应用表面增强激光解析电离-飞行时间质谱对结肠癌组织蛋白质的初步分析

[目的]检测结肠癌组织与正常结肠黏膜组织中差异蛋白质的表达,寻找与结肠癌相关的肿瘤标记物.[方法]采用表面增强激光解析电离-飞行时间质谱仪（SELDI-TOF MS）对42例结肠癌组织及其配对的正常结肠黏膜组织进行蛋白质指纹图谱检测.[结果]结肠癌与正常对照共有38种蛋白质质谱峰强度差异有统计学意义（P＜0.01）,有分类意义的蛋白质有7种,质核比（M/Z）为M1678.47、M2182.19 、M3358.44的蛋白质在大肠癌组织中低表达,M/Z为M1688.43、M2718.72、M3241.60、M3893.13的蛋白质在大肠癌组织中高表达.[结论]利用SELDI-TOF MS技术筛选出的具有分类意义的7种蛋白质,有可能成为大肠癌诊断及预后判断的一组生物学指标.     

表面增强的激光解析电离飞行时间质谱技术在肝细胞癌血清学诊断中的应用

目的:检测HBV感染携带者与肝细胞癌(HCC)患者血清蛋白质的差异性表达,以发现HCC的肿瘤诊断标志物.方法:用表面增强的激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测27例HCC患者,27例HBV感染携带者,25例健康对照血清中的蛋白质谱,并用Biomarker Patterns System 5．0软件分析,建立诊断模型．结果:检测HCC患者与HBV感染携带者。正常对照与HCC患者,正常对照与HBV感染携带者的差异性蛋白分子,据此构建分类模型,得到的灵敏度和特异度分别为93％,85％; 96％,96％;84％,89％．其中相对分子质量为8141 Da的蛋白峰在HCC组明显高于HBV感染组(p<10-5);相对分子质量为3448 Da的蛋白峰在HCC及HBV感染携带组表达均较正常组显著增高(P<10-5),而在HCC-HBV组无明显差异(P>0．05),提示其可能为HBV感染的一种标志蛋白;相对分子质量为777) Da的蛋白峰在3组中均有差异性表达．结论:SELDI蛋白芯片技术检测血清蛋白质谱法诊断HCC具有较高的灵敏度和特异度,操作简单快捷,临床应用前景广阔,为HCC诊断提供了新的血清学方法．     

支气管肺泡灌洗液细胞DNA含量分析在周围型肺癌中的诊断价值

目的探讨支气管肺泡灌洗液细胞DNA含量分析对周围型肺癌的诊断价值。方法采用全自动细胞DNA定量分析系统,对31例周围型肺癌及20例肺良性病变患者的支气管肺泡灌洗液细胞作DNA含量分析,并与纤维支气管镜活检和刷检比较。结果灌洗液细胞DNA含量分析诊断周围型肺癌的敏感性为67．7％,特异性为95．0％,显著高于活检（29．0％）和刷检（32．3％）的阳性率（P均〈0.01）。结论支气管肺泡灌洗液细胞DNA含量分析是诊断周围型肺癌有效的辅助方法。     

肺癌支气管肺泡灌洗液细胞端粒酶测定的临床意义

目的探讨端粒酶活性在肺癌支气管肺泡灌洗液（ＢＡＬＦ）细胞中的表达与肺癌发生机制的关系。方法应用银染多聚酶链方法对肺癌患者和支气管、肺良性疾病患者各３０例的ＢＡＬＦ细胞端粒酶活性的表达进行检测。结果肺癌患者ＢＡＬＦ细胞端粒酶活性８３％阳性表达，支气管、肺良性疾病ＢＡＬＦ细胞表达均阴性（Ｐ＜０．００１）。同一肺癌患者患侧肺端粒酶活性在ＢＡＬＦ细胞８３％表达，健侧肺阳性表达２７％。结论端粒酶活性是肺癌诊断的标志物之一，与肺癌的发生发展密切相关。     

BALF对周围型肺癌的诊断价值

目的探讨支气管肺泡灌洗液(BALF)对内镜检查阴性的周围型肺癌患者的诊断价值。方法选取我科2011年1月至2014年7月住院行支气管镜检查镜下阴性的患者共114例,进行支气管肺泡灌洗和支气管镜下肺活检(TBLB)。根据病理结果 ,将所有患者分为肺癌(周围型肺癌)组和肺良性病变组。对比两组患者BALF中肿瘤指标水平和细胞基质金属蛋白酶-7(matrix metalloproteinase-7,MMP-7)、Survivin mRNA相对表达量。以开胸手术或经皮肺穿刺病理活检结果为金标准,对比IBAL和TBLB检测对于肺癌的灵敏度、特异度。结果肺癌组BALF中CEA、CYFRA21-1、ET-1和NSE水平显著高于肺良性病变组,P0.01。肺癌组的MMP-7、Survivin mRNA相对表达量显著高于肺良性病变组,P0.01。IBAL检测肺癌的灵敏度为72.41%,特异度为62,50%;TBLB检测肺癌的灵敏度为79.31%,特异度为67.86%。两种检测肺癌方法灵敏度和特异度无显著差异,P0.05。结论行支气管肺泡灌洗可以提高内镜检查阴性周围型肺癌患者的诊断率,进而早期手术治疗。     

表面增强激光解析电离飞行时间质谱法筛选肺癌患者血清和肺泡灌洗液中标志蛋白的研究

目的 应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术筛选肺癌患者血清和BALF中的差异性表达蛋白,探讨是否可作为诊断肺癌的肿瘤标志物.方法 应用SELDI-TOF-MS技术通过弱阳离子交换蛋白芯片(WCX-2芯片)分别检测35例肺癌和18例肺部良性病变患者血清和BALF中的蛋白质质谱图,用Biomarker Pattern软件分析肺癌的差异蛋白并初步建立诊断模型,通过盲筛进一步验证诊断模型.结果 在肺癌患者血清中发现5个高表达的蛋白质波峰,选用其中质荷比为5639的差异蛋白波峰建立分类树模型,其诊断的敏感度为80%(28/35),特异度为78%(14/18).盲法验证的敏感度为85%(17/20),特异度为90%(9/10),粗符合率为87%(26/30),Youden指数为0.7.在肺癌患者BALF中发现8个高表达蛋白质波峰,选用其中质荷比为7976和11 809的差异蛋白波峰建立分类树模型,其诊断的敏感度为86%(30/35),特异度为72%(13/18).盲法验证的敏感度为90%(18/20),特异度为90%(9/1O),粗符合率为90%(27/30),Youden指数为0.8.平行试验结果显示两者联合应用时诊断肺癌的敏感度、准确率及特异度均为100%,具有互补作用.结论SELDI-TOF-MS技术可筛选出肺癌患者血清和BALF中差异性表达蛋白,作为一种肿瘤标志物,其诊断敏感度高,特异度好,尤其是BALF中差异性表达蛋白的测定可能具有较好的临床应用前景.

Abstract：

Objective To detect the protein markers in serum and bronchoalveolar lavage fluid (BALF) of the patients with lung cancer by surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) technology, and to explore if they can be used as markers for the diagnosis of lung cancer.Methods SELDI-TOF-MS technology and protein chips weak cation exchange (WCX-2 chip) were used to detect the protein mass spectrum in serum and BALF of 35 patients with lung cancer and 18 cases of benign pulmonary diseases.The different protein markers were analyzed by Biomarker Pattern Software and the initial diagnosis models were set up.The diagnosis models were verified further by blind screen to confirm the efficacy of diagnosis.Results Five protein peaks in the sera of the patients with lung cancer were significantly higher (P ＜ 0.05 ).The protein peak with a mass/charge ratio (M/Z)of 5639 was selected to establish the classification tree model.The sensitivity of diagnosis was 80% (28/35) and the specificity was 78% (14/18).The results verified by blind screen showed a sensitivity of 85% (17/20),a specificity of 90% (9/10), a crude accuracy (CA) of 87% ( 26/30 ) and Youden' s index (γ) of 0.7.Eight protein peaks in the BALF of the patients with lung cancer were significantly higher ( P ＜ 0.05).The different protein peaks with M/Z of 7976 and 11 809 respectively were selected to establish the classification tree model.The sensitivity of diagnosis was 86% (30/35) and the specificity was 72% (13/18).The results verified by blind screen showed a sensitivity of 90% (18/20), a specificity of 90% (9/10), a CA of 90% (27/30) and γof 0.8.There was a complementary role in combination of differential proteins in serum and BALF and the sensitivity, specificity and accuracy of diagnosis for lung cancer were 100% by parallel test.Conclusions The SELDI-TOF-MS technology can screen out the differential protein markers in serum and BALF of the patients with lung cancer, which show high sensitivity and specificity as tumor markers.The differential proteins in the BALF may be more promising for clinical application.     

表面增强激光解析电离飞行时间质谱法筛选肺癌患者血清和肺泡灌洗液中标志蛋白的研究

Objective To detect the protein markers in serum and bronchoalveolar lavage fluid (BALF) of the patients with lung cancer by surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF-MS) technology, and to explore if they can be used as markers for the diagnosis of lung cancer.Methods SELDI-TOF-MS technology and protein chips weak cation exchange (WCX-2 chip) were used to detect the protein mass spectrum in serum and BALF of 35 patients with lung cancer and 18 cases of benign pulmonary diseases.The different protein markers were analyzed by Biomarker Pattern Software and the initial diagnosis models were set up.The diagnosis models were verified further by blind screen to confirm the efficacy of diagnosis.Results Five protein peaks in the sera of the patients with lung cancer were significantly higher (P ＜ 0.05 ).The protein peak with a mass/charge ratio (M/Z)of 5639 was selected to establish the classification tree model.The sensitivity of diagnosis was 80% (28/35) and the specificity was 78% (14/18).The results verified by blind screen showed a sensitivity of 85% (17/20),a specificity of 90% (9/10), a crude accuracy (CA) of 87% ( 26/30 ) and Youden' s index (γ) of 0.7.Eight protein peaks in the BALF of the patients with lung cancer were significantly higher ( P ＜ 0.05).The different protein peaks with M/Z of 7976 and 11 809 respectively were selected to establish the classification tree model.The sensitivity of diagnosis was 86% (30/35) and the specificity was 72% (13/18).The results verified by blind screen showed a sensitivity of 90% (18/20), a specificity of 90% (9/10), a CA of 90% (27/30) and γof 0.8.There was a complementary role in combination of differential proteins in serum and BALF and the sensitivity, specificity and accuracy of diagnosis for lung cancer were 100% by parallel test.Conclusions The SELDI-TOF-MS technology can screen out the differential protein markers in serum and BALF of the patients with lung cancer, which show high sensitivity and specificity as tumor markers.The differential proteins in the BALF may be more promising for clinical application.     

经支气管镜肺活检联合肺泡灌洗对周围性肺癌的诊断价值

目的探讨经支气管肺活检(TBLB)联合支气管肺泡灌洗液(BAL)的脱落细胞学检测对周围性肺癌的诊断价值。方法对65例肺部占位可疑肺癌患者分别行经支气管肺活检,获取标本满意后再行病灶部位的局部肺泡灌洗,将收集的灌洗液及肺活检标本分别送病理室行细胞或组织的病理学检查。病理结果进行整理、分析,将联合检测(TBLB+BAL)结果的阳性率分别同单独的TBLB或BAL进行比较。结果 65例肺部患者中,TBLB确诊肺癌共32例,BAL确诊肺癌41例,联合检测(TBLB+BAL)确诊肺癌共52例,其中TBLB及BAL均找到癌细胞者共27例,TBLB结果阴性而BAL结果阳性者共20例。联合组阳性率(85.2%)高于单独应用组(TBLB 49.2%,BAL 63.1%,P<0.05)。结论经支气管肺活检联合支气管肺泡灌洗较单纯的支气管肺活检或肺泡灌洗能明显提高肺癌的诊断率,值得临床推广。     

支气管肺泡灌洗液癌胚抗原检测对周围型肺癌的诊断价值

目的探讨支气管肺泡灌洗液（BALF）中肿瘤标志物癌胚抗原（CEA）的检测在周围型肺癌诊断中的价值。方法采用发光免疫法测定31例肺癌病人及25例肺部良性疾病者BALF与血清CEA的含量。结果血清中CEA含量肺癌组稍高于肺部良性疾病组,但差异无统计学意义（P〉0．05）。而两组BALF中CEA含量有非常显著差异（P〈0．01）,肺癌组显著高于肺部良性疾病组。结论CEA在BALF中含量的检测比血清敏感,BALF中CEA含量的检测对肺癌的早期诊断价值优于血清,可作为诊断肺癌的有力手段。     

支气管灌洗液抑癌基因过甲基化对肺癌诊断价值的研究

目的探讨支气管灌洗液抑癌基因过甲基化对肺癌诊断的应用价值。方法选取北京市结核病胸部肿瘤研究所收治怀疑肺癌患者78例,包括肺癌51例和良性病变患者27例,从支气管灌洗液上清和细胞提取DNA,对上清游离DNA进行定量分析,并采用NMSP完成对支气管灌洗液上清和细胞的过甲基化分析,所选择的靶基因包括p16,MGMT以及RASSF1A基因。将NMSP结果与临床诊断比较,计算敏感性和特异性。结果中心型肺癌、周围型肺癌和肺部良性病变支气管灌洗液上清游离DNA含量没有显著差异。支气管灌洗液细胞抑癌基因过甲基化诊断中心型肺癌的敏感性和特异性分别为85.2%和81.5%,诊断周围型肺癌的敏感性和特异性分别为54.2%和81.5%。支气管灌洗液上清和细胞抑癌基因过甲基化诊断中心型肺癌的敏感性和特异性为88.9%和70.4%,诊断周围型肺癌的敏感性和特异性为75.0%和70.4%。结论支气管灌洗液抑癌基因的过甲基化可为肺癌诊断提供帮助。     

应用SELDI-TOF-MS技术筛选肺鳞癌和肺腺癌患者差异蛋白的研究

目的 应用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术筛选出不同病理类型肺癌患者血清、支气管肺泡灌洗液(BALF)和肺癌组织中的差异蛋白,并探讨其临床意义.方法 选用WCX-2芯片、SELDI-TOF-MS技术检测20例肺鳞癌、20例肺腺癌和20例肺良性病变患者血清、BALF和肺组织匀浆的蛋白质谱,用Biomarker Pattern软件分析肺鳞癌和肺腺癌的差异蛋白并初步建立诊断模型.结果 ①肺鳞癌组与肺良性病变组:在血清、BALF、肺组织匀浆中发现存在2、9、8个差异蛋白峰(P＜0.05),分别选用其中质荷比为5 124.24、7 967.29联合10 843.45、7914.59联合8709.66的差异蛋白波峰建立分类树模型,其诊断肺鳞癌的灵敏度分别为85％、80％、75％,特异度分别为65％、80％、90％,正确率分别为75％、80％、83％,阳性预测值分别为71％、80％、88％,阴性预测值分别为81％、80％、78％,其相对应ROC曲线下面积分别为0.750、0.916、0.930.②肺腺癌组与肺良性病变组:在血清、BALF、肺组织匀浆中发现存在8、9、7个差异蛋白峰(P＜0.05),分别选用其中质荷比为9295.79、7923.01、2452.49的差异蛋白波峰建立分类树模型,其诊断肺腺癌的灵敏度分别为75％、80％、70％,特异度分别为65％、90％、85％,正确率分别为70％、85％、78％,阳性预测值分别为68％、89％,82％,阴性预测值分别为72％、82％、71％,其相对应ROC曲线下面积分别为0.844、0.933、0.825.结论 肺鳞癌和肺腺癌BALF及肺组织匀浆中的差异蛋白较血清多,诊断效率比血清高,肺鳞癌BALF中质荷比为7967.29和10843.45的差异蛋白峰联合、肺组织匀浆中质荷比为7914.59和8709.66的差异蛋白峰联合建模及肺腺癌BALF中质荷比为7923.01的差异蛋白峰建立分类树诊断模型,其诊断的灵敏度、特异度和正确率可达到75％～90％,有可能作为诊断肺鳞癌和肺腺癌的标志蛋白.     

SELDI技术筛选肺癌患者血清标志蛋白的临床价值

目的探讨表面增强激光解析电离飞行时间质谱（SELDI-TOF-MS）技术筛选肺癌患者血清标志蛋白的临床价值。方法用SELDI-TOF-MS技术、弱阳离子交换蛋白芯片,检测肺癌和肺良性病变患者的血清蛋白质质谱图;用Biomarker Pattern软件分析肺癌差异蛋白并初建其诊断模型,通过盲筛验证诊断模型。结果发现有统计学差异的蛋白峰20个,其中肺癌患者血清高表达蛋白质波峰14个,低表达蛋白质波峰6个;用质荷比2 090.77、2 503.31 Da的差异蛋白峰建立分类树模型,其诊断肺癌的灵敏度88%,特异度95%;盲筛验证灵敏度90%,特异度100%,粗符合率93.33%,Youden指数0.9。结论SELDI-TOF-MS技术筛选的肺癌血清差异性蛋白及分类树模型,诊断肺癌的灵敏度高、特异性好。     

SELDI-TOF MS技术在胰腺癌早期诊断中的应用

胰腺癌是目前预后最差的恶性肿瘤,早期诊断被认为是改善胰腺癌预后的重要出路之一.表面增强激光解吸电离飞行时间质谱(SELDI- TOF MS)技术,是近年来发展起来一种新的高通量蛋白质组学研究方法,本文就SELDI-TOF MS技术相关的原理、特点,在胰腺癌早期诊断中的应用新进展和未来的发展趋势作一综述.     

Study on the quality of recombinant proteins using matrix-assisted laser desorption ionization time of flight mass spectrometry

Subjecxtheadingsrecombinantproteins;molecularweight;flightmassspectrometry;erythroPOietintrypticdigestsINTRODUCTIONSinceHillenkampetal[ljfirstintroducedmatrixassistedlaserdesorption/ionizationtimeofflightmassspectrometry(MALDI-TOFMS)toanalyzeproteinswithmolecularmassesgreaterthanM.10000,~DI--TOFMShasbeenwidelyusedtostudydifferentclassesofbiomoleculessuchasproteins,oligonucleotides,polysaccharidesandpolymers"--".Comparedtothetraditionaltechniques,suchassodiumdodecylsulfatepolyacryl…     

用飞行质谱技术筛选肝棘球蚴病患者血清蛋白质组学标记物

目的 筛选肝棘球蚴病患者血清蛋白质组学标记物,建立血清蛋白质指纹图诊断模型,评价其应用价值.方法 肝棘球蚴病患者68例,对照组73例(其他肝病患者33例,健康人40例),将全部样品分为训练组(37例)和测试组(67例).用弱阳离子交换蛋白芯片结合表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)技术检测肝棘球蚴病患者和对照者的血清蛋白质谱.分别比较训练组中37例肝棘球蚴患者和37例对照者、5例肝囊型棘球蚴病(HCE)患者和5例肝泡球蚴病(HAE)患者、8例肝棘球蚴病患者手术前后两组血清蛋白质峰的差异.利用ZJU-PDAS软件进行数据处理,通过支持向量机(SVM)运算建立蛋白质指纹图诊断模型,并用测试组标本采用盲法对模型的灵敏度和特异度进行验证.结果 肝棘球蚴病组和对照组共筛选出9个差异蛋白峰,其中相对分子质量为1044、1047、1073、1075、1338、6453、6649、8714 m/z的8个蛋白质峰在病例组中下调,相对分子质量为5651 m/z的蛋白峰在病例组中上调(P<0.05),盲法验证表明所建模型对肝棘球蚴病诊断的灵敏度为77.4%(24/31),特异度为66.7%(24/36);阳性预测值为66.7%(24/36),阴性预测值为77.4%(24/31).HCE和HAE患者之间筛选出2个差异表达蛋白峰,质荷比分别为8716和2751 m/z(P<0.05);8例肝棘球蚴病患者手术前后两组共筛选出6个差异表达蛋白质峰,相对分子质量分别为1297、1505、1525、1534、5921,5941 m/z(P<0.05).结论 SELDI-TOF-MS技术结合生物信息学方法能筛选出肝棘球蚴病患者血清蛋白质组学标记物,对肝棘球蚴病诊断及预后判断具有潜在的应用价值.     

DNA sequencing by delayed extraction-matrix-assisted laser desorption/ionization time of flight mass spectrometry.

Matrix-assisted laser desorption/ionization (MALDI) time of flight mass spectrometry was used to detect and order DNA fragments generated by Sanger dideoxy cycle sequencing. This was accomplished by improving the sensitivity and resolution of the MALDI method using a delayed ion extraction technique (DE-MALDI). The cycle sequencing chemistry was optimized to produce as much as 100 fmol of each specific dideoxy terminated fragment, generated from extension of a 13-base primer annealed on 40- and 50-base templates. Analysis of the resultant sequencing mixture by DE-MALDI identified the appropriate termination products. The technique provides a new non-gel-based method to sequence DNA which may ultimately have considerable speed advantages over traditional methodologies.     

The value of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in identifying clinically relevant bacteria: a comparison with automated microbiology system

Background

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been developed as a new-type soft ionization mass spectrometry in the recent year. Increasing number of clinical microbiological laboratories consider it as an innovate approach for bacterial identification.

Methods

A total of 876 clinical strains, comprising 52 species in 27 genus, were obtained from Fudan University Affiliated Zhongshan Hospital. We compared the identification accuracy of the Vitek MS system (bioMerieux, Marcy l’Etoile) to other conventional methods for bacterial identification. 16S rRNA gene sequencing was performed as a reference identification method in cases of discrepant results.

Results

The Vitek MS system consistently produced accurate results within minutes of loading, while conventional methods required several hours to produce identification results. Among the 876 isolates, the overall performance of Vitek MS was significantly better than the conventional method both for correct species identification (830, 94.7% vs. 746, 85.2%, respectively, P=0.000).

Conclusions

Compared to traditional identification methods, MALDI-TOF MS is a rapid, accurate and economical technique to enhance the clinical value of microorganism identification.