CN116539390A - 一种用于单个胚胎痕量蛋白提取的前处理方法 - Google Patents
一种用于单个胚胎痕量蛋白提取的前处理方法 Download PDFInfo
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Abstract
本发明涉及生物技术与蛋白质组学技术领域，尤其涉及一种用于筛选冷冻保存/新鲜体外受精胚胎差异蛋白的方法，包括以下步骤：(1)冷冻保存/新鲜胚胎培养；(2)痕量胚胎蛋白提取与酶解；(3)通过液相色谱‑串联质谱联合技术进行非标定量的胚胎蛋白检测；(4)筛选冷冻保存/新鲜胚胎之间的差异表达蛋白并进行生物信息学分析。所述方法可以对胚胎中的蛋白质组进行非标定量的分析，检测更多胚胎蛋白质；可以对单个的不同发育时期的胚胎样本进行定性定量的痕量蛋白质组学检测分析，更全面的了解分析胚胎中蛋白的表达变化情况；仪器灵敏度更高；前处理方法简单可行，仪器检测时间更短；实验可重复性强、结果准确。
Description
技术领域
本发明涉及生物技术与蛋白质组学技术领域，尤其涉及一种痕量非标记蛋白质组学的前处理新技术，可用于分析受精胚胎间差异表达蛋白进而筛选冷冻保存/新鲜体外胚胎。
背景技术
针对动物植入前胚胎的研究有助于阐明胚胎的细胞功能以及胚胎发育过程中体内生物分子的变化情况。已有很多针对动物胚胎的基因表达分析为研究胚胎活性提供了丰富信息，但是基因并不能准确预测蛋白质在生化水平上发挥功能的信息。作为在生理学过程中发挥核心作用的蛋白质，研究其丰度和活性至关重要。
目前对动物胚胎的蛋白质研究主要集中在识别单个或几个蛋白质上，然而，生理过程涉及多种蛋白质的相互作用，仅针对单个蛋白质进行研究存在很大的局限性。对胚胎进行非标记蛋白质组学可以更全面的了解细胞的功能信息。检测痕量的胚胎蛋白是非常具有挑战性的，经典的蛋白质组学方法涉及2D聚丙烯酰胺凝胶电泳，不但需要大量的起始材料、操作复杂且无法进行高通量分析。
质谱技术作为一种高灵敏度、高通量且低成本的方法使得对胚胎的非标记蛋白质组学的研究成为可能。液相色谱-串联质谱联用(LC-MS/MS)技术和表面增强激光解吸和电离飞行时间质谱(SELDI-TOF MS)已被用于胚胎的非标记蛋白质组学检测并且取得不错的检测效果。但是受限于胚胎的低样本量、低蛋白质丰度以及现有仪器的低灵敏度，目前检测还是使用多个胚胎的混合样本，无法实现对单个胚胎蛋白质组学的检测。不同胚胎存在显著的个体差异，对单个胚胎的蛋白质组学检测可以更准确的反应胚胎生长发育的信息，帮助临床挑选最具生殖潜力的胚胎进行移植。所以我们急需构建一种快速、高效的非标记蛋白质组学检测方法，实现对单个胚胎细胞全面的蛋白质组学检测。
在过去四十年中，辅助生殖技术(ARTs)已日趋成熟，为众多不孕症患者带来福音。但低植入率、低妊娠率以及婴儿的出生缺陷、生长障碍等疾病依旧阻碍着辅助生殖技术的发展。我们无法判断这种疾病风险是胚胎质量不佳导致的还是体外受精技术所带来的副作用。目前大多数的研究集中在如何判别胚胎质量、提高植入率上，对体外受精技术是否影响胚胎发育/植入潜力的研究少之又少。已有部分研究认为体外受精胚胎发育过程中周围环境压力有可能对胚胎的植入潜力和后续生长发育有潜在影响，研究这种影响有助于改进胚胎培养程序、提高胚胎的临床植入率。
根据欧洲人类生殖与胚胎学会的最新报道，冷冻保存胚胎技术是生育治疗中第二常用的技术。这种技术可以实现储存多余胚胎，为植入失败的患者进行二次植入提供了可能。但是冷冻胚胎技术会让胚胎暴露于低温和冷冻保护液的环境中，这可能会对胚胎造成潜在影响。已有研究表明，玻璃化胚胎移植过程涉及的技术对后代发育的影响有累加效应。与新鲜移植的体外受精胚胎相比，经过玻璃化操作的体外受精胚胎移植后所诞生的兔在成年期的生长速度、体重和重要器官重量都有不同[6]。来自冷冻保存胚胎的小牛也表现出特殊的临床和生化特征[7]。
目前的研究主要集中在通过体内/体外冷冻保存胚胎移植后出生的胎儿的生理特征，还没有针对冷冻保存胚胎和新鲜胚胎的蛋白质组学差异的研究。质谱是一种高灵敏度、高通量的蛋白质组学研究工具，可以实现对胚胎不同发育阶段全面的蛋白检测。由于胚胎的体积小、蛋白质丰度低，导致针对单个冷冻/新鲜胚胎的非标记蛋白质组学检测难度大，所以还没有针对冷冻/新鲜体外受精胚胎的蛋白质组学或筛选差异表达蛋白的相关研究报道。
在这里，我们开发了一种基于液相色谱-质谱联用(LC-MS/MS)技术的非标记蛋白质组学检测方法，用于分析单个胚胎的蛋白质组学。该策略适用于发育不同阶段胚胎的蛋白质表达检测，用于确定不同阶段的特异性蛋白以及筛选差异表达的潜在蛋白标志物，最终为临床移植的胚胎选择提供帮助。目前还没有此方面的专利发表。该技术适用于筛选植入前发育不同阶段的冷冻/新鲜胚胎中特异性蛋白以及差异表达蛋白，为临床提供可以实现多指标评估胚胎质量的蛋白标志物。
目前国内外关于胚胎样本差异表达蛋白的研究也有不少，Deisy J.D等人[1]基于液相色谱-质谱联用(LC-MS/MS)技术对体内受精六天龄的处于桑葚和囊胚发育状态的54个绵羊胚胎混合样本进行了蛋白质组学分析。通过四种软件共鉴定出667种蛋白质，JoséRenato S等人[2]利用一维十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)结合液相色谱-质谱联用(LC-MS/MS)技术对植入前六天龄(D6)的绵羊胚胎样本进行了蛋白质组学检测，从45个胚胎的混合样本中共鉴定出2262种蛋白质。Charles Banliat等人[3]利用纳米液相色谱结合串联质谱(nanoLC-MS/MS)分析5个不同阶段的牛胚胎混合样本，共鉴定出2757种蛋白质，其中1950种进行了定量分析。Gao等人[4]基于串联质量标签(TMT)的定量质谱技术监测了植入前六个不同阶段小鼠胚胎的蛋白质表达谱，每个阶段使用8000个胚胎，共检测到4000多种蛋白质。Mandy G等人[5]第一个利用表面增强激光解吸和电离飞行时间质谱(SELDI-TOF MS)分析了单个人类囊胚的蛋白质组，在发育中的囊胚和已退化胚胎之间观察到几种差异表达蛋白。但只展示了差异代谢物，并未展示具体检测出的蛋白质名称和数量。Ximo Garcia-Dominguez等人[7]首次通过液相色谱-质谱联用(LC-MS/MS)技术对经过玻璃化移植胚胎出生的兔和自然受孕的兔的肝组织进行蛋白质组学分析，筛选出与产后结局相关的差异表达蛋白。(Long-Term Phenotypic and Proteomic Changes FollowingVitrified Embryo Transfer in the Rabbit Model)Ximo Garcia-Dominguez等人[8]利用液相色谱-电喷雾电离-高分辨率质谱(LC-ESI-HRMS)和液相色谱-大气压化学电离-高分辨率质谱(LC-APCI-HRMS)对兔胚胎进行了标记和非标记的代谢组学分析。样本来源于自然受孕/新鲜/玻璃化胚胎移植后在体内发育第六天的胚胎混合样本。结果表明，三个实验组中共有40种代谢物的积累量下降，胚胎移植/冷冻操作都发挥了累积效应。
因此，本领域的技术人员致力于开发一种新的单个胚胎蛋白质检测的方法。
参考文献
1.Sanchez,D.J.D.,et al.,Proteomic profile of pre-implantational ovineembryos produced in vivo.Reprod Domest Anim,2021.56(4):p.586-603.
2.JR,S.P.,et al.,Global proteomic analysis of preimplantational ovineembryos produced in vitro.Reprod Domest Anim,2022.57(7):p.784-797.
3.Banliat,C.,et al.,Dynamic Changes in the Proteome of Early BovineEmbryos Developed In Vivo.Frontiers in Cell and Developmental Biology,2022.10.
4.Gao,Y.,et al.,Protein Expression Landscape of Mouse Embryos duringPre-implantation Development.Cell Rep,2017.21(13):p.3957-3969.
5.Katz-Jaffe,M.G.,D.K.Gardner,and W.B.Schoolcraft,Proteomic analysisof individual human embryos to identify novel biomarkers of development andviability.Fertil Steril,2006.85(1):p.101-7.
6.Garcia-Dominguez,X.,et al.,Long-Term Phenotypic and ProteomicChanges Following Vitrified Embryo Transfer in the Rabbit Model.Animals(Basel),2020.10(6).
7.Gomez,E.,et al.,Fitness of calves born from in vitro-produced freshand cryopreserved embryos.Front Vet Sci,2022.9:p.1006995.
8.Garcia-Dominguez,X.,et al.,Developmental and metabolic changesfollowing vitrified embryo transfer in rabbit embryos.Cryobiology,2020.97:p.293-294.
发明内容
有鉴于现有技术的上述缺陷，本发明所要解决的技术问题是对单个的不同发育时期的胚胎样本进行定性定量的痕量蛋白质组学检测分析，更全面的了解分析胚胎中蛋白的表达变化情况。
在本发明的较佳实施方式中，本发明提供了一种用于单个胚胎痕量蛋白提取的前处理方法，其特征在于，包括以下步骤：
(1)采集含有胚胎细胞的悬液；
(2)加入4倍体积的十二烷基β-D-麦芽苷(DDM)裂解缓冲液，在室温下水浴超声裂解1h；
(3)将步骤(2)裂解后的样品在金属浴中加热1h还原蛋白质，室温下黑暗处反应30min使蛋白质发生烷基化反应，向溶液中直接加入胰蛋白酶，用封口膜封口，进行酶解反应，放入37℃的烘箱过夜孵育12-16小时；
(4)向步骤(3)酶解后的样品加入终体积的1％的甲酸(FA)终止反应，涡旋混匀；
(5)通过Ziptip C18微量层析柱对样本进行脱盐处理；
(6)用真空干燥仪干燥样品，放置于-20℃备用。
优选地，所述裂解缓冲液为：40-60mM碳酸氢铵溶液、0.05-0.2％ DDM、0.5-2mM三(2-羧基乙基)磷化氢(TECP)和1-3mM 2-氯乙酰胺(CAA)。
优选地，所述酶解步骤中W胰酶:W蛋白的比例为1:5-20。
在本发明的较佳实施方式中，本发明还提供了上述前处理方法筛选冷冻保存/新鲜体外受精胚胎间差异表达蛋白的方法，其特征在于，包括以下步骤：
(1)冷冻保存/新鲜胚胎培养；
(2)痕量胚胎蛋白提取与酶解；
(3)通过液相色谱-串联质谱联合技术进行非标定量的胚胎蛋白检测；
(4)筛选冷冻保存/新鲜胚胎之间的差异表达蛋白并进行生物信息学分析。
优选地，所述胚胎来自绵羊，牛，小鼠，人，兔；更优选地，所述胚胎来自小鼠。
优选地，步骤(3)对非标定量的胚胎蛋白检测还包括纳米液相色谱系统(NanoElute)分离流动相A溶解后的肽段，所述纳米液相色谱系统(NanoElute)分离流动相A的梯度洗脱方案为：0-75min，2％-22％的流动相B；75-80min，22％-37％的流动相B；85-90min，80％流动相B；流动相A为含0.1％甲酸的水溶液，流动相B为含0.1％甲酸的乙腈溶液，流速设置为300nL/min。
在本发明的又一较佳实施方式中，提供了一种单个胚胎痕量蛋白提取的试剂盒，其特征在于，包含：M16培养液、裂解缓冲液、酶解液和终止反应液；
所述裂解缓冲液包含40-60mM碳酸氢铵溶液、0.05-0.2％ DDM、0.5-2mM三(2-羧基乙基)磷化氢(TECP)和1-3mM 2-氯乙酰胺(CAA)；
所述酶解液包含胰蛋白酶；
所述终止反应液包含甲酸。
在本发明的另一较佳实施方式中，还提供了差异表达蛋白组合在鉴定冷冻保存/新鲜体外受精胚胎的应用，所述差异表达蛋白组合为GLUD1、TF、GAPDH、ANXA2、TXN、PIP、DSP、CASP14、COPS2、UBE2I、DSG1、TGM3、DSC1、BLMH、KBTBD8和SERPINB12中的一种或几种。
优选地，所述差异表达蛋白组合为DSP、DSG1和DSC1。
优选地，所述差异表达蛋白为GLUD1、TF、GAPDH、ANXA2、TXN、PIP、DSP、CASP14、COPS2、UBE2I、DSG1、TGM3、DSC1、BLMH、KBTBD8和SERPINB12。
本发明带来了以下技术效果：
1)可以实现对胚胎中的蛋白质组进行非标定量的分析，检测到更多胚胎蛋白质，更全面的绘制胚胎蛋白质图谱。
2)可以针对单个的胚胎样本进行定性定量的痕量蛋白质组学检测分析。
3)可以实现对不同发育时期单个胚胎中的蛋白质组进行非标定量的分析，更全面的了解分析胚胎中蛋白的表达变化情况。
4)仪器灵敏度更高，样品只需50-200μg的痕量蛋白即可对其蛋白质组学进行分析。
5)前处理方法简单可行，仪器检测时间更短，有效缩短整体样本检测所需的整体时间。
6)实验可重复性强、结果准确。该方法适用于多个物种胚胎蛋白检测。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1是总离子流(TIC)色谱图；
图2是IVF组和FET组的OPLS-DA分析图；
图3是IVF组和FET组的差异表达蛋白的火山图；
图4是差异表达蛋白的层聚类分析图；
图5是差异表达蛋白的GO分析统计图；
图6是差异表达蛋白的PPI网络图。
具体实施方式
以下参考说明书附图介绍本发明的多个优选实施例，使其技术内容更加清楚和便于理解。本发明可以通过许多不同形式的实施例来得以体现，本发明的保护范围并非仅限于文中提到的实施例。
一、材料及试剂
样品制备及预处理所需材料及试剂如表1所示。
表1：材料与试剂
二、具体实施过程
1、冷冻保存/新鲜胚胎培养：
从卵母细胞供体鼠和精子供体鼠中获得卵母细胞与精子。精子获能后与卵母细胞在人输卵管液(HTF培养液)中进行体外受精培养。体外受精后28小时获得二细胞胚胎。取其中一半二细胞胚胎转移至M16培养液中继续培养至桑葚胚，得到体外受精-新鲜胚胎(IVF)组胚胎细胞。取另一半胚胎进行玻璃化冷冻(先用EFS20冷冻液冷冻，再转移到EFS40冷冻液中冷冻)。解冻(先用0.75SU冷冻液冷冻，再转移到0.25SU冷冻液中解冻)后将二细胞胚胎置入M16培养液中洗涤3次。之后将胚胎转移至M16培养液中继续培养至桑葚胚，获得体外受精-冻融胚胎(FET)组胚胎细胞。
2、痕量胚胎蛋白提取与酶解：
采集含有胚胎细胞的悬液后，分别加入4倍体积的十二烷基β-D-麦芽苷(DDM)裂解缓冲液(50mM碳酸氢铵溶液、0.1％ DDM、1mM三(2-羧基乙基)磷化氢(TECP)和2mM 2-氯乙酰胺(CAA))中；在室温下水浴超声裂解1h；裂解后的样品在60℃金属浴中加热1h还原蛋白质，室温下黑暗处反应30min使蛋白质发生烷基化反应。向溶液中直接加入胰蛋白酶(W胰酶:W蛋白＝1:10)，用封口膜封口，进行酶解反应。放入37℃的烘箱过夜孵育12-16小时；之后加入终体积的1％的甲酸(FA)终止反应，涡旋混匀。通过Ziptip C18微量层析柱对样本进行脱盐处理。用真空干燥仪干燥样品，放置于-20℃备用。
3、通过液相色谱-串联质谱联合技术进行非标定量的胚胎蛋白检测：
用含0.1％甲酸的水溶液重悬步骤(2)中酶解所得肽段。通过超微量分光光度计(NanoDrop)定量蛋白浓度，所有样品上样量需一致。通过纳米液相色谱系统(NanoElute)分离流动相A溶解后的肽段；使用90min的梯度洗脱方案：0-75min，2％-22％的流动相B；75-80min，22％-37％的流动相B；85-90min，80％流动相B。流动相A为含0.1％甲酸的水溶液。流动相B为含0.1％甲酸的乙腈溶液。流速设置为300nL/min。电喷雾(ESI)离子源电离分离后的肽段。随后利用捕集离子淌度飞行时间(tims-TOF Pro)质谱进行检测分析。数据采集模式设置为平行累积串行碎裂(PASEF)模式。二级质谱扫描范围设置为100-1700m/z。
通过Maxquant(V2.1.3.0)软件进行定量比库检索分析。参数设置：数据库设置为Uniprot Homo sapiens(20607条序列)；检索类型设置为TIMS-DDA；定量分析模式设置为无标记定量(LFQ)；固定修饰设置为氨基甲基(C)，可变修饰设置为甲硫氨酸的氧化，蛋白N端的乙酰化。酶切方式设置为Trypsin/P；最大漏切位点数设置为为2；一级母离子质量误差容忍度设置为10ppm，二级碎片离子的质量误差容忍度为0.05Da；添加反库计算假阳性率(FDR)，蛋白鉴定、肽谱匹配(PSM)鉴定的(FDR)都设置为1％。添加常见实验室污染物数据库，用于降低鉴定结果中污染蛋白的干扰.
4、筛选冷冻保存/新鲜胚胎之间的差异表达蛋白并进行生物信息学分析：
通过SPSS 24.0进行单变量统计学分析(学生T检验)、SIMCA 14.1软件进行多变量统计学(正交偏最小二乘判别分析(OPLS-DA))，根据所得p值和倍数变化(FC)/变量重要程度(VIP)值确定差异表达蛋白。
通过生物信息学分析对差异表达蛋白进行注释和富集分析。包括蛋白功能注释(包括基因本体论(GO)注释、京都基因与基因组百科全书(KEGG)通路注释、蛋白结构域注释和亚细胞定位)、蛋白质功能富集分析(包括GO、KEGG富集分析)和蛋白-蛋白互作网络分析(PPI)。
三、实施结果
本发明通过体外培养获得小鼠受精卵并培养发育至第四天，获得8个新鲜胚胎(IVF)和9个冷冻保存(FET)的小鼠桑葚胚。通过液相色谱-质谱联用技术实现了对单个小鼠胚胎的非标记定量(DDA、LFQ)的蛋白质组学研究(图1)。通过Maxquant软件进行比库检索分析共定性检测出1388种蛋白质(FDR<0.01,至少存在一个独特肽段)。采用数据依赖性采集和无标记定量(DDA LFQ)方法共定性定量检测到1015种蛋白质(FDR<0.01,至少存在一个独特肽段)。去除常见污染物后筛选出938种蛋白质进行后续的统计学分析和生物信息学分析。
OPLS-DA聚类分析结果显示，IVF组和FET组显著分离，说明两组间的蛋白表达水平存在显著性差异(图2)。通过学生t检验所得p值和OPLS-DA分析所得变量重要程度值VIP，在IVF组和FET组中共筛选出了16种差异表达蛋白(p<0.05,VIP<1)(表2)。其中，有4种差异表达蛋白在FET组中显著性上调，12种差异表达蛋白在FET组中显著性的下调(图3)。对差异表达蛋白进行层聚类分析，结果表明，差异表达蛋白被明显聚为上调和下调两类，结果与统计学分析结果相一致(图4)。
表2：IVF组和FET组之间的16种差异表达蛋白信息
通过基因本体论(GO)数据库对差异表达蛋白进行蛋白功能注释。注释结果如图5所示，在生物进程(BP)方面，胚胎蛋白主要参与了RNA聚合酶Ⅱ对转录的负向调节、信号传导、细胞黏附、细胞蛋白修饰和细胞增殖过程。在细胞成分(CC)方面，胚胎蛋白主要定位到了细胞外泌体、胞浆、细胞核、细胞膜、细胞外空间和核质体。在分子功能方面，胚胎蛋白主要与钙离子结合、酶结合和肌动蛋白酶结合相关，除此之外，还与一些酶的催化活性相关。通过对差异蛋白进行KEGG富集分析，我们发现差异表达蛋白主要集中在HIF-1信号传导途径、碳代谢、精氨酸的生物合成、糖酵解/葡萄糖生成和丙氨酸、天门冬氨酸和谷氨酸的代谢途径。通过String绘制差异表达蛋白的PPI网络图(如图6)，发现DSP、DSG1和DSC1三种蛋白的相互作用得分最高，说明冷冻保存过程中这三种蛋白共同作用调节胚胎发育。
上述结果能够说明，本申请的技术方案可以实现对单个胚胎定性定量的痕量蛋白质组学检测分析。
以上详细描述了本发明的较佳具体实施例。应当理解，本领域的普通技术无需创造性劳动就可以根据本发明的构思作出诸多修改和变化。因此，凡本技术领域中技术人员依本发明的构思在现有技术的基础上通过逻辑分析、推理或者有限的实验可以得到的技术方案，皆应在由权利要求书所确定的保护范围内。
Claims (10)
1.一种用于单个胚胎痕量蛋白提取的前处理方法，其特征在于，包括以下步骤：
(1)采集含有胚胎细胞的悬液；
(2)加入4倍体积的十二烷基β-D-麦芽苷(DDM)裂解缓冲液，在室温下水浴超声裂解1h；
(3)将步骤(2)裂解后的样品在金属浴中加热1h还原蛋白质，室温下黑暗处反应30min使蛋白质发生烷基化反应，向溶液中直接加入胰蛋白酶，用封口膜封口，进行酶解反应，放入37℃的烘箱过夜孵育12-16小时；
(4)向步骤(3)酶解后的样品加入终体积的1％的甲酸(FA)终止反应，涡旋混匀；
(5)通过Ziptip C18微量层析柱对样本进行脱盐处理；
(6)用真空干燥仪干燥样品，放置于-20℃备用。
2.根据权利要求1所述的前处理方法，其特征在于，所述裂解缓冲液为：40-60mM碳酸氢铵溶液、0.05-0.2％DDM、0.5-2mM三(2-羧基乙基)磷化氢(TECP)和1-3mM 2-氯乙酰胺(CAA)。
3.根据权利要求1所述的前处理方法，其特征在于，所述酶解步骤中W胰酶:W蛋白的比例为1:5-20。
4.权利要求1-3所述前处理方法筛选冷冻保存/新鲜体外受精胚胎间差异表达蛋白的方法，其特征在于，包括以下步骤：
(1)冷冻保存/新鲜胚胎培养；
(2)痕量胚胎蛋白提取与酶解；
(3)通过液相色谱-串联质谱联合技术进行非标定量的胚胎蛋白检测；
(4)筛选冷冻保存/新鲜胚胎之间的差异表达蛋白并进行生物信息学分析。
5.根据权利要求4所述的方法，其特征在于：所述胚胎来自绵羊，牛，小鼠，人，兔；更优选地，所述胚胎来自小鼠。
6.根据权利要求4所述的方法，其特征在于：步骤(3)对非标定量的胚胎蛋白检测还包括纳米液相色谱系统(NanoElute)分离流动相A溶解后的肽段，所述纳米液相色谱系统(NanoElute)分离流动相A的梯度洗脱方案为：0-75min，2％-22％的流动相B；75-80min，22％-37％的流动相B；85-90min，80％流动相B；流动相A为含0.1％甲酸的水溶液，流动相B为含0.1％甲酸的乙腈溶液，流速设置为300nL/min。
7.一种单个胚胎痕量蛋白提取的试剂盒，其特征在于，包含：M16培养液、裂解缓冲液、酶解液和终止反应液；
所述裂解缓冲液包含40-60mM碳酸氢铵溶液、0.05-0.2％DDM、0.5-2mM三(2-羧基乙基)磷化氢(TECP)和1-3mM 2-氯乙酰胺(CAA)；
所述酶解液包含胰蛋白酶；
所述终止反应液包含甲酸。
8.差异表达蛋白组合在鉴定冷冻保存/新鲜体外受精胚胎的应用，所述差异表达蛋白组合为GLUD1、TF、GAPDH、ANXA2、TXN、PIP、DSP、CASP14、COPS2、UBE2I、DSG1、TGM3、DSC1、BLMH、KBTBD8和SERPINB12中的一种或几种。
9.根据权利要求8所述的应用，其特征在于，所述差异表达蛋白组合为DSP、DSG1和DSC1。
10.根据权利要求8所述的应用，其特征在于：所述差异表达蛋白为GLUD1、TF、GAPDH、ANXA2、TXN、PIP、DSP、CASP14、COPS2、UBE2I、DSG1、TGM3、DSC1、BLMH、KBTBD8和SERPINB12。
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