肝硬化患者血清蛋白组学研究

目的比较肝硬化组与健康对照组以及肝硬化A、B、C级之间的血清蛋白组学,寻找用于肝硬化早期诊断、疗效预测的特异性蛋白标志物。方法应用美国CipherGen公司表面增强激光解析电离飞行时间质谱仪和蛋白芯片获得肝硬化患者及健康人血清中的蛋白质指纹图谱,用Biomarker Wizard软件进行分析,筛选出差异表达的蛋白峰。结果肝硬化组与健康对照组共有16种蛋白质差异有统计学意义,A级与B级、C级之间的差异蛋白分别为1、9种,B级与C级之间无差异蛋白表达。结论表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)在肝硬化的诊断及病情判断等方面具有一定价值。     

胃癌血清蛋白质谱的差异性研究

目的对比胃癌患者手术前后与正常人血清蛋白质谱的差异,筛选出诊断胃癌的特异性蛋白标志物。方法应用表面增强激光解吸离子化飞行时间质谱（SELDI—TOF—MS）技术和CM10蛋白质芯片,对22例胃癌患者和18名正常人的血清蛋白质谱进行检测。结果通过对胃癌术前血清与正常人血清蛋白质谱分析,发现由5个蛋白质峰组成的生物标记物可将胃癌术前患者与正常人准确分组。结论建立胃癌的血清蛋白质谱,为胃癌蛋白质组学研究奠定一定的基础．建立了以8961．2404M／Z、3947．264M／Z、5919．7541M／Z、4103．348M／Z、8711．0363M/Z5个蛋白质峰组成的生物标记物检测胃癌。     

飞行质谱技术筛选有家族史食管癌患者的血清标志蛋白

目的探讨有家族史食管癌患者血清蛋白的表达差异及其与食管癌遗传易患性之间的关系。方法应用表面增强激光解析电离飞行时间质谱技术结合铜离子蛋白芯片获得20例有家族史和26例无家族史食管癌患者的血清蛋白质谱。将获得的蛋白质谱采用Ciphergen公司的Biomarker Wizard和Biomarker Pattern软件分析。结果共找到10个表达差异蛋白,且此10个蛋白的含量在家族史阳性组均显著上调。其中,以质荷比为5344.77和7571.19建立的分类树模型能有效区别家族史阳性者和阴性者。在学习模式下分组正确率达到100%,在测试模式下分组正确率分别为100%(20/20)和96.2%(25/26)。结论质荷比为5344.77和7571.19的两个蛋白可能是影响食管癌遗传易患性的重要血清相关蛋白。     

单耐利福平结核分枝杆菌株早中期培养滤液蛋白质／多肽谱分析

目的对比分析单耐利福平（RFP）结核分枝杆菌株与敏感结核分枝杆菌株早中期培养滤液蛋白质／多肽谱的差异,以寻找检测RFP耐药的检测标志物。方法利用表面增强激光解析电离飞行时间质谱（SELDI-TOF-MS）技术对结核分枝杆菌参考株（ATCC27294）、10株单耐RFP结核分枝杆菌株的Middlebrook 7H9液体培养第7天和第14天培养滤液进行蛋白质／多肽谱分析。结果与Middlebrook7H9培养液蛋白质／多肽谱相比．结核分枝杆菌参考株、单耐RFP结核分枝杆菌株的第7、14天培养滤液中分别存在144、152、131和140种差异蛋白质／多肽;与结核分枝杆菌参考株的第7、14天培养滤液相比,单耐RFP结核分枝杆菌株的第7、14天培养滤液中分别存在39、72种差异蛋白质／多肽;与结核分枝杆菌参考株、单耐RFP结核分枝杆菌株的第7天培养滤液相比,其各自第14天培养滤液中分别存在82、64种差异蛋白质／多肽。结论RFP耐药分枝杆菌株的早中期培养滤液中存在与敏感分枝杆菌早中期培养滤液中相异的蛋白质／多肽,有望从中筛选到RFP耐药的检测标志物。     

表面加强激光解析电离-飞行时间-质谱技术在急性重症胰腺炎预后相关蛋白质谱检测中的应用

目的 应用蛋白芯片表面加强激光解析电离-飞行时间-质谱(SELDI-TOF-MS)技术检测急性重症胰腺炎不同预后相关蛋白质谱,探讨急性重症胰腺炎轻重程度及预后评估的新方法.方法 用SELDI-TOF-MS方法检测浙江省人民医院2008年3月至2010年10月23例急性重症胰腺炎患者的蛋白质谱,按是否发生器官功能衰竭、胰腺脓肿、腹内压异常、死亡将全部患者分组进行蛋白指纹图谱比较.结果 蛋白质峰谱特征显示,在1094 u、2751 u、5904 u处并发胰腺脓肿(13例)的重症胰腺炎患者峰值(13.21±3.73,45.62±10.31,48.37±9.24)明显高于未并发胰腺脓肿组(70例)(4.33±1.79,8.87±3.21,4.45±1.59),差异有统计学意义,均P<0.05.在635 u并发器官功能衰竭组(11例)峰值(8.56±3.21)明显低于未并发器官功能衰竭组(12例)(37.82±12.65);而在4103 u、4187 u处并发器官功能衰竭组(21.63±8.23.9.81±2.32)峰值则高于未并发器官功能衰竭组(3.32±1.29,1.14±0.49).在4173 u处并发腹内压增高组(10例)峰值(8.94±3.58)明显高于无腹内压增高组(13例)(1.97±0.73);而在5635 u处并发腹内压增高组峰值(0.62±0.23)则低于无腹内压增高组(15.78±6.34).2例死亡患者蛋白指纹表现为血清蛋白全面合成功能减退,峰值降低.结论 蛋白指纹图谱技术可筛选出有意义的生物标记蛋白,为急性重症胰腺炎并发症的早期诊断与治疗提供了新的路径,对急性重症胰腺炎预后评估具有重要意义.

Abstract：

Objective To analyze the serum proteomic pattern in different severe acute pancreatitis complication by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) for evaluating the stage and prognosis of severe acute pancreatitis. Methods Serum samples were collected from 23 severe a-cute pancreatitis. Serum from organ nonfunction, pancreatic abscess, intra-abdominal hypertension and death were profiled using WCX Proteinchip and analyzed by mass spectrometry. Results Protein peak in the spectrum characteristics showed that at 1094u, 2751u, 5904u, the peak value of concurrent pancreas abscess pancreatitis [13.21 ± 3.73, 45.62±10.31, 48.37±9.24] were significantly higher than those in none-pancreas abscess group [4.33 ± 1.79, 8. 87 ±3.21, 4.45 ±1.59] (P<0.05). At 635u, the peak value of concurrent organ failure group(8.56 ± 3.21) was obviously lower than those in none-organ failure group(37.82 ± 12.65). At 4103u and 4187u, the peak value of in complicated organ failure group [21.63 ±8.23, 9.81±2.32] were higher than those in none-organ failure group [3.32±1.29, 1.14 ±0.49], At4173u, the peak value of concurrent increased intraabdominal pressure group(8.94 ±3.58) was significantly higher than that in none-intraabdominal pressure group(1.97 ±0. 73) , while at 5635u the peak value of concurrent increased intraabdominal pressure groups ( 0. 62 ± 0. 23 ) was lower than that without intraabdominal pressure group (15.78 ±6.34). Two cases died. Protein fingerprints showed that overall serum protein composite function dropped and the peak value reduced. Conclusion Proteomic technology can significantly identify novel significant biomarkers in the serum, which provides a new way to diagnose and treat the different complication of severe acute pancreatitis early and has clinical significance in evaluating the prognosis of severe acute pancreatitis.     

顺铂循环热灌注化疗治疗原发性肝癌恶性腹腔积液43例

目的 观察顺铂循环热灌注化疗治疗原发性肝癌恶性腹腔积液的临床疗效及安全性.方法 选取2018年6月—2019年7月在江汉大学附属湖北省第三人民医院肿瘤科收治的原发性肝癌伴腹腔积液患者86例,按随机数字表法分为治疗组、对照组各43例,所有患者均行中心静脉导管双侧腹腔穿刺引流置管术,均以0.9％氯化钠溶液3000 mL+顺...     

应用基质辅助激光解析电离飞行时间质谱源后衰变技术鉴定蛋白质

目的应用源后衰变(PSD)技术结合数据库检索鉴定二维蛋白质电泳(2DE)胶上的蛋白质斑点。方法 用已知多肽(ACTH)18~39肽段和TPA被胰酶降解后的1个肽段建立了PSD-MALDI-TOF-MS方法。结果应用已建立的PSD-MALDI-TOF-MS法分别将2DE分离后胶上的2个未知蛋白质斑点鉴定为40S核糖体蛋白S12和dnaK抑制蛋白。结论PSD技术在蛋白质组学研究中有较大的应用前景。     

应用SELDI—TOFMS技术筛选乳腺癌蛋白标志物

目的：应用表面增强激光解吸电离飞行时间质谱技术（ SELDI-TOF MS）筛选乳腺癌的特异性蛋白标志物,建立诊断模型。方法用表面增强激光解吸电离飞行时间质谱仪及CM10蛋白芯片检测35例乳腺癌患者标本及53例对照组标本（包括35例乳腺良性病变和18例正常人）的血清蛋白指纹图谱,Ciphergen Proteinchip 软件自动采集数据,Ciphergen Biomaker Wizard 软件筛选差异蛋白,Biomarker Pattern软件建立乳腺癌的分类树诊断模型。结果乳腺癌组及对照组血清蛋白质谱图共检测到59个蛋白质峰,其中19个蛋白峰表达差异具有显著性（P＜0．01）。以2个蛋白质峰（质荷比分别为M6636．62,M13889．6）建立的诊断模型,诊断的准确率达到94．3％,灵敏度和特异度分别为80．0％和71．7％。结论 SELDI-TOF MS技术可用于乳腺癌特异性蛋白的筛选,为其快速诊断奠定基础。     

结核性胸腔积液蛋白质组学初步研究

目的本实验利用二维凝胶电泳-肽质量指纹谱的蛋白质组学方法,对结核性、恶性和漏出性胸腔积液的相关蛋白质二维凝胶电泳（2-DE）图谱识别、鉴定,寻求结核性胸腔积液的特异蛋白质及其对临床诊断的意义。方法收集我院2010年5月～2011年6月收治的符合要求的胸腔积液患者50例,其中结核性胸腔积液20例,恶性胸腔积液17例,漏出性胸腔积液13例。利用二维凝胶电泳分离技术,基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）分析到相应的肽质量指纹谱（PMF）,最后借助Mascot软件结合NCBInr数据库进行检索鉴定相关结构蛋白质。结果通过对结核性、恶性和漏出性胸腔积液的蛋白质肽质量指纹图比较、分析发现3个存在明显差异蛋白,它们分别是：C1-抑制蛋白、甲状腺三级结构A链和补体C3b。C1-抑制蛋白在结核性胸腔积液高表达,在恶性胸腔积液中低表达。甲状腺素三级结构A链和补体C3b在恶性腔积液中高表达,在漏出液中低表达。结论利用二维凝胶电泳技术和基质辅助激光解吸电离飞行时间质谱技术对胸腔积液进行蛋白质组学研究,得到结核性、恶性和漏出性胸腔积液的蛋白质图谱,获得差异蛋白质,为结核性胸腔积液的鉴别诊断提供新的思路和方法。     

SELDI蛋白质芯片技术在医学中的应用现状

随着全球基因组计划的推进，人类基因组工作草图得以公布，标志人类染色体总DNA有32亿碱基对，包含有大约3万个基因，远远少于原来约10万个基因的估计。据报道，人类结构与功能各异的蛋白质有10万余种，与功能基因（3万-4万个）相比显然表现了更强的多样性和可变性，以及它们在产生和代谢方面受到多因素调节和相互之间的作用更是构成了一个复杂多变的体系。因此，随着人类基因组测序的完成，研究不同细胞或组织表达的全部蛋白质数据库的构建与细胞在不同状态的蛋白质表达差异已成研究热点，标志着蛋白质组学时代的到来。     

基体辅助激光解吸质谱法在生物大分子质量研究中的应用

研究了基体辅助激光解析飞行时间质谱法(MALDI-TOF MS)测定蛋白和糖蛋白的实验条件,实测了重组人白细胞介素2、肿瘤坏死因子、粒细胞巨噬细胞集落刺激因子、白细胞干扰素α2b、白细胞干扰素α₁、促红细胞生成素、钙调蛋白及其片段、大鼠脑型一氧化氮合成酶和天然牛精液蛋白提取物的分子量和纯度,鉴定了蛋白质混合物中各单个成分,结果表明MALDI-TOF MS可有效地研究基因工程蛋白质和天然蛋白提取物等生物大分子的质量。     

MALDI／TOF MS法测定蛇毒纤溶酶及磷脂酶A2的分子量和纯度

目的： 分析测定长白山白眉蝮蛇蛇毒纤溶酶和磷脂酶Ａ２ 的分子量和纯度。方法与结果： 应用ＭＡＬＤＩ／ＴＯＦ ＭＳ 法测定纤溶酶的分子量为２３ ３３３ ±９０ , 磷脂酶Ａ２ 的分子量为１４ ０００ ±２０ , 相对偏差在０－３８ ％ 以内。结论： 应用此方法未检测到杂蛋白质谱峰的存在, 酶的纯度较好, 测得结果要比电泳法准确。ＭＡＬＤＩ／ＴＯＦ ＭＳ提供了一种测定蛋白质药物纯度快速准确的新方法。     

Laser Ablation Remote-Electrospray Ionisation Mass Spectrometry (LARESI MSI) Imaging—New Method for Detection and Spatial Localization of Metabolites and Mycotoxins Produced by Moulds

To date, no method has been developed to assess the distribution of mycotoxins on the surface of grains, or other plant material, and the depth of their penetration into the interior. The Infrared (IR) Laser Ablation-Remote-Electrospray Ionization (LARESI) platform coupled to a tandem mass spectrometer (MS/MS), measuring in selected reaction monitoring (SRM) mode, was employed for the targeted imaging of selected metabolites of Aspergillus fumigatus, including mycotoxins in biological objects for the first time. This methodology allowed for the localisation of grain metabolites and fungal metabolites of grain infected by this mould. The distribution of metabolites in spelt grain was differentiated: fumigaclavine C, fumitremorgin C, and fumiquinazoline D were located mainly in the embryo, brevianamide F in the seed coat, and fumagillin in the endosperm. The LARESI mass spectrometry imaging method can be used in the future for the metabolomic analysis of mould metabolites in various plants and agricultural products.     

重组人粒细胞巨噬细胞集落刺激因子的生物质谱和毛细管电泳法

目的：为准确分析重组人粒细胞巨噬细胞集落刺激因子(rHuGM-CSF)半成品及其制剂的纯度和表达的正确性。方法：用毛细管区带电泳测定了2批rHuGM-CSF半成品和3批rHuGM-CSF注射液的纯度。用基体辅助激光解吸飞行时间质谱和电喷雾质谱测定了2批rHuGM-CSF半成品的分子量。用毛细管等电聚集法测定了rHuGM-CSF半成品的等电点。结果：2批半成品中1批含有少量二聚体,且含有4个不同等电点的组分,另1批表达不正确;2批成品均存在大量杂质。结论：发现有些rHuGM-CSF样品有生物学活性,但表达不正确,纯度也不高;各分析方法是互补的,不能相互替代。     

Molecular weights of poly(butyl cyanoacrylate) nanoparticles determined by mass spectrometry and size exclusion chromatography

Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and size exclusion chromatography (SEC) were employed to elucidate the chemical composition, mean number average molecular weight (M_n), mean weight average molecular weight (M_w), and polydispersity (PD) of poly(butyl cyanoacrylate) (PBCA) manufactured by emulsion polymerisation. Both methods gave similar results for M_n, but substantial differences were observed for M_w and PD, with MALDI producing consistently lower values which could not be improved by off-line coupling of SEC and MALDI. MALDI gave a more detailed view on the chemical composition of the cyanoacrylate and revealed the presence of two additional polymer series with different end groups besides the expected PBCA series, which showed different retention in SEC. Their formation is explained by the secession/addition of formaldehyde from/to the regular polymer via (reverse) Knoevenagel reaction. In additional experiments, the influence of different pH on PBCA-NP during polymerisation was examined by comparison of polymerisation yield and particle diameter to their chemical composition as revealed by the MALDI spectra. The most uniform nanoparticles, with the highest polymerisation yield, narrowest particle size, and mass distribution were produced at pH 1.     

重组人粒细胞巨噬细胞集落刺激因子 的生物质谱和毛细管电泳法

目的：为准确分析重组人粒细胞巨噬细胞集落刺激因子（ｒＨｕＧＭＣＳＦ）半成品及其制剂的纯度和表达的正确性。方法：用毛细管区带电泳测定了２批ｒＨｕＧＭＣＳＦ半成品和３批ｒＨｕＧＭＣＳＦ注射液的纯度。用基体辅助激光解吸飞行时间质谱和电喷雾质谱测定了２批ｒＨｕＧＭＣＳＦ半成品的分子量。用毛细管等电聚集法测定了ｒＨｕＧＭＣＳＦ半成品的等电点。结果：２批半成品中１批含有少量二聚体,且含有４个不同等电点的组分,另１批表达不正确;２批成品均存在大量杂质。结论：发现有些ｒＨｕＧＭＣＳＦ样品有生物学活性,但表达不正确,纯度也不高;各分析方法是互补的,不能相互替代。     

Combined treatment of human MCF-7 breast carcinoma with antibody,cationic lipid and hyaluronic acid using ex vivo assays

The effective targeting of malignant cell surface antigens is essential in cancer therapy. Resistance to treatment and rapid invasion of cancer cells are the main causes of cancer mortality. Despite intense research efforts, treatments often have demonstrated insufficient outcomes in clinical applications.     

Searching for novel biomarkers of centrally and peripehrally-acting neurotoxicants, using surface-enhanced laser desorption/ionisation-time-of-flight mass spectrometry (SELDI-TOF MS).

The neurotoxicity of chemicals to humans is difficult to monitor as there are no suitable methods of detecting early neuronal dysfunction. Here, a proof of principle study was designed to assess the potential of identifying protein biomarkers in accessible biofluids for this purpose. Groups of rats were treated with a range of doses of the model neurotoxicants, acrylamide (0, 2, 10, 50mg/kg) and methylmercury (0, 0.2, 1, 5mg/kg) for up to 3 weeks and samples of serum, urine, and cerebral spinal fluid analysed by surface-enhanced laser desorption/ionisation-time-of-flight mass spectrometry. There was no neuropathology up to the highest dose tested. Protein profiles were obtained from all samples and changes in the levels of many proteins were detected in both serum and urine, although not cerebral spinal fluid. In serum, the combination of three protein ion levels with m/z values of 4968, 9402 and 12,948 was able to correctly classify the treatment groups thus: 88% control, 100% acrylamide, 92% methylmercury. In urine, three protein ions with m/z values of 4944, 12,966 and 21,992 classified correctly the groups: 67% control, 94% acrylamide, 97% methylmercury. Similar classifications using other serum and urinary protein ions were also possible. This indicates the potential of serum and urine protein biomarkers for the assessment of sub-clinical neurotoxicity.     

生物质谱技术及其在核酸领域的应用

生物质谱技术具有准确、快速、灵敏度高等特点,因此在生物大分子领域得到了广泛应用。本文介绍了电喷雾电离和基质辅助激光解吸/电离质谱技术的基本原理,并综述了生物质谱在核昔酸的鉴定与定量分析、单核苷酸多态性分型以及寡聚核苷酸片断序列测定中的应用。     

基质辅助激光解析电离飞行时间质谱检测耐甲氧西林金黄色葡萄球菌δ-毒素的应用

目的 本研究采用基质辅助激光解析电离飞行时间质谱(MALDI-TOF MS)检测δ-毒素,评估其在耐甲氧西林金黄色葡萄球菌(MRSA)的分型和毒力表达中的作用。方法 使用Bruker microflex MALDI-TOF仪器,采集2000~20000Da质量范围内,以正线性模式采集图谱,使用仪器配套的MALDI Biotyper 2.0软件分析MRSA菌株的原始图谱,产生的峰列表直接使用flexAnalysis、clinProTools3.0软件分析。结果 本研究中共检测83株MRSA,共有39(47.0%)株MRSA检出(3005±5)m/z信号峰,其中HA-MRSA 19(22.9%)株,CA-MRSA 20(26.5%)株,P=0.766,两者之间无显著性差异;33(39.8%)株MRSA检出(3035±5)m/z信号峰,其中HA-MRSA 9 (10.8%)株,CA-MRSA 24(28.9%)株,P=0.003,两者之间有显著性差异;11(13.%)株MRSA既未检出(3005±5)m/z信号峰也未检出(3035±5)m/z信号峰,全部为HA-MRSA菌株。spa分型中检出(3005±5)m/z信号峰,15(18.1%)株为t062型,8株为t015(9.6%)型,4株为t030(4.8%)株,P=0,有显著性差异;检出(3035±5)m/z信号峰,31(37.3%)株为t437型,2(2.4%)株t8660型,P=0,有显著性差异;(3035±5)m/z作为spa t437型特征信号ROC曲线下面积0.89,P=0。11株未检出δ-毒素,6株分离自骨关节标本,3株分离自呼吸道标本,1株分离自慢性溃疡的分泌物标本,1株分离自血液;在血平板的菌落特征,6株MRSA菌落形态发生改变,5株菌落形态正常。结论 MALDI-TOF MS使用常规方法即可快速检测MRSA的δ-毒素,其质谱峰为(3005±5)m/z和(3035±5)m/z两种;(3035±5)m/z是δ-毒素的同基因变异体(HldG10S)的质谱峰,该峰可快速鉴别spa t437型;不产生δ-毒素的菌株是agr调控系统失调的表现,与慢性感染、小菌落形成、无明显β-溶血有关。