKFVSTSYSRGTR。
进一步，用来对羊绒羊毛进行鉴别的差异表达蛋白的肽段包括：FSGGVCGGGAGGGFGSR、QSLLQPLNVEIDPEIQK、SLNNQFASFIDKVRFLEQQNQVLQTKWELLQQINTSTRTYSLEPFFEAYIS NLR、NMQDLVEDYR、TNAENEFVGIKKDVDAAYITK、FDNLLQEIDFFKTLFQAELSQMQTHISDTNVILSMDNNRNLDLNSIIDEVK、YEELQITAGQHGDSLK、KMEISELNR、QISSLQQAISDAEQR、LNELEDALQKAK、DYQELMNTKLALDVEIATYR或者GGGGFSSGGGGYSSGGGGGYGSGGGGGYGSGGGSSYGSR。
进一步，采用肽段组合进行鉴别羊毛、羊绒，或者采用肽段单独进行鉴别羊毛、羊绒。
应用于所述的山羊绒蛋白标记方法的差异表达蛋白的肽段序列为：MSRHFSSRSGYRCGGGGGGFSSGSAGVVSYQRRSTSSSVRRSGGGGGRFSGGVCGGGAGGGFGSRSLINLGGSKSISISVAGGGGRGGFGGGYGGGYGGGGFGGGGFGGGGFGGGGFGGGGIGGGFGGGGFGGGGFGGGGFPVCPGGIQEVTINQSLLQPLNVEIDPEIQKIKSQEREQIKSLNNQFASFIDKVRFLEQQNQVLQTKWELLQQINTSTRTYSLEPFFEAYISNLRRNVDQLKSDRSRLDSELKNMQDLVEDYRSKYEDEINKRTNAENEFVGIKKDVDAAYITKVDLQAKFDNLLQEIDFFKTLFQAELSQMQTHISDTNVILSMDNNRNLDLNSIIDEVKNQYEEIAQRSKAEAEALYQTKYEELQITAGQHGDSLKNTKMEISELNRVIQRLRSEIDSVKKQISSLQQAISDAEQRGENAIKDAQNKLNELEDALQKAKEDMARLLRDYQELMNTKLALDVEIATYRTLLEGEESRMSGECVPNVSVSVSTSHTSISGGGGRGGGGFSSGGGGYSSGGGGGYGSGGGGGYGSGGGSSYGSRGGGSYGSGGGSSGSRRGGSGGGGGGSGGSFSSSGGRASSTKTSGGSSSVKFVSTSYSRGTR。
优选的，用来对羊绒羊毛进行鉴别的差异表达蛋白的肽段包括：QSLLQPLNVEIDPEIQK、
SLNNQFASFIDKVRFLEQQNQVLQTKWELLQQINTSTRTYSLEPFFEAYIS NLR、NMQDLVEDYR、TNAENEFVGIKKDVDAAYITK、FDNLLQEIDFFKTLFQAELSQMQTHISDTNVILSMDNNRNLDLNSIIDEVK、YEELQITAGQHGDSLK、KMEISELNR、QISSLQQAISDAEQR、LNELEDALQKAK、DYQELMNTKLALDVEIATYR或者
GGGGFSSGGGGYSSGGGGGYGSGGGGGYGSGGGSSYGSR。
本发明技术效果包括：
相对于标记定量方法，本发明具有以下优势：
1、不需要在样品分析前对肽段进行标记，避免了标记环节可能造成的样品损失。
2、检测肽段的数量多、蛋白质的覆盖率大和分析通量高。
3、不受样品来源和数量的限制。
4、具有高度敏感性、动态范围广、扫描速度快、较高覆盖率、成本相对较低，试验操作简便和不受限制的特点。
附图说明
图1是本发明中山羊绒蛋白标记方法的流程图；
图2是本发明中离子色谱图；
图3是本发明中主成分分析示意图；
图4是本发明中火山图；
图5是本发明中LI-COR Odyssey近红外成像仪成像扫描结果图。
具体实施方式
以下描述充分地示出本发明的具体实施方案，以使本领域的技术人员能够实践和再现。
本发明采用液相色谱串联质谱技术(LC-MS/MS)对绒山羊的绒和毛的蛋白进行定量差异分析，找到了影响绒和毛性状差异的关键蛋白，一方面，对未来提高绒山羊的绒毛品质打下基础，对绒和毛的真伪鉴定、成分检测奠定了理论依据和数据支持；另一方面，解决了目前技术所出现的问题，比如受到主观因素干扰导致结果误差大、判定精确度有限、鉴别效果不明显、成本高、耗时长、依赖性大、操作复杂等问题。
如图1所示，是本发明中山羊绒蛋白标记方法的流程图。
山羊绒蛋白标记方法，具体步骤如下：
步骤1：提取绒山羊的绒和毛的蛋白质，测定总蛋白浓度；
1、蛋白提取及含量测定。
作用：从绒、毛的样本山提取出其含有的蛋白质。
目的：为后续实验的开展做准备。
(1)、分别称取0.5g绒和毛作为样品，用水冲洗，置于1.5ml离心管中；
本试验样品选取3只、2.5周岁，内蒙古绒山羊(阿尔巴斯型)体测的同一部位绒毛样本(内蒙古鄂尔多斯市亿维白绒山羊养殖场)。
(2)、离心管中加入1ml二氯甲烷和甲醇混合物，在50℃下摇动2小时，清洗样品携带的油脂和杂质；
(3)、样品在1000rpm和4℃下离心10分钟，弃去含有脂质的上清液。
(4)加1ml纯水，在10000rpm和4℃下再次离心样品15分钟，取出样品并在50℃下干燥20分钟；
(5)、将清洗后的样品放入裂解液(尿素Urea、Tris-Hcl、DTT)中并放入烘箱中48小时；
(6)、取出样品并在转速10000rpm和4℃下离心20分钟；
(7)、收集上清液进行进一步研究，使用北京Bioteke公司的BCA(bicinchoninicacid)方法对蛋白质浓度进行测定。
2、SDS-PAGE(十二烷基硫酸钠聚丙烯酰胺)。
作用及目的：分离开不同长度的蛋白质以找到所需要长度的蛋白质。
按照伯乐预制胶的说明书的方法具体操作如下：
(1)、浓缩胶制备：3mL ResolverA+3mL ResolverB+30μL 10％APS+3μL TEMED；
(2)、涡旋混匀倒入胶板中；
(3)、分离胶制备：1mL StockerA+1mL StockerB+10μL 10％APS+2μLTEMED，涡旋混匀倒入胶板中；
(4)、插上10孔梳子，聚合反应30分钟；
(5)、将步骤1提取出来的蛋白质，取30μg(浓度)样品，加入4μL5×buffer超纯水补足至20μL，混匀，煮沸10min(变性)；
(6)、跑胶：在电泳槽中加入1×电泳缓冲液，接通电源，浓缩胶及分离胶电压均为200V，照胶。
步骤2：总蛋白的酶解，记录酶解情况及其蛋白含量；
作用及目的：将大片段的蛋白质切成小片段，为进行液相色谱串联质谱做准备。
将100微克蛋白质(步骤1中取提出并测完浓度的蛋白质)添加到200μL 8M尿素和10mM二硫苏糖醇中，并在37℃下孵育1小时。在12000rpm下离心40分钟后，向每个滤液管中添加200μL尿素，涡旋。接下来，滤液管以12000rpm离心两次，每次30分钟。然后，将200μL50mM碘乙酸添加到每个滤液管中；在黑暗中反应30分钟，离心，去除液体。接下来，向每个过滤管中添加100μL碳酸氢铵，并以12000rpm离心20分钟。此步骤执行3次，将液体除去。样品在37℃下与胰蛋白酶孵育过夜。次日12000rpm下离心30分钟。然后，向每个过滤管中添加50μL碳酸氢铵，在12000rpm下离心30分钟，并重复此步骤。收集滤液，冷冻干燥(成分：小肽段的蛋白质)，并在-20℃下储存。
步骤3：采用HPLC(高效液相色谱)-MS/MS(二级质谱)分析肽段组成和含量；
在液相色谱串联质谱技术(LC-MS/MS)系统上，采用两种方法，即IDA(informationdependent acquisition)模式的无标记定量方法和SWATH(sequential windowedacquisition ofall theoretical fragment ions)所有理论碎片离子的顺序窗口采集。
取约2μg肽并在C18 HPLC柱(75μm×15cm)上分离。使用0.1％甲酸水中和0.1％甲酸乙腈中的线性梯度(120分钟，以500nL/min从5％到80％B)分离肽。
IDA质谱的条件为：分辨率：30000；飞行时间(TOF)-MS采集范围：350～1800m/z；二级质谱与IDA自动碰撞能量扫描范围：400～1800m/z。
SWATH质谱的条件为：MS1采集范围：150～1200m/z；MS2质量范围：100～1500m/z。
SWATH数据内容：依据Transition峰面积计算出肽段含量，通过肽段含量推理出绒和毛中蛋白质的含量
步骤4：将SWATH质谱获取的肽段数据作为非目标数据，IDA模式的无标记定量方法获取的肽段数据作为目标数据，进行数据分析处理。
使用Protein Pilot 4.5软件以UniProt/SWISS-PROT/Caprahircus为数据库来鉴定蛋白。结果在1％假阳性(FDR)过滤。选定的搜索参数：使用胰蛋白酶，设置最多允许两个缺失的切割位点，肽质量耐受性为±15ppm，片段质量耐受性为20mmu。
步骤5：测定差异表达蛋白含量，利用差异表达蛋白KRT1(属于角蛋白1)含量进行绒和毛的鉴别。
1、KRT1(角蛋白1)蛋白免疫沉淀，进一步验证其数据的可靠性。
(1)、将SureBeads磁珠重悬混匀后，取20μL SureBeads到1.5mL eppendorf管中。使用配套磁力架吸附磁珠，待液体澄清后，弃上清。(加入SureBeads体积可根据具体实验来调节，可使用不同的体积如20μL，40μL，100μL等来摸索哪个条件最合适。蛋白样品上样量可根据实验室以往经验加入，如常见的500μL或者500μg-2mg)。
(2)、使用1mLPBST(PBS+0.1％Tween 20)来清洗磁珠，将磁珠充分重悬，可使用涡旋振荡或者枪头上下吹打，但注意涡旋振荡后要小离心，以确保盖子和管壁上的磁珠被充分离心到管底。使用磁力架吸附去除上清。重复6次。
(3)、加入23.1μL IP抗体(1-10μg抗体，加入到实验室常用的抗体稀释液中，如不清楚用PBST也可)到磁珠中，充分重悬。
(4)、旋转摇床上室温孵育2h，注意隔一段时间需将其充分混匀。使用磁力架吸附去除上清。
(5)、使用1mLPBST(PBS+0.1％Tween 20)来清洗磁珠,步骤同2，清洗4次。加入含靶蛋白的裂解液，10-200μL，根据实验室经验可调节。
(6)、4℃孵育过夜。(孵育时间可根据IP抗体的性能来优化)。
(7)、使用磁力架吸附去除上清。使用1mLPBST(PBS+0.1％Tween 20)来清洗磁珠，清洗5次。(磁珠的最后一次清洗时，注意更换一个新的EP管)。
(8)、小离心,使管壁上的磁珠都离心至管底，用磁力架吸附磁珠，弃上清液体。
(9)、洗脱：加入8ul 5Buffer和32ul PBS，混匀后，70℃加热孵育10分钟，磁化，取上清。
(10)、蛋白酶解(方法同步骤2的总蛋白酶解)。
(11)、液质联用上机检测获取IDA数据(方法同步骤3)。
2、Westernblot，检测KRT1蛋白的表达量并进一步验证质谱分析结果。
(1)、总蛋白提取及测定(方法同步骤1)
(2)、蛋白变性：取200μg(浓度)样品，加入4μL 5×buffer，超纯水补足至20，混匀，煮沸10min(变性)。
(3)、跑胶：在电泳槽中加入1×电泳缓冲液，接通电源，浓缩胶电压为80V，分离胶电压为120V。
(4)、泡膜/滤纸：滤纸浸入转膜液中，将PVDF膜浸入甲醇30s(不超过1min)，再浸入转膜液。
(5)、配TPBS:PBS中Tween 20含量为0.05％。
(6)、转膜(功率：25V电压：2.5mA时间：18min)(滤纸+膜+胶+滤纸：从下到上)
(7)、丽春红染5min(4℃)
(8)、TPBS洗10mins×4次
(9)、封闭液封闭2h
(10)、稀释一抗:KRT1蛋白一抗按说明书进行稀释。
(11)、一抗孵育4℃过夜,shi wen室温2h。
(12)、TPBS洗10mins 4次
(13)、稀释二抗：奥德赛二抗按说明书进行稀释
(14)、二抗孵育37℃，2h
(15)、TPBS洗5mins，4次
(16)、使用LI-COR Odyssey近红外成像仪成像扫描，并进行灰度值的计算。
如图2所示，是本发明中离子色谱图；如图3所示，是本发明中主成分分析示意图；如图4所示，是本发明中火山图。
下面以差异表达基因分析来筛选差异表达蛋白，差异表达基因分析包括：差异倍数(fold change)，差异的显著性(P-value)、火山图。
将ProteinPilot软件生成的数据加载到PeakView软件中，对非目标数据、目标数据生成并提取离子色谱图，PeakView软件在处理目标数据和非目标数据后生成提取离子色谱图(XICs)。然后，利用MarkerView软件对结果进行了定量分析。
采用主成分分析(参考图3主成分分析示意图)和火山图分析相结合的方法，将差异倍数(倍数变化法的FC值)和t检验法(假设检验方法，P值、FDR值)相结合。使用差异倍数(fold change)>2或<0.5，以及P-Value(差异的显著性)<0.05为阈值筛选差异表达蛋白。
差异表达蛋白KRT1的肽段序列：
MSRHFSSRSGYRCGGGGGGFSSGSAGVVSYQRRSTSSSVRRSGGGGGRFSGGVCGGGAGGGFGSRSLINLGGSKSISISVAGGGGRGGFGGGYGGGYGGGGFGGGGFGGGGFGGGGFGGGGIGGGFGGGGFGGGGFGGGGFPVCPGGIQEVTINQSLLQPLNVEIDPEIQKIKSQEREQIKSLNNQFASFIDKVRFLEQQNQVLQTKWELLQQINTSTRTYSLEPFFEAYISNLRRNVDQLKSDRSRLDSELKNMQDLVEDYRSKYEDEINKRTNAENEFVGIKKDVDAAYITKVDLQAKFDNLLQEIDFFKTLFQAELSQMQTHISDTNVILSMDNNRNLDLNSIIDEVKNQYEEIAQRSKAEAEALYQTKYEELQITAGQHGDSLKNTKMEISELNRVIQRLRSEIDSVKKQISSLQQAISDAEQRGENAIKDAQNKLNELEDALQKAKEDMARLLRDYQELMNTKLALDVEIATYRTLLEGEESRMSGECVPNVSVSVSTSHTSISGGGGRGGGGFSSGGGGYSSGGGGGYGSGGGGGYGSGGGSSYGSRGGGSYGSGGGSSGSRRGGSGGGGGGSGGSFSSSGGRASSTKTSGGSSSVKFVSTSYSRGTR
肽段序列中，用来对羊绒羊毛进行鉴别的肽段如下：
1.FSGGVCGGGAGGGFGSR
2.QSLLQPLNVEIDPEIQK
3.SLNNQFASFIDKVRFLEQQNQVLQTKWELLQQINTSTRTYSLEPFFEAYISNLR
4.NMQDLVEDYR
5.TNAENEFVGIKKDVDAAYITK
6.FDNLLQEIDFFKTLFQAELSQMQTHISDTNVILSMDNNRNLDLNSIIDEVK
7.YEELQITAGQHGDSLK
8.KMEISELNR
9.QISSLQQAISDAEQR
10.LNELEDALQKAK
11.DYQELMNTKLALDVEIATYR
12.GGGGFSSGGGGYSSGGGGGYGSGGGGGYGSGGGSSYGSR
以上12个肽段是差异表达蛋白KRT1的肽段序列的部分肽段，其与其他部分肽段序列相比较，吻合度更高。即可以组合在一起进行鉴别，也可以单独拿出来鉴别使用。
如图5所示，是本发明中LI-COR Odyssey近红外成像仪成像扫描结果图。
A表示实验结果，B表示数据分析结果。
LI-COR Odyssey近红外成像仪成像扫描结果验证结果显示，羊绒中角蛋白1的含量显著高于羊毛。
本发明所用的术语是说明和示例性、而非限制性的术语。由于本发明能够以多种形式具体实施而不脱离技术方案的精神或实质，所以应当理解，上述实施例不限于任何前述的细节，而应在随附权利要求所限定的精神和范围内广泛地解释，因此落入权利要求或其等效范围内的全部变化和改型都应为随附权利要求所涵盖。
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序列表
<110> 内蒙古农业大学
<120> 山羊绒蛋白标记方法及其差异表达蛋白的肽段序列
<130> 2021
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 614
<212> PRT
<213> 山羊（Capra hircus）
<400> 1
Met Ser Arg His Phe Ser Ser Arg Ser Gly Tyr Arg Cys Gly Gly Gly
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Gly Gly Gly Phe Ser Ser Gly Ser Ala Gly Val Val Ser Tyr Gln Arg
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Arg Ser Thr Ser Ser Ser Val Arg Arg Ser Gly Gly Gly Gly Gly Arg
35 40 45
Phe Ser Gly Gly Val Cys Gly Gly Gly Ala Gly Gly Gly Phe Gly Ser
50 55 60
Arg Ser Leu Ile Asn Leu Gly Gly Ser Lys Ser Ile Ser Ile Ser Val
65 70 75 80
Ala Gly Gly Gly Gly Arg Gly Gly Phe Gly Gly Gly Tyr Gly Gly Gly
85 90 95
Tyr Gly Gly Gly Gly Phe Gly Gly Gly Gly Phe Gly Gly Gly Gly Phe
100 105 110
Gly Gly Gly Gly Phe Gly Gly Gly Gly Ile Gly Gly Gly Phe Gly Gly
115 120 125
Gly Gly Phe Gly Gly Gly Gly Phe Gly Gly Gly Gly Phe Pro Val Cys
130 135 140
Pro Gly Gly Ile Gln Glu Val Thr Ile Asn Gln Ser Leu Leu Gln Pro
145 150 155 160
Leu Asn Val Glu Ile Asp Pro Glu Ile Gln Lys Ile Lys Ser Gln Glu
165 170 175
Arg Glu Gln Ile Lys Ser Leu Asn Asn Gln Phe Ala Ser Phe Ile Asp
180 185 190
Lys Val Arg Phe Leu Glu Gln Gln Asn Gln Val Leu Gln Thr Lys Trp
195 200 205
Glu Leu Leu Gln Gln Ile Asn Thr Ser Thr Arg Thr Tyr Ser Leu Glu
210 215 220
Pro Phe Phe Glu Ala Tyr Ile Ser Asn Leu Arg Arg Asn Val Asp Gln
225 230 235 240
Leu Lys Ser Asp Arg Ser Arg Leu Asp Ser Glu Leu Lys Asn Met Gln
245 250 255
Asp Leu Val Glu Asp Tyr Arg Ser Lys Tyr Glu Asp Glu Ile Asn Lys
260 265 270
Arg Thr Asn Ala Glu Asn Glu Phe Val Gly Ile Lys Lys Asp Val Asp
275 280 285
Ala Ala Tyr Ile Thr Lys Val Asp Leu Gln Ala Lys Phe Asp Asn Leu
290 295 300
Leu Gln Glu Ile Asp Phe Phe Lys Thr Leu Phe Gln Ala Glu Leu Ser
305 310 315 320
Gln Met Gln Thr His Ile Ser Asp Thr Asn Val Ile Leu Ser Met Asp
325 330 335
Asn Asn Arg Asn Leu Asp Leu Asn Ser Ile Ile Asp Glu Val Lys Asn
340 345 350
Gln Tyr Glu Glu Ile Ala Gln Arg Ser Lys Ala Glu Ala Glu Ala Leu
355 360 365
Tyr Gln Thr Lys Tyr Glu Glu Leu Gln Ile Thr Ala Gly Gln His Gly
370 375 380
Asp Ser Leu Lys Asn Thr Lys Met Glu Ile Ser Glu Leu Asn Arg Val
385 390 395 400
Ile Gln Arg Leu Arg Ser Glu Ile Asp Ser Val Lys Lys Gln Ile Ser
405 410 415
Ser Leu Gln Gln Ala Ile Ser Asp Ala Glu Gln Arg Gly Glu Asn Ala
420 425 430
Ile Lys Asp Ala Gln Asn Lys Leu Asn Glu Leu Glu Asp Ala Leu Gln
435 440 445
Lys Ala Lys Glu Asp Met Ala Arg Leu Leu Arg Asp Tyr Gln Glu Leu
450 455 460
Met Asn Thr Lys Leu Ala Leu Asp Val Glu Ile Ala Thr Tyr Arg Thr
465 470 475 480
Leu Leu Glu Gly Glu Glu Ser Arg Met Ser Gly Glu Cys Val Pro Asn
485 490 495
Val Ser Val Ser Val Ser Thr Ser His Thr Ser Ile Ser Gly Gly Gly
500 505 510
Gly Arg Gly Gly Gly Gly Phe Ser Ser Gly Gly Gly Gly Tyr Ser Ser
515 520 525
Gly Gly Gly Gly Gly Tyr Gly Ser Gly Gly Gly Gly Gly Tyr Gly Ser
530 535 540
Gly Gly Gly Ser Ser Tyr Gly Ser Arg Gly Gly Gly Ser Tyr Gly Ser
545 550 555 560
Gly Gly Gly Ser Ser Gly Ser Arg Arg Gly Gly Ser Gly Gly Gly Gly
565 570 575
Gly Gly Ser Gly Gly Ser Phe Ser Ser Ser Gly Gly Arg Ala Ser Ser
580 585 590
Thr Lys Thr Ser Gly Gly Ser Ser Ser Val Lys Phe Val Ser Thr Ser
595 600 605
Tyr Ser Arg Gly Thr Arg
610
MSRHFSSRSG YRCGGGGGGF SSGSAGVVSY QRRSTSSSVR RSGGGGGRFS GGVCGGGAGG 60
GFGSRSLINL GGSKSISISV AGGGGRGGFG GGYGGGYGGG GFGGGGFGGG GFGGGGFGGG 120
GIGGGFGGGG FGGGGFGGGG FPVCPGGIQE VTINQSLLQP LNVEIDPEIQ KIKSQEREQI 180
KSLNNQFASF IDKVRFLEQQ NQVLQTKWEL LQQINTSTRT YSLEPFFEAY ISNLRRNVDQ 240
LKSDRSRLDS ELKNMQDLVE DYRSKYEDEI NKRTNAENEF VGIKKDVDAA YITKVDLQAK 300
FDNLLQEIDF FKTLFQAELS QMQTHISDTN VILSMDNNRN LDLNSIIDEV KNQYEEIAQR 360
SKAEAEALYQ TKYEELQITA GQHGDSLKNT KMEISELNRV IQRLRSEIDS VKKQISSLQQ 420
AISDAEQRGE NAIKDAQNKL NELEDALQKA KEDMARLLRD YQELMNTKLA LDVEIATYRT 480
LLEGEESRMS GECVPNVSVS VSTSHTSISG GGGRGGGGFS SGGGGYSSGG GGGYGSGGGG 540
GYGSGGGSSY GSRGGGSYGS GGGSSGSRRG GSGGGGGGSG GSFSSSGGRA SSTKTSGGSS 600
SVKFVSTSYS RGTR 614
Claims (3)
1.一种山羊绒蛋白标记方法，包括：
提取绒山羊的绒和毛的蛋白质，测定总蛋白浓度；总蛋白的酶解，记录酶解情况及其蛋白含量，采用高效液相色谱二级质谱方法分析肽段组成和含量；
在液相色谱串联质谱技术系统上，采用IDA模式的无标记定量方法和SWATH所有碎片离子的顺序窗口采集；依据Transition峰面积计算出肽段含量，通过肽段含量得出绒和毛中蛋白质的含量；将SWATH质谱获取的肽段数据作为非目标数据，IDA模式的无标记定量方法获取的肽段数据作为目标数据；
测定差异表达蛋白含量，以差异表达基因分析来筛选差异表达蛋白，利用差异表达蛋白含量进行绒和毛的鉴别；差异表达蛋白中，选用其中的肽段FSGGVCGGGAGGGFGSR、QSLLQPLNVEIDPEIQK、SLNNQFASFIDKVRFLEQQNQVLQTKWELLQQINTSTRTYSLEPFFEAYISNLR、NMQDLVEDYR、TNAENEFVGIKKDVDAAYITK、FDNLLQEIDFFKTLFQAELSQMQTHISDTNVILSMDNNRNLDLNSIIDEVK、YEELQITAGQHGDSLK、KMEISELNR、QISSLQQAISDAEQR、LNELEDALQKAK、DYQELMNTKLALDVEIATYR和GGGGFSSGGGGYSSGGGGGYGSGGGGGYGSGGGSSYGSR进行绒和毛的鉴别。
2.如权利要求1所述的山羊绒蛋白标记方法，其特征在于，差异表达蛋白的肽段序列为：MSRHFSSRSGYRCGGGGGGFSSGSAGVVSYQRRSTSSSVRRSGGGGGRFSGGVCGGGAGGGFGSRSLINLGGSKSISISVAGGGGRGGFGGGYGGGYGGGGFGGGGFGGGGFGGGGFGGGGIGGGFGGGGFGGGGFGGGGFPVCPGGIQEVTINQSLLQPLNVEIDPEIQKIKSQEREQIKSLNNQFASFIDKVRFLEQQNQVLQTKWELLQQINTSTRTYSLEPFFEAYISNLRRNVDQLKSDRSRLDSELKNMQDLVEDYRSKYEDEINKRTNAENEFVGIKKDVDAAYITKVDLQAKFDNLLQEIDFFKTLFQAELSQMQTHISDTNVILSMDNNRNLDLNSIIDEVKNQYEEIAQRSKAEAEALYQTKYEELQITAGQHGDSLKNTKMEISELNRVIQRLRSEIDSVKKQISSLQQAISDAEQRGENAIKDAQNKLNELEDALQKAKEDMARLLRDYQELMNTKLALDVEIATYRTLLEGEESRMSGECVPNVSVSVSTSHTSISGGGGRGGGGFSSGGGGYSSGGGGGYGSGGGGGYGSGGGSSYGSRGGGSYGSGGGSSGSRRGGSGGGGGGSGGSFSSSGGRASSTKTSGGSSSVKFVSTSYSRGTR。
3.如权利要求1所述的山羊绒蛋白标记方法，其特征在于，采用差异表达蛋白的肽段序中的肽段组合进行鉴别羊毛、羊绒。
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| WO1987003503A1 (en) * | 1985-12-06 | 1987-06-18 | Memtec Limited | Treatment of emulsions |
| CN104897790A (zh) * | 2015-03-23 | 2015-09-09 | 中国科学院天津工业生物技术研究所 | 一种羊绒与羊毛的鉴别方法、用于该方法的标志物肽段 |
| CN107014929A (zh) * | 2017-05-26 | 2017-08-04 | 中国科学院天津工业生物技术研究所 | 一种鉴别山羊绒与细羊毛的方法 |
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| CN104897790A (zh) * | 2015-03-23 | 2015-09-09 | 中国科学院天津工业生物技术研究所 | 一种羊绒与羊毛的鉴别方法、用于该方法的标志物肽段 |
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