CN113655144A - 一种基于丙酮酸激酶和α-2-HS-糖蛋白的早期预警重症急性胰腺炎的预测模型 - Google Patents
一种基于丙酮酸激酶和α-2-HS-糖蛋白的早期预警重症急性胰腺炎的预测模型 Download PDFInfo
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Abstract
本发明公开了一种基于丙酮酸激酶和α‑2‑HS‑糖蛋白的早期预警重症急性胰腺炎的预测模型,属于生物检测技术领域。所述预测模型为血清中丙酮酸激酶和α2‑HS‑糖蛋白,通过检测血清中丙酮酸激酶和α2‑HS‑糖蛋白的含量变化,在患者患病48h内,区分中重症急性胰腺炎和重症急性胰腺炎,具有良好的指导意义。
Description
技术领域
本发明一种基于丙酮酸激酶和α-2-HS-糖蛋白的早期预警重症急性胰腺炎的预测模型,属于生物检测技术领域。
背景技术
急性胰腺炎是常见的消化系统疾病之一,其特点是发病快、进展迅速,并且发病率逐年增高。早期有效的液体复苏与合理的支持治疗,是目前主要的治疗策略。2012年最新亚特兰大分类根据急性胰腺炎病情严重程度,将急性胰腺炎分为轻症急性胰腺炎(MAP)、中度重症急性胰腺炎(MSAP)、重症急性胰腺炎(SAP)。轻度急性胰腺炎是一种自限性疾病,经对症治疗后患者预后较好。值得注意的是,约20%的患者会进展为重症急性胰腺炎,死亡率高达10%-30%,即使幸存的患者也伴有严重的近期与远期并发症。很大部分原因在于目前尚无精确反应胰腺炎严重程度的血液标志物或评分量表,延迟了治疗时间窗。重症急性胰腺炎的早期预警仍然是临床诊疗过程中的一项重大挑战。因此,需要进一步的发掘具有高诊断效能标志物,对具有重症转化风险的患者早期预警,选择合适的治疗策略,具有重要的临床意义。
丙酮酸激酶(PK)是负责糖酵解最后限速反应的酶,催化磷酸烯醇式丙酮酸(PEP)和ADP生成丙酮酸和ATP,并在调节细胞代谢中发挥重要作用。PK存在两种同工酶:M型(PKM)和L型,PKM有M1和M2亚型,广泛分布于心脏、肝脏、大脑和免疫细胞。PKM1含有高活性的酶,能有效地将PEP转化为丙酮酸,通常在终末分化组织中表达。PKM2主要存在于增殖细胞或具有合成代谢功能的组织中,近年来引起广泛关注。研究表明,慢性胰腺炎或胰腺导管腺癌(PDAC)患者PKM2明显升高,其他研究表明PKM2在免疫代谢和炎症中起重要作用。
α2-HS-糖蛋白(AHSG),一种存在于血清中的急性期磷酸化糖蛋白,几乎完全由肝脏合成和分泌。AHSG在骨矿化、胰岛素信号、炎症和可能的肿瘤发生中起着多方面的作用。研究表明,AHSG对各种炎症刺激和损伤发生急性期反应,它经常作为负急性期蛋白发挥作用。
在蛋白质组学研究领域,基于液相色谱-质谱联用(LC-MS/MS)技术的发现蛋白质组学可在生物样本中检测和相对定量上千个蛋白,在过去十几二十年中已得到广泛的应用,这些方法都是基于数据依赖采集模式(data-dependent acquisition,DDA)来采集蛋白质谱数据的。然而,DDA数据采集模式具有重复性差、定量不精准、数据缺失多、低丰度蛋白难以检测等缺点。
基于MRM/SRM/PRM的靶标蛋白质组学,可以为几十到上百个目标蛋白提供精确的绝对定量结果,并且重复性很高,是质谱绝对定量的金标准。但通量相对较低,无法开展高通量蛋白的精确定量。
发明内容
本发明提供了一种基于丙酮酸激酶和α-2-HS-糖蛋白的早期预警重症急性胰腺炎的预测模型,所述预测模型为血清中丙酮酸激酶和α2-HS-糖蛋白,通过检测血清中丙酮酸激酶和α2-HS-糖蛋白的含量变化,在患者患病48h内,区分中重症急性胰腺炎和重症急性胰腺炎。
进一步地,上述技术方案中,所述检测血清中丙酮酸激酶和α2-HS-糖蛋白的含量变化为:相较于健康人,重症急性胰腺炎患者血清中丙酮酸激酶上升的倍数>3.94,α2-HS-糖蛋白下降的倍数<0.41,中重症急性胰腺炎患者血清中丙酮酸激酶上升的倍数为1.38~3.94,α2-HS-糖蛋白下降的倍数为0.41~0.56。
进一步地,上述技术方案中,取血浆进行蛋白质酶解,得到酶解肽段,对酶解肽段进行肽段脱盐,再采用DIA蛋白质组学分析法对丙酮酸激酶和α2-HS-糖蛋白含量进行检测,在患者患病起48h内,区分中重症急性胰腺炎和重症急性胰腺炎。
进一步地,上述技术方案中,所述蛋白质酶解包括:
(1)将血浆稀释后加入到孔板中;
(2)加入0.8~1.2wt%SDC,Tris-HCl,pH 8.5,混匀,至SDC终浓度0.4~0.6wt%;
(3)加入2μL 0.5M TCEP,8μL 0.5M CAA,70℃反应10min;
(4)在10min内冷却至室温,按照1:18~1:22(Lys C:总蛋白量)加入Lys C,再按1:8~1:12(Trypsin:总蛋白量)加入Trypsin的混合酶,Lys C和Trypsin分别为1μg和2μg,37℃反应2h;
(5)酶切溶液中,加入100μL iPA 0.8%~1.2%TFA溶液;得到酶切肽段。
进一步地,上述技术方案中,对酶解肽段进行肽段脱盐的方法包括:
(1)将所得酶切肽段分别用甲醇和0.1~0.3wt%TFA活化;
(2)上样
(3)分别用iPA 0.8~1.2wt%TFA和0.1~0.3wt%TFA淋洗;
(4)加入0.8~1.2wt%ammonia(氨)和75~85wt%acetonitrile(乙氰)进行洗脱;
(5)洗脱液离心冻干;
(6)用0.08~0.12wt%甲酸-水将多肽复溶,备用。
进一步地,上述技术方案中,DIA蛋白质组学分析法为使用液相色谱-质谱联用仪对丙酮酸激酶和α2-HS-糖蛋白含量进行检测,检测时使用双柱模式:Trap Column和分析柱;流动相:A:0.05~0.2wt%甲酸-水;B:0.05~0.2wt%甲酸-80wt%乙腈-水;梯度:2分钟内B相从起始3wt%上升至8wt%,接着在137分钟内B相从8wt%升至30wt%,最后B相在4分钟内从30wt%变为99wt%,并保持3分钟,流速为0.2~0.4mL/min。
上述预测模型在早期预警重症急性胰腺炎中的应用,用于48h内区分中重症急性胰腺炎和重症急性胰腺炎。
发明有益效果
本检测方法所采用的DIA技术(data-independent acquisition,数据非依赖采集),兼具高通量和定量精准且重复性高等特点。
本发明发现SAP患者血清PKM水平较MSAP显著升高,在SAP早期诊断中具有较高的有效性(AUC 0.824)。
本发明的结果也证实AHSG水平与急性胰腺炎的严重程度呈负相关,具有早期预测SAP的潜力(auc0.789)。二者结合预测力更强。
然而,本检测方法所采用的DIA技术(data-independent acquisition,数据非依赖采集),兼具高通量和定量精准且重复性高,应用前景广阔。
本发明所述急性胰腺炎(AP)严重程度分级是根据2012急性胰腺炎亚特兰大分类标准,但该分类标准对于中重症和重症的区分存在48h的等待时间窗:即AP患者伴有器官衰竭,48h内恢复即为中重症,48h内未恢复即为重症。而本发明的联合测定丙酮酸激酶和α-2-HS-糖蛋白能够在相对早期(48h内)辅助区分中重症和重症(见ROC曲线结果),具有良好的指导意义。
附图说明
图1为丙酮酸激酶在不同组的丰度。
图2为α2-HS-糖蛋白在不同组的丰度。
图3为丙酮酸激酶和α2-HS-糖蛋白的ROC曲线。
具体实施方式
下述非限定性实施例可以使本领域的普通技术人员更全面地理解本发明,但不以任何方式限制本发明。
实施例1
基本试剂:脱氧胆酸钠、三(羟甲基)氨基甲烷、三(2-羧乙基)膦盐酸盐、2-氯乙酰胺、赖氨酰肽链内切酶、胰酶、甲醇、乙腈、异丙醇。
试剂配制:
·1%SDC:1%SDC in 0.1M Tris-base pH 8.5
1)称取0.2g SDC(Sodium deoxycholate脱氧胆酸钠)和242.28mg Tris-base加入到50ml离心管中;
2)加入超纯水,溶液总体积约19mL,震荡使得SDC和Tris-base完全溶解;
3)加入90μL 6N HCl调节pH,使得溶液pH值为8.5左右;
4)补加超纯水,使得溶液总体积为20mL;
5)存储在20℃,待用,使用时在37℃振荡使其溶解。
·0.5M TCEP(Tris(2-carboxyethyl)phosphine三(2-羧乙基)膦盐酸盐)溶液:
1)分装成0.1mL/管,存储在-80℃冰箱,待用。
·0.5M CAA(2-Chloroacetamide 2-氯乙酰)溶液:0.5M CAA in ddH2O
1)称取467.55mg CAA到15mL离心管中;
2)加入10mL ddH2O,震荡,使得CAA全部溶解;
3)分装成0.1mL/管,存储在-80℃冰箱,待用。
·LysC(赖氨酰肽链内切酶)溶液:0.2μg/μL LysC in超纯水
1)LysC每瓶为20μg,加入100μL超纯水,用枪头吹打数次;
2)现配现用。
·Trypsin(胰蛋白酶)溶液:0.2μg/μL Trypsin in超纯水
1)Trypsin每瓶为20μg,加入100μL超纯水,用枪头吹打数次;
2)现配现用。
·10%TFA(三氟乙酸)
1)加入9ml超纯水和1ml TFA到15ml离心管中;
2)存储在2~8℃,待用。
·Wash 1:1%TFA in iPA(异丙醇)
1)加入49.5ml iPA和0.5mL TFA到50ml离心管中;
2)存储在2~8℃,待用。
·Wash 2:0.2%TFA in超纯水
1)加入49mL超纯水和1mL 10%TFA到50mL离心管中;
2)存储在2~8℃,待用。
·Elute buffer(洗涤缓冲液):1%氢氧化氨溶液in 80%ACN
1)加入9.5mL超纯水,0.5mL氢氧化氨溶液和40mL ACN(乙腈)到50mL离心管中;
2)存储在2~8℃,待用。
前处理步骤:
蛋白质酶解(SDC法)
(1)取2μL血浆(约120μg),加入238μL ddH2O稀释到0.5μg/μL,混匀;
(2)取40μL稀释血浆(约20μg)到96孔板中,
(3)加入50μL的1%SDC,100mM Tris-HCl,pH 8.5,混匀,体积为90μL(SDC终浓度0.5%);
(4)加入2μL 0.5M TCEP,8μL 0.5M CAA,70℃反应10min;
(5)约10min冷却至室温,按照1:20(酶:蛋白)加入Lys C和1:10(酶:蛋白)加入Trypsin的混合酶,Lys C和Trypsin分别为1μg和2μg,37℃反应2h;
(6)酶切溶液中,加入100μL iPA 1%TFA溶液;得到酶切肽段。
肽段脱盐(SCX)
(1)SCX活化1:SCX分离孔板中加入400μL甲醇,1000g,离心1min(样品量少时可以把脱盐柱拔出放到2ml离心管中进行脱盐操作);
(2)SCX活化2:步骤(1)活化处理后的SCX分离孔板中加入400μL 0.2%TFA,1000g,离心1min;
(3)上样:加入200μL上述蛋白质酶解所得酶切肽段,1000g,离心2min,上样2次;
(4)淋洗1:加入400μL iPA 1%TFA,1000g,离心2min;
(5)淋洗2:加入400μL 0.2%TFA,1000g,离心2min;
(6)洗脱:加入200μL 1%ammonia(氨)80%acetonitrile(乙氰),1000g,离心2min;
(7)离心冻干;
(8)0.1%甲酸-水将多肽复溶至0.5μg/μL,上样2μL进行质谱分析。
蛋白检测:
DIA蛋白质组学分析使用Thermo Ultimate RSL Cnano LC和Orbitrap FusionLumos Tribrid高分辨率质谱仪((Thermo Fisher Scientific,USA)。主要参数如下:双柱模式:Trap Column(Acclaim PepMap C18,3μm,100A,75μm x 2cm),分析柱(AcclaimPepMap C18,2μm,100A,75μm x 25cm)。流动相:A:0.1%甲酸-水;B:0.1%甲酸-80%乙腈-19.9%水。梯度:2分钟内B相从起始3%上升至8%,接着在137分钟内B相从8%升至30%,最后B相在4分钟内从30%变为99%,并保持3分钟,流速为0.3ml/min。
ESI喷雾电压:2.1kV;毛细管温度:300℃;S-lens:50%;碰撞能:32%HCD。分辨率设置:一级60000FWHM@200m/z,二级30000FWHM@200m/z。最大进样时间:一级全扫描20ms,二级全扫描54ms。母离子扫描范围:m/z 350-1200,子离子扫描范围:起点为m/z 200。窗口数为70,根据洗脱肽段的m/z调整可变隔离窗。
利用该检测方法分别对102例受试者血清样本中丙酮酸激酶和α2-HS-糖蛋白进行检测,包括轻度(39例)、中重症(20例)、重症(19例)急性胰腺炎患者和健康对照者(24例)。结果显示:丙酮酸激酶均随着疾病严重程度的增加而上升(如图1);α2-HS-糖蛋白(AHSG)随着疾病严重程度的增加而降低(如图2)。且二者对SAP具有较好的区分效力,丙酮酸激酶的AUC值为0.824,特异性和敏感性分别为100%和58.8%;α2-HS-糖蛋白的AUC值为0.789,特异性和敏感性分别为68.4%,88.2%。两者结合的AUC值为0.833,特异性和敏感性分别为94.7%和70.6%,二者结合可早期(48h内)区分中重症急性胰腺炎和重症急性胰腺炎(如图3)。
Claims (7)
1.一种基于丙酮酸激酶和α-2-HS-糖蛋白的早期预警重症急性胰腺炎的预测模型,其特征在于,所述预测模型为血清中丙酮酸激酶和α2-HS-糖蛋白,通过检测血清中丙酮酸激酶和α2-HS-糖蛋白的含量变化,在患者患病48h内,区分中重症急性胰腺炎和重症急性胰腺炎。
2.根据权利要求1所述的预测模型,其特征在于,所述检测血清中丙酮酸激酶和α2-HS-糖蛋白的含量变化为:相较于健康人,重症急性胰腺炎患者血清中丙酮酸激酶上升的倍数>3.94,α2-HS-糖蛋白下降的倍数<0.41,中重症急性胰腺炎患者血清中丙酮酸激酶上升的倍数为1.38~3.94,α2-HS-糖蛋白下降的倍数为0.41~0.56。
3.权利要求1或2所述的预测模型的预测方法,其特征在于,取血清(或血浆)进行蛋白质酶解,得到酶解肽段,对酶解肽段进行肽段脱盐,再采用DIA蛋白质组学分析法对丙酮酸激酶和α2-HS-糖蛋白含量进行检测,在患者患病48h内,区分中重症急性胰腺炎和重症急性胰腺炎。
4.根据权利要求3所述的方法,其特征在于,所述蛋白质酶解包括:
(1)将血浆稀释后加入到孔板中;
(2)加入0.8~1.2wt%SDC,Tris-HCl,pH 8.5,混匀,至SDC终浓度0.4~0.6wt%;
(3)加入2μL 0.5M TCEP,8μL 0.5M CAA,70℃反应10min;
(4)在10min内冷却至室温,按照1:18~1:22(Lys C:总蛋白量)加入Lys C,再按1:8~1:12(Trypsin:总蛋白量)加入Trypsin的混合酶,Lys C和Trypsin分别为1μg和2μg,37℃反应2h;
(5)酶切溶液中,加入100μL iPA 0.8%~1.2%TFA溶液;得到酶切肽段。
5.根据权利要求3所述的方法,其特征在于,对酶解肽段进行肽段脱盐的方法包括:
(1)将所得酶切肽段分别用甲醇和0.1~0.3wt%TFA活化;
(2)上样
(3)分别用iPA 0.8~1.2wt%TFA和0.1~0.3wt%TFA淋洗;
(4)加入0.8~1.2wt%ammonia(氨)和75~85wt%acetonitrile(乙氰)进行洗脱;
(5)洗脱液离心冻干;
(6)用0.08~0.12wt%甲酸-水将多肽复溶,备用。
6.根据权利要求3所述的方法,其特征在于,DIA蛋白质组学分析法为使用液相色谱-质谱联用仪对丙酮酸激酶和α2-HS-糖蛋白含量进行检测,检测时使用双柱模式:Trap Column和分析柱;流动相:A:0.05~0.2wt%甲酸-水;B:0.05~0.2wt%甲酸-80wt%乙腈-水;梯度:2分钟内B相从起始3wt%上升至8wt%,接着在137分钟内B相从8wt%升至30wt%,最后B相在4分钟内从30wt%变为99wt%,并保持3分钟,流速为0.2~0.4mL/min。
7.权利要求1或2所述的预测模型在早期预警重症急性胰腺炎中的应用,其特征在于,用于48h内区分中重症急性胰腺炎和重症急性胰腺炎。
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