CN114577956A - 二氧化钛富集-在线酶切-分步洗脱糖肽和磷酸化肽方法 - Google Patents
二氧化钛富集-在线酶切-分步洗脱糖肽和磷酸化肽方法 Download PDFInfo
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- CN114577956A CN114577956A CN202011398706.XA CN202011398706A CN114577956A CN 114577956 A CN114577956 A CN 114577956A CN 202011398706 A CN202011398706 A CN 202011398706A CN 114577956 A CN114577956 A CN 114577956A
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- titanium dioxide
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- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
本发明为二氧化钛富集‑在线酶切‑分步洗脱糖肽和磷酸化肽方法,通过一种在线酶切的方法,在二氧化钛材料上实现了糖肽和磷酸化肽的同时富集和顺序洗脱。该方法是将糖肽和磷酸化肽样品与二氧化钛材料特异性结合后,先不加入洗脱溶液对糖肽/磷酸化肽进行洗脱(两种修饰肽段容易出现在同一个洗脱馏分中、即同时被洗脱出),直接加入糖苷酶和酶切溶液,即材料、样品、酶及酶切溶液同时存在的条件下‑在线切除糖肽(结合在材料上的)的糖链,再通过两种洗脱溶液,分别从材料上得到含去糖链糖肽的馏分、含磷酸化肽的馏分。在蛋白标准品和复杂生物样品(如鼠脑)中实现了糖肽和磷酸化肽的同时、高效、高选择性的分步富集。
Description
技术领域
本发明涉及二氧化钛材料在翻译后修饰蛋白组学的应用,具体说是一种利用在线酶切实现糖肽和磷酸化肽在二氧化钛上的同时富集和分步洗脱。
背景技术
蛋白质的糖基化和磷酸化修饰是两种非常重要翻译后修饰。这两种常见的翻译后修饰因修饰位点相同可产生相互影响,与信号转导、阿尔兹海默症中Tau 蛋白缠结等生理病理活动密切相关,同时研究它们的生物学意义重大。目前蛋白质的糖基化和磷酸化修饰研究主要依赖质谱。由于复杂生物样品中糖肽和磷酸化肽含量很低,且受高丰度非修饰肽的信号抑制,所以在质谱分析前必须对目标肽段进行富集。而已有报道能富集糖肽和磷酸化肽的材料中有自制二氧化钛微球、键合有二氧化钛的氨基硅胶、键合上硼酸和二氧化钛的磁性纳米片、固定钛离子 -聚合壳聚糖的纳米聚合物材料和植酸修饰固定钛离子的磁性石墨烯等等。这些材料都能实现糖肽/磷酸化肽的高效选择性富集,其中二氧化钛用得较多。而在能同时富集糖肽和磷酸化肽的材料中,大部分是共富集共洗脱。这样处理的样品存在两个问题:1.样品复杂,需要采用二维分离降低样品复杂度,才能在质谱分析获取更多的信息2.磷酸化肽在碱性条件下(共洗脱馏分中糖肽需要糖苷酶处理后才能进质谱分析)会水解。因此,为了使这些材料能实现同时富集分步洗脱糖肽和磷酸化肽,从而能广泛应用于复杂生物样品的分离分析中,急需发明一种简洁通用,能保证糖肽和磷酸化肽的选择性却又能将两者分步洗脱的富集方法。
发明内容
为解决上述技术问题,发展了一种在线酶切法实现二氧化钛同时富集分步洗脱糖肽和磷酸化肽。其关键步骤即在二氧化钛与糖肽、磷酸化肽结合后,混入糖苷酶在线酶解,通过将糖肽上的糖链从糖肽上切除,实现糖肽和磷酸化肽的分步分离。
本发明涉及的材料为表面成分是纳米材料、微球材料以及金属氧化物涂覆或包覆的球形或无定形二氧化钛材料。其中二氧化钛由金红石和锐钛矿组成、合成后材料pH 1.5-11.5、密度1.74~4.3范围内。
材料所用方法的操作模式为分散固相萃取(dSPE)/或固相萃取(SPE)/或超滤管过滤,具体方案如下:
⑴在60-90%有机相(V/V,后同)、pH在0.5-5.范围内的溶液条件下将含糖肽、磷酸化肽样品上样至二氧化钛材料(材料质量/样品质量>1),采用5-2000 柱体积内的上样溶液条件,淋洗去除非修饰肽段;
⑵加入2-100柱体积、pH在3-7之间、5-200mM的盐溶液(含或不含酸、 50%以下比例的有机相),再混入一种或多种糖苷酶,于20-60℃下在线酶切,时间控制在48h以内。
⑶加入2-200柱体积、20-55%有机相、pH在0.5-5范围内溶液洗脱去糖链肽段。
⑷加入2-200柱体积、0-55%有机相、pH在8-12范围内溶液洗脱磷酸化肽。
本专利通过引入在线酶切步骤,在二氧化钛材料上进行了糖肽和磷酸化肽的同时富集和顺序洗脱。同时,对在线酶切步骤中酶切溶液的pH和盐溶液浓度、酶切时间、温度、体积等条件进行优化,最终将该方法用于蛋白标准品和复杂生物样品(如鼠脑),实现了糖肽和磷酸化肽的同时、高效、高选择性的分步富集。
本发明具有如下优势:
1.在二氧化钛材料上实现糖肽和磷酸化肽的同时上样和分步洗脱;
2.该方法不仅可用于蛋白标准品的富集分离,也可应用于实际样品的分离检测;
3.本发明比无在线酶切共洗脱糖肽和磷酸化肽方法,仅增加了糖肽洗脱一个步骤,且富集得到的糖肽获得的糖肽更多,能显著提高二氧化钛富集糖肽的效率;
4.可用于微量样品的分离检测。
附图说明
图1为SPE模式下经二氧化钛共富集后,a-酪蛋白酶解产物中的磷酸化肽质谱信号示意图。
图2为SPE模式下经二氧化钛共富集后,牛胎球蛋白和a-酪蛋白酶解产物中的糖肽和磷酸化肽质谱信号示意图。
图3为dSPE&SPE混合模式下,经二氧化钛同时富集分步洗脱后,牛胎球蛋白和a-酪蛋白酶解产物中的糖肽质谱信号示意图。
图4为dSPE&SPE混合模式下经二氧化钛同时富集分步洗脱后,牛胎球蛋白和a-酪蛋白酶解产物中的磷酸化肽质谱信号示意图。
图5为不同溶液作为酶切溶液时,二氧化钛在线酶切法同时富集牛胎球蛋白和a-酪蛋白酶解产物中的糖肽质谱信号示意图。
图6为不同浓度碳酸氢铵作为去糖链糖肽洗脱溶液时,二氧化钛在线酶切法同时富集牛胎球蛋白和a-酪蛋白酶解产物中的糖肽质谱信号示意图。
图7为50%乙腈/体积浓度1%甲酸的水溶液(V/V)去糖链糖肽洗脱溶液、每次用40μL洗脱三次时,二氧化钛在线酶切法同时富集牛胎球蛋白和a-酪蛋白酶解产物中的糖肽质谱信号示意图。
具体实施方式
以下结合具体实施例进一步说明,这些实施例仅用于说明本发明,而本发明不仅限于以下实施例。
实施例材料和样品准备
本发明以商品化二氧化钛(GLScience,5020-75000)为富集材料、糖蛋白和磷酸化蛋白酶解液为样品,分别富集、同时富集同时洗脱、同时富集分步洗脱糖肽和磷酸化肽。
糖肽和磷酸化肽标准品的制备:每1mg的蛋白(糖蛋白或磷酸化蛋白)溶解在100μL含6mol/L尿素的50mM碳酸氢铵中,加入5μL 200mmol/L的二硫苏糖醇(Dithiothreitol;简称DTT)溶液,56℃震荡45min,然后加入20μ L200mmol/L碘代乙酸(iodoacetic acid;简称IAA),室温避光静置30min。加入725μL 50mM碳酸氢铵后,按蛋白与胰蛋白酶质量比1∶40(w/w)加入胰蛋白酶, 37℃酶解过夜,最后加入终浓度为0.5%(v/v)纯甲酸终止酶解,获得浓度为1 mg/mL的蛋白酶解液即为肽标准品(糖蛋白牛胎球蛋白酶解后得到糖肽标准品,磷酸化蛋白a-酪蛋白酶解后得到磷酸化肽标准品),分装冻存用于后续富集。
实施例1
二氧化钛材料富集糖肽标准品。将5ug(5μL)牛胎球蛋白酶解液去盐[1](作为分析样品),调整乙腈和酸浓度至体积浓度80%乙腈/5%三氟乙酸水溶液(含1M羟基乙酸)40μL,与1mg二氧化钛材料(购自GL Science公司,粒径5um) 混匀上样,装入eppendorf吸头中制成微型SPE小柱(SPE模式),用40μL的体积浓度80%乙腈/5%三氟乙酸的水(含1M羟基乙酸)溶液淋洗一次,再用 40μL体积浓度60%乙腈/0.1%甲酸的水溶液淋洗一次;最后用40μL含5%氨水(质量浓度)的水溶液洗脱两次;洗脱液旋干重溶至20μL入质谱进行分析。洗脱液中能看见较多数量的糖肽峰,二氧化钛材料可单独富集糖肽。
实施例2
二氧化钛材料富集磷酸化肽标准品。将2ug(2μL)a-酪蛋白酶解液去盐(作为分析样品),操作过程如实施例1条件进行富集。洗脱液中能看见明显的磷酸化肽质谱峰,二氧化钛单独富集磷酸化肽效果也很稳定(见图1)。
实施例3
二氧化钛材料同时富集同时洗脱糖肽和磷酸化肽标准品。将5ug(5μL)牛胎球蛋白酶解液和5ug(5μL)a-酪蛋白酶解液混合去盐(作为分析样品),操作过程如实施例1条件进行富集。洗脱液中能看见磷酸化肽及少量糖肽。二氧化钛同时富集糖肽和磷酸化肽同时洗脱会导致糖肽的损失。
实施例4
二氧化钛材料同时富集同时洗脱糖肽和磷酸化肽标准品、洗脱后的样品经糖苷酶处理。将5ug(5μL)牛胎球蛋白酶解液和5ug(5μL)a-酪蛋白酶解液混合去盐(作为分析样品),操作过程如实施例1步骤进行富集。洗脱馏分(含糖肽和磷酸化肽)再经PNGase F酶5U(购自NEB)37℃酶切16h,去盐后进质谱分析(见图2)。二氧化钛同时富集糖肽和磷酸化肽和共同洗脱,所得馏分再经糖苷酶酶切处理后进质谱分析。结果中能看见磷酸化肽(绿色圆圈标记)和去糖链的糖肽(绿色星星标记),还有一些干扰肽段(未标记质谱峰)。
实施例5
二氧化钛材料同时富集-在线酶切-分步洗脱糖肽和磷酸化肽标准品。将5ug (5μL)牛胎球蛋白和5ug(5μL)a-酪蛋白混合去盐(作为分析样品),调整至 40μL的体积浓度80%乙腈/5%三氟乙酸的水(含1M羟基乙酸)溶液。然后转移至放有1mg二氧化钛材料(购自GL Science公司,粒径5um)的EP管中,即在dSPE模式下,混匀上样。再用体积浓度80%乙腈/5%三氟乙酸的水(含 1M羟基乙酸)溶液淋洗40μL,加入50μL50 mM乙酸铵溶液,再加入PNGase F酶500U(购自NEB)在线酶切16h。酶解结束后混匀,装填成微型SPE小柱。洗脱去掉糖链的糖肽:用体积浓度40%乙腈/体积浓度5%三氟乙酸水溶液(V/V) 洗脱两次。洗脱磷酸化肽:用40μL含体积浓度5%氨水的水溶液洗脱两次。洗脱馏分旋干重溶至20μL入质谱进行分析(结果见图3和图4)。40%乙腈/体积浓度5%三氟乙酸水溶液洗脱馏分中主要包含去掉糖肽的糖肽,氨水溶液洗脱馏分中大部分为磷酸化肽。跟图2结果比较,在线酶切不仅使富集后的谱图更干净、还可以让糖肽和磷酸化肽出现在两个不同的馏分,实现分步洗脱。
实施例6
本发明可在dSPE、SPE或dSPE&SPE混合模式下进行,操作灵活且通量高。
同实施例5平行两组,一组离心,一组装填成SPE小柱,即一组dSPE&SPE 混合模式(过程同实施例5)、一组SPE进行富集。
首先装填二氧化钛微型小柱,过程方法同实施例1。将5ug(5μL)牛胎球蛋白和5ug(5μL)a-酪蛋白混合去盐(作为分析样品),调整至40μL的体积浓度80%乙腈/5%三氟乙酸的水(含1M羟基乙酸)溶液,注入微柱进行上样,再用体积浓度80%乙腈/5%三氟乙酸的水(含1M羟基乙酸)溶液淋洗40μL,加入45μL 50mM乙酸铵溶液,再加入PNGase F酶500U(购自NEB),用封口膜封住微柱上下入口,在线酶切16h。酶切结束后打开封口膜,分别用体积浓度40%乙腈/体积浓度5%三氟乙酸水溶液(V/V)洗脱两次,再用40μL含体积浓度5%氨水的水溶液洗脱两次。洗脱馏分旋干重溶至20μL入质谱进行分析。将实施例6两组洗脱馏分与实施例5两组馏分的质谱图进行对比,结果无太大差别。说明该方法在各种模式下均能获得较好的富集选择性
实施例7-8
本发明可以在不同种类二氧化钛材料上进行。分别称取GL Science二氧化钛(实施例5)、NP二氧化钛和自身合成二氧化钛材料1mg,三组平行,共富集5ug (5μL)牛胎球蛋白和5ug(5μL)a-酪蛋白(作为分析样品),过程如实施例5 条件同时富集分步洗脱,所得结果磷酸化肽仅有丰度差异,糖肽数量和丰度均有差异。实验结果说明在线酶切法可应用于不同的二氧化钛材料,获得的糖肽和磷酸化肽个数与材料本身性质相关。
实施例9-12
在线酶切法适用的材料使用量考察:平行四组,分别称取二氧化钛材料1mg、2 mg、4mg、8mg,每组都共富集10ug(10μL)牛胎球蛋白和10ug(10μL)a- 酪蛋白(作为分析样品),过程其他条件同实施例5,富集后得到的糖肽进行质谱分析。质谱结果表明,1mg材料即可有效富集10ug牛胎球蛋白和10uga-酪蛋白。
实施例13-16
考察不同种类酶切溶液对在线酶切法中酶切步骤的影响:平行四组,在线酶切前步骤同实施例5,调整实施例5中在线酶切溶液分别为50mM甲酸铵(pH=3)、 50mM乙酸铵(pH=6.86)、50mM碳酸氢铵(pH=8.34)和0.1%氨水(pH=11.45),室温孵化3h后终止酶解,离心取上清液去盐入质谱分析(图5)。使用50mM 甲酸铵和乙酸铵作为酶切溶液条件的上清液(图5A-5B)中没有出现糖肽和磷酸化肽,而50mM碳酸氢铵和0.1%氨水作为酶切溶液的上清液(图5C-5D)中不仅出现了糖肽(星星标记),还出现了磷酸化肽(圆形标记)。结果(见图5)显示50mM甲酸铵和乙酸铵作为酶切溶液符合实验要求,进一步考虑到乙酸铵溶液的pH值更接近PNGase F酶的最适pH,最终选用乙酸铵作为酶切溶液继续优化条件。
实施例17-21
考察不同浓度乙酸铵溶液对在线酶切法中酶切步骤的影响:平行五组,在线酶切前步骤同实施例5,调整实施例5中在线酶切溶液为5mM乙酸铵、10mM乙酸铵、20mM乙酸铵、25mM乙酸铵、50mM乙酸铵,在线酶切过夜(20h) 后按实施例5中条件进行洗脱。所得数据经分析比较,随着盐浓度的升高,获得的糖肽和磷酸化肽总丰度也升高,所以50mM乙酸铵是合适的在线酶切溶液浓度。
实施例22-26
考察不同酶切时间对在线酶切法中酶切步骤的影响:平行五组,在线酶切前步骤同实施例5,调整在线酶切时间为6h、9h、12h、24h、48h,其他条件同实施例5,富集后得到的糖肽和磷酸化肽进质谱分析,通过比较糖肽和磷酸化肽的相对丰度,发现磷酸化肽信号丰度在9h最高,超过12h丰度会减半,而糖肽信号在24h时丰度最高,9-12h时丰度差别不大,综上,9-12h是在线酶切最佳的酶切时长。
实施例27
考察糖苷酶联用对在线酶切法中酶切步骤的影响:调整在线酶切的酶为PNGase F酶和Endo H酶各500U(均购自NEB),其他步骤同实施例5。所得两个馏分质谱分析图与实施例5结果类似,证明在线酶切步骤中可将两种糖苷酶联合使用。
实施例28-29
考察不同洗脱条件对在线酶切法的影响:平行两组,在线酶切前步骤同实施例5,一组调整酶切后洗脱糖肽的条件依次为:5mM碳酸氢铵80μL、10mM碳酸氢铵80μL、 20mM碳酸氢铵80μL各一次(图6);另一组调整酶切后洗脱糖肽的条件为:体积浓度50%乙腈/体积浓度1%甲酸的水溶液(V/V)40μL洗三次(图7),其他条件同实施例5。结果显示这几种条件不是无法洗脱糖肽,就是将磷酸化肽和糖肽一起洗出,跟实施例5相比,体积浓度40%乙腈/体积浓度5%三氟乙酸水溶液(V/V)和体积浓度5%氨水的水溶液才分别是洗脱糖肽、磷酸化肽的最优条件。
实施例30-31
二氧化钛在线酶切法在实际样品中应用:平行两组,调整样品为74ug鼠脑蛋白酶解液((作为分析样品,酶解步骤同标准蛋白酶解),分别用共富集(同实施例 4)和在线酶切法(同实施例5)进行富集。富集得到的糖肽和磷酸化肽入 LTQ-Orbitrap质谱检测。所得数据经MaxQuant数据库搜库处理后,在线酶切法富集到的磷酸化肽和磷酸化位点的数量与共富集差别不大,但在线酶切法富集到的糖肽和糖基化位点的数量是共富集结果的7.7倍。
综上所述,本发明提出的在线酶切法能实现二氧化钛材料对糖肽、磷酸化肽的同时富集和分布洗脱,并可用于复杂生物样品如鼠脑等的蛋白质糖基化和磷酸化研究。经实验证明,在线酶切法还能显著提高二氧化钛对糖肽的富集选择性,可以用于其它已知或未知的、具有共富集能力的材料的富集过程中,将有助于更多生物标志物的发现。
引用文献:
[1]Qing GY,Li XL,Xiong P,Chen C,Zhan MM,Liang XM,Sun TL.Dipeptide-Based Carbohydrate-Receptors and Polymers for Glyco-peptide Enrichment andGlycan Discrimination, ACS Appl.Mater.Interfaces,2016,8(34) 。
Claims (9)
1.二氧化钛富集-在线酶切-分步洗脱糖肽和磷酸化肽方法,其特征在于:选择表面成分为二氧化钛的材料作为富集材料,采用分散固相萃取(dSPE)、固相萃取(SPE)、超滤管过滤等一种或多种分离模式结合,对含有糖肽和磷酸化肽的样品进行糖肽和磷酸化肽同时富集、在线糖苷酶酶切、分步洗脱糖肽和磷酸化肽。
2.按照权利要求1所述的方法,其特征在于,富集过程包含以下五个步骤:
①上样:样品在高有机相低pH值(有机相体积比例60%-90%,pH值0.5-5)水溶液条件下与材料混合,孵化10s-15min;
②淋洗:用高有机相低pH值(有机相体积比例60%-90%,pH值0.5-5)的淋洗水溶液处理材料,以去除非修饰肽段,糖肽和磷酸化肽仍结合在材料;
③在线酶切:按样品中糖蛋白质量为A计,1h内能处理糖蛋白质量A的糖苷酶的活力单位量为B,于体系中加入1-100倍B的糖苷酶、pH值在4-7范围内的盐溶液(酶切溶液),在20-60℃温度范围内酶切0.1-48h,在线切除糖肽上的糖链;
④通过低有机相低pH值(有机相体积比例20-55%,pH值0.5-3)的洗脱水溶液1处理材料,获得已被切掉糖链的糖肽;
⑤最后通过高pH值低有机相或不含有机相(有机相体积比例0-55%,pH值8-12)的洗脱水溶液2处理材料,获取仍保留在材料上的磷酸化肽。
3.按照权利要求1或2所述的方法,其特征在于:所述表面成分为二氧化钛的材料为二氧化钛微球材料(粒径1-10um)、二氧化钛纳米材料、表面带有二氧化钛涂覆层或包覆层的球形(粒径1-10um)或无定形材料,球形或无定形材料为二氧化硅或聚合物。
4.按照权利要求1或2所述的方法,其特征在于:所述富集过程采用固相萃取、分散固相萃取、超滤管过滤中一种或多种模式结合进行;
固相萃取:将材料和乙腈溶液混合均匀后、加压装填至装有筛板的小柱上,制成微型SPE小柱;上样、淋洗、在线酶切及分步洗脱都在小柱上完成;溶液注入小柱后,通过加压与材料分离;
分散固相萃取:将材料放入EP管中,上样、淋洗、在线酶切及分步洗脱都在EP管中完成;每一步加入的溶液与材料混合均匀后通过离心的方式将溶液和材料分层,移除上层溶液,再加入下一步所需的溶液,继续混合均匀后离心、移除上层溶液,直至最后一步结束;
超滤管过滤是将材料置于超滤管中,上样、淋洗、在线酶切及分步洗脱都在超滤管中完成;每一步加入超滤管中的溶液通过高速或超高速离心与材料分离,材料留在超滤膜上方,而溶液进入衬管;
混合模式指权利要求1中5个步骤分别以上述三种模式中二种以上模式的对应步骤中完成;如:上样、淋洗、在线酶切用分散固相萃取模式进行,分步洗脱用固相萃取/超滤管过滤完成。
5.按照权利要求1或2所述的方法,其特征在于:所述方法中所适用样品包括:⑴含糖肽和磷酸化肽的肽和/或蛋白标准品;⑵含糖肽和磷酸化肽的组织、细胞、体液、混合蛋白等复杂样品;⑶以上(1)和(2)样品的混合样品;
样品中糖蛋白质量过程为:标准品配制的样品按样品配制时加入糖蛋白标准品的量,或实际样品按文献含量估计实际样品中糖蛋白的量。
6.按照权利要求1或2所述的方法,其特征在于:在线酶切使用的酶如下:⑴PNGase F;⑵PNGase H;⑶唾液酸糖苷酶;⑷以上(1)、(2)和(3)三种酶中二种以上的组合使用。
7.按照权利要求1所述的方法,其特征在于:
步骤①-②中使用的溶液为有机溶剂和酸水溶液混合溶液;有机溶剂的体积比例为60-90%,酸的体积比例为0.1-10%,pH范围0.5-5;
步骤③中使用的溶液为缓冲盐水溶液、酸水溶液、或缓冲盐水溶液和有机溶剂混合溶液、或酸水溶液和有机溶剂混合溶液、或缓冲盐及酸水溶液和有机溶剂混合溶液;缓冲盐浓度在0-200mM,优选5-200mM,酸的体积比例0-10%,有机溶剂的体积比为0-50%,pH范围4-7;
步骤④中使用的溶液为有机溶剂和酸水溶液混合溶液;有机溶剂的体积比例为20-55%,酸的体积比例为0.1-10%,pH范围0.5-3;
步骤⑤中使用的溶液为碱性溶液或碱性溶液/有机溶剂混合溶液;碱溶液的质量浓度0.1-10%,有机溶剂的体积比为0-55%,pH范围8-12。
8.按照权利要求2或7所述的方法,其特征在于:
有机溶剂包括但不局限于乙腈、甲醇、乙醇等中的一种或二种及以上;
缓冲盐为pH值缓冲范围在4-8之间有机/或无机盐,包括但不局限于甲酸铵、乙酸铵、碳酸氢铵等中的一种或二种及以上;
酸包括pH值在0.5-5.5的有机/或无机酸中的一种后两种及以上,包括但不局限于甲酸、乙酸、三氟乙酸、乳酸、羟基乙酸、二羟基苯甲酸,邻苯二甲酸等中的一种或二种及以上;
碱性溶液为碳酸钠水溶液、碳酸氢钠水溶液、碳酸氢铵水溶液、氨水中的一种或二种及以上。
9.按照权利要求2或7所述的方法,其特征在于:富集材料和上样样品的质量比范围1-50;上样溶液体积和材料装柱后体积比范围5-2000,淋洗溶液和材料柱体积比范围5-2000,在线酶切溶液和材料柱体积比范围2-100,洗脱溶液和材料柱体积比范围2-200。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070269895A1 (en) * | 2002-06-03 | 2007-11-22 | The Institute For Systems Biology | Methods for quantitative proteome analysis of glycoproteins |
CN102070701A (zh) * | 2009-11-25 | 2011-05-25 | 中国科学院大连化学物理研究所 | 用金属氧化物富集糖肽及同时富集糖肽和磷酸化肽的方法 |
CN105300783A (zh) * | 2014-07-13 | 2016-02-03 | 复旦大学 | 一种糖基化肽段固相富集并质谱分析的方法 |
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Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20070269895A1 (en) * | 2002-06-03 | 2007-11-22 | The Institute For Systems Biology | Methods for quantitative proteome analysis of glycoproteins |
CN102070701A (zh) * | 2009-11-25 | 2011-05-25 | 中国科学院大连化学物理研究所 | 用金属氧化物富集糖肽及同时富集糖肽和磷酸化肽的方法 |
CN105300783A (zh) * | 2014-07-13 | 2016-02-03 | 复旦大学 | 一种糖基化肽段固相富集并质谱分析的方法 |
Non-Patent Citations (9)
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