WO2023103437A1 - 一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法 - Google Patents
一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法 Download PDFInfo
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Definitions
- the invention belongs to the technical field of biomolecular analysis reagents, and in particular relates to a solid-phase coupling glycopeptide and an analysis method for enzymatic cleavage and enrichment of Tn antigen by O-glycopeptide.
- Tn antigen (O-GalNAc), a tumor-associated carbohydrate, is highly expressed on the surface of most cancerous tissues. It has a small molecular weight and simple structure, and is usually expressed on the surface of cells, where it can be recognized as foreign by the immune system. Therefore, detecting the expression of Tn antigen not only reflects the situation of immunosuppressive microenvironment, but also can be used as a diagnostic marker of cancer—especially the characteristics of early stage, and can also show the progress and prognosis of tumor.
- the detection methods for Tn antigen mainly include monoclonal antibody, lectin enrichment, and click chemistry.
- the purpose of the present invention is to provide a method for analyzing Tn antigen based on solid phase enrichment combined with O-glycopeptidase digestion.
- a kind of technical scheme of the present invention is:
- a method for analyzing Tn antigen based on solid-phase enrichment combined with O-glycopeptidase digestion comprising steps:
- the protein extraction and concentration measurement include: after sequentially adding RIPA lysate, protease inhibitor and EDTA to the cells, use an ultrasonic breaker to break, cool, repeat the operation until the sample solution is clarified, centrifuge , Take the supernatant and dilute it with 1 ⁇ Tris, and use the Pierce BCA Protein Quantitative Assay Kit to test the protein concentration.
- step 2) the proteolysis includes steps:
- step 3 the purification of the polypeptide includes the steps of:
- step 4 the solid phase binding includes the steps of:
- the sialic acid treatment includes: adding 1 ⁇ PBS to the solid phase sample, and adjusting the pH to 5.5 with 1% TFA, then adding neuraminidase, incubating, vortexing, The solution was removed by centrifugation and the solid phase samples were eluted with 10% ACN + 0.1% TFA.
- step 5 the sialic acid treatment includes the steps of:
- step 6 the N-glycan excision includes the steps of:
- the OpeRATOR enzyme digestion includes: adding Tris-HCl to the solid phase sample, adjusting the pH to 6.8, then adding OpeRATOR for incubation, vortex centrifuging and collecting the liquid, using 5% ACN+ 0.1% TFA elutes and collects the liquid, combines all the collected liquids, purifies on a C18 silica gel column, and vacuum freeze-dries the solid phase sample;
- the O-Glycoprotease digestion includes: adding to the solid phase sample Tris-HCl, adjust the pH to 8.0, then add O-Glycoprotease, incubate, vortex centrifuge and collect the liquid, then use 5% ACN + 0.1% TFA to elute and collect the liquid, combine all the collected liquids, and then collect the liquid O-glycopeptide containing only Tn antigen, purified by C18 silica gel column, vacuum freeze-dried O-glycopeptide sample containing only Tn antigen.
- the LC-MS analysis includes: re-dissolving the O-glycopeptide sample containing only Tn antigen in 50% ACN+0.2% formic acid, and adding it to LC-MS, The obtained mass spectrometry data was combined with bioinformatics software to determine the Tn glycosylation site.
- the invention provides a method based on solid-phase enrichment combined with O-glycopeptidase cleavage to analyze Tn antigen, which can specifically enrich and analyze Tn antigen from complex protein samples, and can be used for Tn antigen in normal cells and cancer cells.
- Qualitative and quantitative analysis of antigens, analysis of Tn antigen glycosylation sites can also be used for qualitative and quantitative analysis of Tn antigens and sites in normal tissues and cancer tissues.
- This approach avoids the false positive and false negative rates associated with antibody and lectin enrichment and click chemistry.
- this method can be used for high-throughput processing and analysis of samples, and improves the processing efficiency of samples.
- Fig. 1 is a schematic diagram (asialic acid) based on solid-phase spherical aldehyde-binding glycopeptide in the present invention
- Fig. 2 is a schematic diagram of a solid-phase spherical aldehyde group-binding glycopeptide (protected sialic acid) in the present invention.
- one embodiment or “embodiment” referred to herein refers to a specific feature, structure or characteristic that may be included in at least one implementation of the present invention. "In one embodiment” appearing in different places in this specification does not all refer to the same embodiment, nor is it a separate or selective embodiment that is mutually exclusive with other embodiments.
- TFA 1000 ⁇ L TFA+1mL HPLC water
- a method for analyzing Tn antigen based on solid-phase enrichment combined with O-glycopeptidase digestion comprising the following steps:
- sample protein amount 400-600ug (referred to as the sample protein amount) and dissolve it in 8M urea, shake slightly until the protein is completely dissolved.
- FIG. 1 is a schematic diagram of a solid-phase spherical aldehyde group-binding glycopeptide (asialo) based on the present invention.
- asialo a solid-phase spherical aldehyde group-binding glycopeptide based on the present invention.
- 1 ⁇ PBS a solid-phase spherical aldehyde group-binding glycopeptide
- TFA neuraminidase
- the O-glycopeptide sample containing only Tn antigen was redissolved in 50% ACN+0.2% formic acid, added to LC-MS, and the mass spectrometry data obtained was combined with bioinformatics software (Byonic, Protein Metrics, USA) for Tn glycosyl Determination of chemical sites.
- a method for analyzing Tn antigen based on solid-phase enrichment combined with O-glycopeptidase digestion comprising the following steps:
- sample protein amount 400-600ug (referred to as the sample protein amount) and dissolve it in 8M urea, shake slightly until the protein is completely dissolved.
- FIG. 2 is a schematic diagram of a solid-phase spherical aldehyde group-binding glycopeptide (protected sialic acid) in the present invention.
- 1 wash the solid-phase sample once with 500 ⁇ L of ethanol
- 2 modify 2,6 sialic acid add 200 ⁇ L 0.5M EDC (1-(3-dimethylaminopropyl)- 3-Ethylcarbonyldiamine methyl iodide) and 0.5M HBot (1-hydroxybenzotriazole monohydrate), incubate at 37°C for 1-2h, vortex centrifuge to remove the supernatant, then wash once with 400 ⁇ L ethanol and 500 ⁇ L of deionized water to wash both sides of the solid phase; 3 2,3 sialic acid modification: add 400 ⁇ L 1M PT (p-phenylmethylamine) to the solid phase sample, then add 50 ⁇ L EDC and 11 ⁇ L 36-38% HCl (adjust
- the O-glycopeptide sample containing only Tn antigen was redissolved in 50% ACN+0.2% formic acid, added to LC-MS, and the mass spectrometry data obtained was combined with bioinformatics software (Byonic, Protein Metrics, USA) for Tn glycosyl Determination of chemical sites.
- the beneficial effect of the present invention is: the present invention provides a method based on solid phase enrichment combined with O-glycopeptidase digestion analysis of Tn antigen, coupling glycopeptides on the solid phase, using two O-glycopeptides - Glycopeptidase cleavage of O-glycopeptide to identify the O-glycosylation site of Tn antigen and the expression level of Tn antigen, which are used as biomarkers for cancer diagnosis.
Abstract
一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,包括步骤:蛋白提取和浓度测量;蛋白酶解;多肽纯化;固相结合;唾液酸处理;N聚糖切除;OpeRATOR酶切;O-Glycoprotease酶切;LC-MS分析。基于固相富集结合O-糖肽酶切分析Tn抗原的方法能够在固相上耦合糖肽,使用两种O-糖肽酶切O-糖肽来鉴定Tn抗原的O-糖基化位点和Tn抗原的表达量,用作癌症诊断的生物标志物。
Description
本发明属于生物分子分析试剂技术领域,具体涉及固相偶联糖肽和采用O-糖肽酶切富集Tn抗原的分析方法。
Tn抗原(O-GalNAc)是一种与肿瘤相关的碳水化合物,在大多数癌组织表面高表达。它的分子量小、结构简单、通常表达在细胞表面,可被免疫系统识别为外来物。因此,检测Tn抗原的表达不仅反映免疫抑制微环境的情况,并且可作为癌症的诊断标志物-特别是早期阶段的特征,也可显示肿瘤的进展以及预后状况。目前对于Tn抗原的检测手段主要有单克隆抗体、凝集素富集以及点击化学等。这些方法大多缺乏特异性和灵敏度,易造成结果的假阳性和假阴性率,且步骤繁琐、成本高。点击化学的催化效率低且重现性差。无铜点击化学生物素方法,所用试剂较为昂贵且叠氮化物剧毒、易爆炸。目前缺乏一种高效便捷的方法能够检测Tn抗原的表达及鉴定其在标志物中的特异性位点。
因此,有必要研发一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法来解决现有技术中鉴定Tn抗原糖蛋白标志物的问题。
发明内容
本发明目的是提供一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法。
本发明的一种技术方案是:
一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,包括步骤:
1)蛋白提取和浓度测量;
2)蛋白酶解;
3)多肽纯化;
4)固相结合;
5)唾液酸处理;
6)N聚糖切除;
7)OpeRATOR酶切;
8)O-Glycoprotease酶切;
9)LC-MS分析。
进一步的,在步骤1)中,所述蛋白提取和浓度测量包括:在细胞中依次加入RIPA裂解液、蛋白酶抑制剂和EDTA后,使用超声破碎仪破碎,冷却,重复操作直至样本溶液澄清,离心,取上层清液并用1×Tris稀释,使用Pierce BCA蛋白定量分析试剂盒测试蛋白的浓度。
进一步的,在步骤2)中,所述蛋白酶解包括步骤:
(1)根据测定的蛋白的浓度,从上层清液中取蛋白,使得所述蛋白的总量在400-600ug,溶于尿素,轻微振荡,直至蛋白完全溶解,获得样品溶液;
(2)向所述样品溶液中加入总体积1/10的TCEP,孵育;
(3)向所述样品溶液中加入总体积1/10的IAA,置于暗箱中孵育;
(4)向所述样品溶液中加入HPLC水,使得所述样品溶液中尿素的浓度<1.6M,调节所述样品溶液的pH至7-9;
(5)向所述样品溶液中加入胰蛋白酶,使得所述样品溶液的蛋白量与所述胰蛋白酶的体积比为50:1~40:1,孵育过夜。
进一步的,在步骤3)中,所述多肽纯化包括步骤:
(1)向所述样品溶液中加入甲酸,使所述样品溶液的pH<3;
(2)将所述样品溶液加入预先处理的C18萃取柱中,重复操作;
(3)使用0.1%TFA多次清洗萃取柱;
(4)使用50%ACN+0.1%TFA洗脱C18萃取柱中的多肽并收集洗脱液,重复操作;
(5)将多次多肽的洗脱液合并真空冷冻干燥获得多肽样品。
进一步的,在步骤4)中,所述固相结合包括步骤:
(1)配置缓冲液体系,测试pH 10-11,将所述缓冲液体系稀释;
(2)取表面具有醛基的球状树脂的固相,加入所述缓冲液体系预处理,去掉过滤液,重复操作两次;
(3)将所述多肽样品重新溶于去离子水中,涡旋振荡,并加入具有缓冲液体系的固相中,涡旋,并孵育、离心去除溶液,获得固相样品;
(4)向所述固相样品中加入NaCNBH
3,涡旋,孵育后离心去除溶液;
(5)使用PBS涡旋离心去除溶液,清洗;
(6)向所述固相样品中加入PBS和NaCNBH
3,孵育,涡旋离心去除溶液;
(7)使用Tris-HCl清洗所述固相样品,再加入NaCNBH
3和1×Tris,孵育,分别使用NaCl和去离子水清洗所述固相样品。
进一步的,在步骤5)中,所述唾液酸处理包括:向所述固相样品中加入1×PBS,并用1%TFA调节pH为5.5,再加入神经氨酸苷酶,孵育,涡旋,离心去除溶液,并用10%ACN+0.1%TFA洗脱所述固相样品。
进一步的,在步骤5)中,所述唾液酸处理,包括步骤:
(1)用乙醇清洗所述固相样品;
(2)进行2,6唾液酸修饰:向所述固相样品中加入EDC和HBot,孵育,涡旋离心去除上清液后,再用乙醇和去离子水清洗所述固相样品;
(3)进行2,3唾液酸修饰:向所述固相样品中加入PT,再加入EDC和HCl,调节pH为4-6,在室温下孵育,分别使用甲酸、乙腈、NaCl和去离子水清洗。
进一步的,在步骤6)中,所述N聚糖切除包括步骤:
(1)向所述固相样品中先加入NH
4HCO
3,再加入N-糖苷内切酶,孵育,涡旋离心去除上清液;
(2)向所述固相样品中加入ACN,涡旋并离弃上清液。
进一步的,在步骤7)中,所述OpeRATOR酶切包括:向所述固相样品中加入Tris-HCl,调节pH为6.8,再加入OpeRATOR孵育,涡旋离心并收集液体,使用5%ACN+0.1%TFA洗脱并收集液体,将所有收集液体合并,C18硅胶柱纯化,真空冷冻干燥固相样品;在步骤8)中,所述O-Glycoprotease酶切包括:向所述固相样品中加入Tris-HCl,调节pH为8.0,再加入O-Glycoprotease,孵育,涡旋离心并收集液体,再使用5%ACN+0.1%TFA洗脱并收集液体,将所有收集液体合并,此时收集液体中只含有Tn抗原的O-糖肽,C18硅胶柱纯化,真空冷冻干燥只含有Tn抗原的O-糖肽样品。
进一步的,在步骤9)中,所述LC-MS分析包括:将所述只含有Tn抗原的O-糖肽样品重新溶于50%ACN+0.2%甲酸,并加入至LC-MS中,将得到的质谱数据结合生物信息学软件进行Tn糖基化位点的确定。
本发明提供了一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,能从复杂的蛋白样本中,特异性富集Tn抗原并分析,可用于正常细胞和癌细胞中Tn抗原的定性和定量分析,Tn抗原糖基化位点的分析,也可用于正常组织与癌症组织中Tn抗原以及位点的定性和定量分析。这种方法可避免抗体和凝集素富集以及点击化学带来的假阳性率和假阴性率。同时此方法可用于高通量处理和分析样本,提高样本的处理效率。
为了更清楚地说明本发明实施例的技术方案,下面将对实施例描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动性的前提下,还可以根据这些附图获得其它的附图。其中,
图1为本发明中基于固相球形醛基结合糖肽的示意图(去唾液酸);
图2为本发明中基于固相球形醛基结合糖肽的示意图(保护唾液酸)。
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合附图和实施例进一步说明本发明的技术方案。但是本发明不限于所列出的实施例,还应包括在本发明所要求的权利范围内其他任何公知的改变。
首先,此处所称的“一个实施例”或“实施例”是指可包含于本发明至少一个实现方式中的特定特征、结构或特性。在本说明书中不同地方出现的“在一个实施例中”并非均指同一个实施例,也不是单独的或选择性的与其他实施例互相排斥的实施例。
其次,本发明利用结构示意图等进行详细描述,在详述本发明实施例时,为便于说明,示意图会不依一般比例作局部放大,而且所述示意图只是实例,其在此不应限制本发明保护的范围。此外,在实际制作中应包含长度、宽度及深度的三维空间。
最后,本发明中涉及到的百分比(%)均为体积比,M是指浓度mol/L,
例如:0.1%TFA=1000μL TFA+1mL HPLC水;
50%ACN=500mL ACN+500mL HPLC水;
1M NaCNBH
3=62.8mg NaCNBH
3+1mL HPLC水;
5mL 50%ACN+0.1%TFA=2.5mL ACN+5uL TFA+2495uL HPLC水。
实施例1
一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,包括如下步骤:
(1)蛋白提取和浓度测量
在细胞中依次加入900-1000μL的1X RIPA裂解液(Cell Signaling,上海),8-12μL的50X蛋白酶抑制剂(Promega,Madison,WI,美国),8-12μL的EDTA后,使用30-40%能量的超声破碎仪(最大能量为100%)破碎30s,在冰上冷却30s,重复操作4-6次,直至样本溶液澄清。接着,以12000rpm,4℃条件下离心10min,取上层清液并用1×Tris稀释5-10倍。最后使用Pierce BCA蛋白定量分析试剂盒(Thermo Fisher Scientific,Walham,MA,美国)测试蛋白的浓度。
(2)蛋白酶解
①根据测定的蛋白的浓度,从上层清液中取一定体积的蛋白,使得蛋白总量在400-600ug(称之为样品蛋白量)溶于8M尿素,轻微振荡,直至蛋白完全溶解。
②向样品溶液中加入总体积1/10的120mM TCEP,37℃孵育1h;
③向样品溶液中加入总体积1/10的160mM IAA,置于暗箱中,室温孵育30-60min;
④向样品溶液中加入5倍HPLC水(使得样品溶液中尿素的浓度<1.6M),再使用现配1M NH
4HCO
3去调节样品溶液的pH至7-9;
⑤向样品溶液中加入10μg/μL的胰蛋白酶(Promega,Madison,WI,美国),按样品溶液的蛋白量与胰蛋白酶体积比为50:1至40:1。并在37℃条件下,孵育过夜。
(3)多肽纯化
①向样品溶液中加入10-15μL的甲酸(纯度>98%),将样品溶液调至pH<3;
②将样品溶液加入预先处理的C18萃取柱中,重复操作1次;
③使用1mL的0.1%TFA(三氟乙酸)清洗萃取柱6-9次;
④使用300μL的50%ACN+0.1%TFA洗脱C18萃取柱中的多肽并收集 洗脱液,重复1次;
⑤将两次多肽的洗脱液合并真空冷冻干燥多肽样品。
(4)固相结合
①配置10M的缓冲液体系,即11.76g柠檬酸钠和2.12克g碳酸钠加入400mL去离子水,得到100mM柠檬酸钠和50mM碳酸钠,测试pH 10-11。使用时,将该缓冲液稀释10倍;
②取150-200μL表面具有醛基的球状树脂(Thermo Fisher Scientific,Waltham,MA,美国)的固相,加入到1.5-2.0mL离心管,加入500μL 1M的缓冲液预处理,去掉过滤液,重复操作两次;
③将干燥的多肽样品重新溶于425μL的去离子水中,涡旋振荡,再加入具有50μL 10M的缓冲液体系的固相中,涡旋,并孵育4h后离心去除溶液;
④接着,再向固相样品中加入25μL 1M NaCNBH
3,涡旋,孵育4h后离心去除溶液;
⑤再使用500μL 1M PBS涡旋离心去溶液,清洗两次;
⑥再向固相样品中加入475μL 1M PBS和25μL 1M NaCNBH
3,孵育4h,涡旋离心去除溶液;
⑦使用1M Tris-HCl清洗固相样品两次,再加入25μL 1M NaCNBH
3和475μL 1×Tris,孵育30-60min,其目的是为了阻断固相上醛基未结合的部分。最后再分别使用1M NaCl和去离子水清洗固相样品两遍。
(5)唾液酸处理(去唾液酸)
请参阅图1,图1为本发明中基于固相球形醛基结合糖肽的示意图(去唾液酸)。如图1所示,向固相样品中加入一定量的1×PBS,并用1%TFA调节pH为5.5后,再加入5-10μL神经氨酸苷酶(NA),37℃,孵育1h,涡旋,离心去除溶液。并用10%ACN(0.1%TFA)洗固相一次。
(6)N聚糖切除
①向固相样品中先加入200μL 25mM NH
4HCO
3,再加入0.5μL N-糖苷内切酶(PNGaseF),37℃条件下孵育2h或者过夜后,涡旋离心去除上清液。
②向固相样品中加入100μl 10%ACN,涡旋并离心去除上清液。
(7)OpeRATOR酶切
向固相样品中加入150-200μL的20mM Tris-HCl(pH=6.8),再加入 20-30U的OpeRATOR(Genovis,瑞典),37℃,孵育2h后,涡旋离心并收集液体。再使用5%ACN+0.1%TFA洗脱两次并收集液体。将这两收集的液体合并,C18硅胶柱纯化(重复(3)步骤),真空冷冻干燥样品。
(8)O-Glycoprotease酶切
再向固相样品中加入150-200μL 20mM Tris-HCl(pH=8.0),再加入20-30U的O-Glycoprotease(IMPa,New England BioLabs,MA,美国),37℃,孵育5h后,涡旋离心并收集液体。再使用5%ACN+0.1%TFA洗脱两次并收集液体。将这两次收集的液体合并(此种溶液中只含有Tn抗原的O-糖肽),C18硅胶柱纯化(重复(3)步骤),真空冷冻干燥只含有Tn抗原的O-糖肽样品。
(9)LC-MS分析
将只含有Tn抗原的O-糖肽样品重新溶于50%ACN+0.2%甲酸,加入至LC-MS,得到的质谱数据并结合生物信息学软件(Byonic,Protein Metrics,USA)进行Tn糖基化位点的确定。
实施例2
一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,包括如下步骤:
(1)蛋白提取和浓度测量
在细胞中依次加入900-1000μL的1X RIPA裂解液(Cell Signaling,上海),8-12μL的50X蛋白酶抑制剂(Promega,Madison,WI,美国),8-12μL的EDTA后,使用30-40%能量的超声破碎仪(最大能量为100%)破碎30s,在冰上冷却30s,重复操作4-6次,直至样本溶液澄清。接着,以12000rpm,4℃条件下离心10min,取上层清液并用1×Tris稀释5-10倍。最后使用Pierce BCA蛋白定量分析试剂盒(Thermo Fisher Scientific,Walham,MA,美国)测试蛋白的浓度。
(2)蛋白酶解
①根据测定的蛋白的浓度,从上层清液中取一定体积的蛋白,使得蛋白总量在400-600ug(称之为样品蛋白量)溶于8M尿素,轻微振荡,直至蛋白完全溶解。
②向样品溶液中加入总体积1/10的120mM TCEP,37℃孵育1h;
③向样品溶液中加入总体积1/10的160mM IAA,置于暗箱中,室温孵 育30-60min;
④向样品溶液中加入5倍HPLC水(使得样品溶液中尿素的浓度<1.6M),再使用现配1M NH
4HCO
3去调节样品溶液的pH至7-9;
⑤向样品溶液中加入10μg/μL的胰蛋白酶(Promega,Madison,WI,美国),按样品溶液的蛋白量与胰蛋白酶体积比为50:1至40:1。并在37℃条件下,孵育过夜。
(3)多肽纯化
①向样品溶液中加入10-15μL的甲酸(纯度>98%),将样品溶液调至pH<3;
②将样品溶液加入预先处理的C18萃取柱中,重复操作1次;
③使用1mL的0.1%TFA(三氟乙酸)清洗萃取柱6-9次;
④使用300μL的50%ACN+0.1%TFA洗脱C18萃取柱中的多肽并收集洗脱液,重复1次;
⑤将两次多肽的洗脱液合并真空冷冻干燥多肽样品。
(4)固相结合
①配置10M的缓冲液体系,即11.76g柠檬酸钠和2.12克g碳酸钠加入400mL去离子水,得到100mM柠檬酸钠和50mM碳酸钠,测试pH 10-11。使用时,将该缓冲液稀释10倍;
②取150-200μL表面具有醛基的球状树脂(Thermo Fisher Scientific,Waltham,MA,美国)的固相,加入到1.5-2.0mL离心管,加入500μL 1M的缓冲液预处理,去掉过滤液,重复操作两次;
③将干燥的多肽样品重新溶于425μL的去离子水中,涡旋振荡,再加入具有50μL 10M的缓冲液体系的固相中,涡旋,并孵育4h后离心去除溶液;
④接着,再向固相样品中加入25μL 1M NaCNBH
3,涡旋,孵育4h后离心去除溶液;
⑤再使用500μL 1M PBS涡旋离心去溶液,清洗两次;
⑥再向固相样品中加入475μL 1M PBS和25μL 1M NaCNBH
3,孵育4h,涡旋离心去除溶液;
⑦使用1M Tris-HCl清洗固相样品两次,再加入25μL 1M NaCNBH
3和475μL 1×Tris,孵育30-60min,其目的是为了阻断固相上醛基未结合的部分。 最后再分别使用1M NaCl和去离子水清洗固相样品两遍。
(5)唾液酸处理(唾液酸保护)
请参阅图2,图2为本发明中基于固相球形醛基结合糖肽的示意图(保护唾液酸)。如图2所示,①用500μL的乙醇清洗固相样品一遍;②进行2,6唾液酸修饰:向固相样品中加入200μL 0.5M EDC(1-(3-二甲基氨丙基)-3-乙基羰基二胺甲碘)和0.5M HBot(1-羟基苯并三唑一水物),37℃,孵育1-2h,涡旋离心去上清后,再用400μL乙醇洗一次和500μL的去离子水洗两边固相;③进行2,3唾液酸修饰:向固相样品中加入400μL 1M PT(对苯甲胺),再加入50μL EDC和11μL 36-38%HCl(调节pH为4-6),室温下孵育4h或过夜。接着,分别使用500μL 10%甲酸洗3次,10%乙腈洗3次,1M NaCl洗两次,去离子水洗3次。
(6)N聚糖切除
①向固相样品中先加入200μL 25mM NH
4HCO
3,再加入0.5μL N-糖苷内切酶(PNGaseF),37℃条件下孵育2h或者过夜后,涡旋离心去除上清液。
②向固相样品中加入100μl 10%ACN,涡旋并离心去除上清液。
(7)OpeRATOR酶切
向固相样品中加入150-200μL的20mM Tris-HCl(pH=6.8),再加入20-30U的OpeRATOR(Genovis,瑞典),37℃,孵育2h后,涡旋离心并收集液体。再使用5%ACN+0.1%TFA洗脱两次并收集液体。将这两收集的液体合并,C18硅胶柱纯化(重复(3)步骤),真空冷冻干燥样品。
(8)O-Glycoprotease酶切
再向固相样品中加入150-200μL 20mM Tris-HCl(pH=8.0),再加入20-30U的O-Glycoprotease(IMPa,New England BioLabs,MA,美国),37℃,孵育5h后,涡旋离心并收集液体。再使用5%ACN+0.1%TFA洗脱两次并收集液体。将这两次收集的液体合并(此种溶液中只含有Tn抗原的O-糖肽),C18硅胶柱纯化(重复(3)步骤),真空冷冻干燥只含有Tn抗原的O-糖肽样品。
(9)LC-MS分析
将只含有Tn抗原的O-糖肽样品重新溶于50%ACN+0.2%甲酸,加入至LC-MS,得到的质谱数据并结合生物信息学软件(Byonic,Protein Metrics, USA)进行Tn糖基化位点的确定。
与现有技术相比,本发明的有益效果是:本发明提供了一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,在固相上耦合糖肽,使用两种O-糖肽酶切O-糖肽来鉴定Tn抗原的O-糖基化位点和Tn抗原的表达量,用作癌症诊断的生物标志物。
应说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的精神和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (10)
- 一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,包括步骤:1)蛋白提取和浓度测量;2)蛋白酶解;3)多肽纯化;4)固相结合;5)唾液酸处理;6)N聚糖切除;7)OpeRATOR酶切;8)O-Glycoprotease酶切;9)LC-MS分析。
- 根据权利要求1所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤1)中,所述蛋白提取和浓度测量包括:在细胞中依次加入RIPA裂解液、蛋白酶抑制剂和EDTA后,使用超声破碎仪破碎,冷却,重复操作直至样本溶液澄清,离心,取上层清液并用1×Tris稀释,使用Pierce BCA蛋白定量分析试剂盒测试蛋白的浓度。
- 根据权利要求2所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤2)中,所述蛋白酶解包括步骤:(1)根据测定的蛋白的浓度,从上层清液中取蛋白,使得所述蛋白的总量在400-600ug,溶于尿素,轻微振荡,直至蛋白完全溶解,获得样品溶液;(2)向所述样品溶液中加入总体积1/10的TCEP,孵育;(3)向所述样品溶液中加入总体积1/10的IAA,置于暗箱中孵育;(4)向所述样品溶液中加入HPLC水,使得所述样品溶液中尿素的浓度<1.6M,调节所述样品溶液的pH至7-9;(5)向所述样品溶液中加入胰蛋白酶,使得所述样品溶液的蛋白量与所述胰蛋白酶的体积比为50:1~40:1,孵育过夜。
- 根据权利要求3所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤3)中,所述多肽纯化包括步骤:(1)向所述样品溶液中加入甲酸,使所述样品溶液的pH<3;(2)将所述样品溶液加入预先处理的C18萃取柱中,重复操作;(3)使用0.1%TFA多次清洗萃取柱;(4)使用50%ACN+0.1%TFA洗脱C18萃取柱中的多肽并收集洗脱液,重复操作;(5)将多次多肽的洗脱液合并真空冷冻干燥获得多肽样品。
- 根据权利要求4所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤4)中,所述固相结合包括步骤:(1)配置缓冲液体系,测试pH 10-11,将所述缓冲液体系稀释;(2)取表面具有醛基的球状树脂的固相,加入所述缓冲液体系预处理,去掉过滤液,重复操作两次;(3)将所述多肽样品重新溶于去离子水中,涡旋振荡,并加入具有缓冲液体系的固相中,涡旋,并孵育、离心去除溶液,获得固相样品;(4)向所述固相样品中加入NaCNBH 3,涡旋,孵育后离心去除溶液;(5)使用PBS涡旋离心去除溶液,清洗;(6)向所述固相样品中加入PBS和NaCNBH 3,孵育,涡旋离心去除溶液;(7)使用Tris-HCl清洗所述固相样品,再加入NaCNBH 3和1×Tris,孵育,分别使用NaCl和去离子水清洗所述固相样品。
- 根据权利要求5所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤5)中,所述唾液酸处理包括:向所述固相样品中加入1×PBS,并用1%TFA调节pH为5.5,再加入神经氨酸苷酶,孵育,涡旋,离心去除溶液,并用10%ACN+0.1%TFA洗脱所述固相样品。
- 根据权利要求5所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤5)中,所述唾液酸处理,包括步骤:(1)用乙醇清洗所述固相样品;(2)进行2,6唾液酸修饰:向所述固相样品中加入EDC和HBot,孵育,涡旋离心去除上清液后,再用乙醇和去离子水清洗所述固相样品;(3)进行2,3唾液酸修饰:向所述固相样品中加入PT,再加入EDC和HCl,调节pH为4-6,在室温下孵育,分别使用甲酸、乙腈、NaCl和去离子水清洗。
- 根据权利要求6或7所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤6)中,所述N聚糖切除包括步骤:(1)向所述固相样品中先加入NH 4HCO 3,再加入N-糖苷内切酶,孵育,涡旋离心去除上清液;(2)向所述固相样品中加入ACN,涡旋并离弃上清液。
- 根据权利要求8所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤7)中,所述OpeRATOR酶切包括:向所述固相样品中加入Tris-HCl,调节pH为6.8,再加入OpeRATOR孵育,涡旋离心并收集液体,使用5%ACN+0.1%TFA洗脱并收集液体,将所有收集液体合并,C18硅胶柱纯化,真空冷冻干燥固相样品;在步骤8)中,所述O-Glycoprotease酶切包括:向所述固相样品中加入Tris-HCl,调节pH为8.0,再加入O-Glycoprotease,孵育,涡旋离心并收集液体,再使用5%ACN+0.1%TFA洗脱并收集液体,将所有收集液体合并,此时收集液体中只含有Tn抗原的O-糖肽,C18硅胶柱纯化,真空冷冻干燥只含有Tn抗原的O-糖肽样品。
- 根据权利要求9所述的一种基于固相富集结合O-糖肽酶切分析Tn抗原的方法,其特征在于,在步骤9)中,所述LC-MS分析包括:将所述只含有Tn抗原的O-糖肽样品重新溶于50%ACN+0.2%甲酸,并加入至LC-MS中,将得到的质谱数据结合生物信息学软件进行Tn糖基化位点的确定。
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