克山病患者血清蛋白二级质谱鉴定及生物标志物研究

Secondary order mass spectrometry identification and biomarker of serum proteins in patients with Keshan disease

目的探讨血清蛋白鉴定对克山病临床诊断与发病机制研究的价值。方法在克山病病区选择65例慢型克山病患者作为克山病（KD）组；同时以29例扩张型心肌病（DCM）患者作为DCM组、62例克山病病区健康人及28例非病区健康人作为对照。采集受检者血清，采用液体芯片飞行时间质谱技术（ClinProtTMMALDI—TOF—MS）检测差异蛋白表达，应用ClinProTools2．2软件分析确定差异蛋白，以基因遗传（GA）、快速分类（QC）和神经网络（SNN）3种算法筛选标志蛋白。采用MALDI串联飞行时间质谱技术（MALDI—TOF／TOF）鉴定差异多肽：使用flexAnalysis软件标注峰图，MASCOT搜索引擎检索结果。结果①KD组与非病区健康对照组比较获得34个差异蛋白／多肽及5个标志蛋白；KD组与病区健康对照组比较获得52个差异蛋白／多肽及5个标志蛋白：KD组与DCM组比较获得167个差异蛋白／多肽及5个标志蛋白，标志蛋白对组间区分的敏感性与特异性分别达98．07％-99．24％、97．15％-99．12％。②二级质谱鉴定获得2个阳性结果，质荷比（m／z）为2079的肽段匹配p珠蛋白，m／z为1465的肽段匹配纤维蛋白原。B珠蛋白在克山病呈低表达，纤维蛋白原呈高表达。结论慢型克山病血清标志蛋白可作为诊断与鉴别的生物标志物，B珠蛋白与纤维蛋白原在克山病心肌损伤的发生发展中扮演了重要角色。

Objective To investigate the clinical diagnostic value and pathogenesis of serum protein identification in Keshan disease （KD）. Methods A total of 65 chronic KD patients were selected as the patient group in KD endemic areas, while 29 cases of dilated cardiomyopathy （the DCM group）, 62 healthy cases from KD endemic areas （control 1 group） and 28 healthy cases from non--endemic areas （control 2 group） were selected as controls. Liquid chip time of flight mass spectrometry （ClinProtTM MALDI-TOF-MS） was used to determine the expression of proteins/ peptide peaks. ClinProTools 2.2 software was used to analyze the protein profiles to determine differentially expressed proteins/peptide peaks. The Genetic Algorithm （GA）, QuickClassifer Algorithm （QC） and Supervised Neural Network Algorithm （SNN） methods were used to screen marker proteins. Matrix-assisted laser desorption/ionization time-of-flight Mass Spectrometry technique （MALDI-TOF/TOF） was also used as a secondary mass spectrometry to identify differentially expressed peptides. Results Between the KD and control 1 groups, 34 differentially expressed proteins/peptides and 5 marker proteins were identified, while 52 differentially expressed proteins/peptides and 5 marker proteins were identified between the KD and control 2 groups, and there were 67 differentially expressed proteins/peptides and 5 marker proteins between the KD and DCM groups. During secondary mass spectrometry, two peptides for mass-to-charge ratio （m/z） 2 079 and 1 465 were obtained, peptide of matching β- globin showed low expression while peptide of matching fibrinogen showed high expression in the KD patients. Conclusions Serum marker proteins can be used as biomarkers for diagnosis and differentiation of KD. β-globin and fibrinogen play an important role in the development of KD myocardial injury.