SELDI蛋白质芯片检测技术

从基因组学到蛋白质组学,到当前有关小RNA对蛋白质合成调控的研究无一例外地说明蛋白质是直接发挥对生命活动调控的物质。同基因研究相比较,由于蛋白质分子种类繁多,有复杂的修饰成份和空间结构,使得蛋白质研究比较困难。新近发展起来的蛋白质芯片技术为蛋白质的检测和研究提供了新的技术平台,比如荧光标记技术,蛋白质指纹图谱．飞行时间．质谱联用技术（SELDI蛋白质芯片）,表面等离子基元共振生物传感器技术（SPR芯片）以及初步应用的光学蛋白质芯片技术,其中,后三种是新兴的无需标记进行蛋白质检测的技术。就SELDI蛋白质芯片及其新近研究作一综述。     

血清SELDI蛋白质指纹图谱在乳腺癌术前分级中的应用

应用SELDI技术和生物信息学方法从血清中筛选能反映乳腺癌术前分期的蛋白质峰并构建检测模型。采用CM10芯片,对34例Ⅰ～Ⅱ期乳腺癌患者和31例Ⅲ～Ⅳ期乳腺癌的血清进行了检测,发现11个蛋白质峰在两组患者之间表达量有显著性差异（P<0.05）,M/Z为 M2042.87、M2459.83、M3881.37、M4804.47、M6683.24和 M6706.06的6个蛋白质峰被选为分类变量构成决策树分类模型,该模型的交叉验证（测试组）总准确率为80.0%,Ⅰ～Ⅱ期乳腺癌检出率为82.4%,Ⅲ～Ⅳ期检出率为77.4%。SELDI技术在乳腺癌患者术前分级的判断方面具有一定的应用价值。     

以SELDI芯片进行细胞标本蛋白分析的方法学研究

目的:探讨以SELDI芯片技术进行细胞标本蛋白分析的最适方法及条件,筛选细胞标本蛋白表达差异。方法:对细胞标本分别用超声裂解法,U9细胞裂解缓冲液配方和自配细胞裂解液提取蛋白,以BCA法测定蛋白浓度;分别以磁珠活化后点样和生物芯片处理器点样使蛋白样品与芯片结合;并对提取蛋白进行检测,比较不同蛋白浓度梯度点样及WCX2,SAX2,IMAC-Cu,H50芯片捕获蛋白差异,用WCX2芯片筛选蛋白差异表达。结果:相同培养条件细胞以上述三种不同蛋白提取方法获得的蛋白浓度分别为:0.25±0.034μg/μl,0.6±0.06μg/μl,1.02±0.077μg/μl;生物芯片处理器点样法操作简单,要求样本量较少,点样时间短;SELDI芯片蛋白质峰图谱与蛋白浓度呈较好的正相关;WCX2,SAX2,H50,IMAC-Cu芯片捕获的蛋白质种类有较大区别;在分子量1000～300000Da范围内,以WCX2芯片共检测到87个差异蛋白峰,其中17个呈趋势变化。结论:上述三种方法比较,选用自配的细胞裂解液提取蛋白的浓度较高且更适于芯片研究;生物芯片处理器能较好地使蛋白与芯片结合;SELDI芯片能准确定位蛋白,且其蛋白质峰与被测蛋白浓度呈正相关变化;SELDI各芯片捕获蛋白类型不同,选择适宜芯片或联合运用芯片检测更易获得较理想蛋白差异表达结果。     

尿样在SELDI技术中的优化研究

目的：建立表面增强激光解吸离子化飞行时间质谱（SELDI．TOF／MS）技术检测尿液样本的方法。方法：采用SELDI技术及弱阳离子交换表面蛋白芯片（CM10）对糖尿病病例组和正常对照组的尿液样本进行分析,从尿液标本的采集、样品保存、上样浓度的控制、实验仪器的内外校准、实验结果重复性验证与分析等方面进行实验条件的优化。结果：反复冻融3次以上的样品经SELDI检测的出峰数量和峰强度情况较差;以1：1或1：2的浓度作倍比稀释后的样本经芯片检测得到的蛋白峰强度和蛋白数量最优;对两组样本重复检测3次,蛋白丰度的平均变异系数（CV值）分别为0．121和0．095;两组样本共检测到202个蛋白峰,其中差异表达蛋白29个,在肝纤维化组中表达上调13个,表达下调16个。结论：初步建立了SELDI技术检测尿液样本的方法,提高了SELDI技术检测尿液样本的质量。     

血清蛋白质指纹图谱诊断早期胃癌临床意义

目的：应用SELDI蛋白质芯片检测胃癌患者血清蛋白质指纹图谱,筛选候选肿瘤标志物以建立诊断模型。方法：表面加强激光解吸电离-飞行时间质谱（SELDI-TOF-MS）技术及其配套蛋白质芯片检测34例胃癌患者（Ⅰ／Ⅱ期12例与Ⅲ／Ⅳ期22例）和30例健康人的血清蛋白质组图谱,运用判别分析处理数据筛选标志物并建立诊断模型。结果：2046m／z、1179m／z、1817m／z、1752m／z和1588m／z等5个蛋白质峰组合所构建的诊断模型能达到鉴别胃癌患者和健康人的最佳诊断效果,特异度94．1％（32／34）,灵敏度93．3％（28／30）。单个4665m／z蛋白质峰诊断模型可达到鉴别Ⅰ／Ⅱ期与Ⅲ／Ⅳ期胃癌效果,其特异度91．6％（11／12）,灵敏度95．4％（21／22）。结论：该方法在胃癌的诊断尤其是早期诊断方面具有一定价值,值得进一步研究。     

蛋白质芯片-飞行质谱技术

蛋白质芯片-飞行质谱技术(Proteinchip—Surface Enhanced Laser Desorption／Ionization,SELDI)是21世纪出现的一种新型生物芯片技术,是研究表达蛋白的有力工具。本对该技术的原理、构成、使用方法、应用等做了简要介绍。     

表面增强激光解吸电离质谱芯片在蛋白质组学研究中的应用

蛋白质组学是当今生命科学研究中最活跃的前沿领域。其主要技术手段是双向凝胶电泳,质谱技术和生物信息学,但各有其局限性。芯片技术和质谱技术相结合产生的表面增强激光解析离子化－飞行时间－质谱9SELDI－TOF－MS）技术,将蛋白质样品的制备,生化反应到检测分析的整个过程集成在芯片上进行,实现了新型,高效,快速,高通量的检测,不仅促进了蛋白质组学研究的迅速发展,而且为阐明生命活动的规律和疾病的发病机理以及寻找合适的医药提供提供了有力的工具。     

电针对大鼠胃黏膜损伤修复的血清蛋白质差异表达研究

目的：观察电针胃经穴对大鼠胃黏膜损伤修复的血清蛋白质差异表达,为进一步阐明针刺效应的体液机理提供理论依据。方法：用表面增强激光解吸离子化飞行时间质谱（SELDI—TOF—MS）技术和WCX2（弱阳离子交换芯片）对正常大鼠血清和电针大鼠血清进行蛋白质指纹图谱检测分析,通过Biomarker Wizard和Biomarker Patterns System软件判别分析处理数据并结合生物信息学方法筛选差异表达蛋白质。结果：与正常大鼠血清比较,电针大鼠血清蛋白质在质荷比为2000-50000有25个蛋白质峰差异有显著意义,其中有19个标志蛋白在电针胃经大鼠血清中呈现高表达,6个标志蛋白在电针胃经大鼠血清中呈现低表达。结论：电针可促进胃黏膜损伤大鼠血清蛋白质差异表达,这种差异蛋白质可能与电针促进胃黏膜损伤修复效应密切相关。     

蛋白质芯片技术的发展与应用

蛋白质芯片（proteinchip）,狭义上可以称为蛋白质微阵列（proteinarray）,是继基因芯片后发展起来的生物检测技术,是蛋白质组学研究中除了酵母双杂交、双向电泳技术、质谱技术等之外的一种重要的工具。蛋白质芯片是一种高通量的蛋白质功能分析技术,具有平行、快速、自动化的优点,     

采用SELDI蛋白质芯片技术研究氧化亚铁硫杆菌对磷酸盐缺失的反应

氧化亚铁硫杆菌(At.f)是能够利用Fe2 和硫化矿来获取能量的一种化能自养菌.这种细菌在金属硫化矿的生物浸出中起着重要的作用.在硫化矿的生物浸出过程中,浸矿细菌通常会遇到多种胁迫条件,如温度的变化、营养成分的缺失和pH值的变化等,这些因素会影响到细菌的活性.因此对在胁迫条件下这类细菌的应急反应生理机制的研究具有重要的意义.SELDI蛋白质芯片技术是近年一种高通量的蛋白质组学研究技术.测定了以Fe2 为能源正常条件培养的At.f和磷酸盐缺失培养At.f的生长情况,绘制了相应的生长曲线;采用NP20蛋白质芯片,对At.f总蛋白的蛋白质芯片上样量进行了优化.在此基础上,采用IMAC-Cu、SAX2、WCX2三种特异性SELDI蛋白质芯片技术,获取了磷酸盐缺失培养At.f与正常条件培养的At.f的比较蛋白质图谱,采用软件对比较蛋白质图谱进行分析,发现了磷酸盐缺失培养At.f的13个明显差异表达的蛋白质分子,为进一步分离鉴定这些差异表达蛋白质奠定了基础.     

On-chip Escherichia coli culture, purification, and detection of expressed proteins

In a recent study, we reported the results of a rapid high-throughput expression analysis of the affinity-tagged proteins present in total cell lysates, using a surface plasmon resonance (SPR) imaging protein chip system. In this paper, we describe a novel method, which is able to sequentially carry out a recombinant Escherichia coli culture, as well as the detection and purification of the expressed proteins on a single microwell chip, fabricated on a two-dimensional thin gold film. Following the induction of the protein on the microwell chip, the E. coli cells were lysed on the chip via the addition of lysozymes, and the expressed glutathione S-transferase-fused green fluorescent protein (GST–GFP) was then purified on the chip via affinity interaction with the glutathionylated gold surface of the chip. Finally, the expressed protein was directly detected using the surface plasmon resonance (SPR) imaging system. This system saves a substantial amount of time, experimental resources, and labor, by allowing for the complicated and labor-intensive procedures inherent to the production of recombinant proteins to be conducted on a single microwell chip, simply and economically.M. Kim and S. Y. Lee contributed equally to this work.     

Search for a novel biomarker for the brain cancer astrocytoma by using surface enhanced laser desorption/ionisation (SELDI) technique.

The protein chip surface enhanced laser desorption/ionisation (SELDI) technique is a highly versatile analytical mass spectrometry system with considerable potential for detection, identification and quantitation of protein complex mixtures. Astrocytoma is a tumour of the astrocytes with a very poor prognosis. There is no effective biomarker system for detection of astrocytoma. The SELDI technique was used to study differential protein expression in astrocytoma cells in comparison to normal brain astrocytes. Several novel proteins were found to be expressed in the astrocytoma cells, not present in the astrocytes.     

Surface plasmon resonance imaging-based protein arrays for high-throughput screening of protein-protein interaction inhibitors

The E7 protein produced by high-risk human papillomavirus (HPV) induces a degradation of the retinoblastoma tumor suppressor RB through direct interaction, which suggests that an inhibitor for the interaction can be a potential anticancer drug. A surface plasmon resonance (SPR) imaging-based protein array chip was developed for the high-throughput screening of inhibitor molecules targeting RB-E7 interaction. The glutathione S-transferase-fused E7 protein (GST-E7) was first layered onto a glutathionylated gold chip surface that had been designed to specifically bind to GST-fused proteins. Subsequently, a microarrayer was used to spot the hexa-histidine-tagged RB proteins (His(6)-RB) onto the GST-E7-layered gold chip surface, and the resulting SPR image was analyzed. Upon increased His(6)-RB concentration in the spotting solution, the SPR signal intensity increased proportionally, indicating that His(6)-RB bound to GST-E7 in a concentration-dependent manner. The His(6)-RB/GST-E7 interaction was challenged by spotting the His(6)-RB solution in the presence of a RB binding peptide (PepC) derived from a motif on E7. The SPR imaging data showed that PepC inhibited the His(6)-RB/GST-E7 interaction in a concentration-dependent manner. Our results show that the SPR imaging-based protein array chip can be applied to screen small molecule inhibitors that target protein-protein interaction.     

Proteomics profiling of urine with surface enhanced laser desorption/ionization time of flight mass spectrometry

Background  

Urine consists of a complex mixture of peptides and proteins and therefore is an interesting source of biomarkers. Because of its high throughput capacity SELDI-TOF-MS is a proteomics technology frequently used in biomarker studies. We compared the performance of seven SELDI protein chip types for profiling of urine using standard chip protocols.     

Rapid coupling of Surface Plasmon Resonance (SPR and SPRi) and ProteinChip based mass spectrometry for the identification of proteins in nucleoprotein interactions

We compared coupling approaches of SPR to LC-MS and ProteinChip-based mass spectrometry (SELDI) as a means of identifying proteins captured on DNA surfaces. The approach we outline has the potential to allow multiple, quantitative analysis of macromolecular interactions followed by rapid mass spectrometry identification of retained material.     

Analysis of differential protein expression in Acidithiobacillus ferrooxidans grown under different energy resources respectively using SELDI-ProteinChip technologies

Surface-enhanced laser desorption/ionization (SELDI)-time of flight is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a chip, which allows proteomic analysis with limited material requirements. This characteristic makes it a valuable technique for microbiologists handling problematic samples, such as low cell number cultures. In this study, we explored differential-expressed proteome of Acidithiobacillus ferrooxidans cultivated with Fe(2+) and elemental sulfur separately by adopting the protein biochip SELDI approach. The cell lysates of A. ferrooxidans were applied onto Ciphergen ProteinChip WCX2, SAX2 and IMAC-Cu arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. A summary of the molecular masses of the differentially regulated proteins found on WCX2, IMAC-Cu and SAX2 was obtained and 28 differentially expressed proteins were found on the molecular weight range of 5.0 to 25 kDa.     

Nanodiscs allow the use of integral membrane proteins as analytes in surface plasmon resonance studies

Nanodiscs are small-sized and flat model membranes that provide a close to native environment for reconstitution of integral membrane proteins. Incorporation of membrane proteins into nanodiscs results in water-soluble proteolipid particles making the membrane proteins amenable to a multitude of bioanalytical techniques originally developed for soluble proteins. The transmembrane domain of the human CD4 receptor was fused to ubiquitin with a preceding N-terminal decahistidine tag. The resulting integral membrane protein was incorporated into nanodiscs. Binding of the nanodisc-inserted histidine-tagged protein to a monoclonal anti-pentahistidine antibody was quantified using surface plasmon resonance (SPR) experiments. For the first time, a membrane-inserted transmembrane protein was employed as analyte while the antibody served as ligand immobilized on the sensor chip surface. SPR experiments were conducted in single-cycle mode. We demonstrate that the nanodisc-incorporated membrane protein showed nearly identical affinity toward the antibody as did the soluble decahistidine-tagged ubiquitin studied in a comparative experiment. Advantages of the new experimental setup and potential applications are discussed.     

Identification of the SELDI ProteinChip human serum retentate by microcapillary liquid chromatography-tandem mass spectrometry

Surface-enhanced laser desorption/ionization (SELDI) time-of-flight (TOF) mass spectrometry (MS) has been widely applied for conducting biomarker research with the goal of discovering patterns of proteins and/or peptides from biological samples that reflect disease status. Many diseases, ranging from cancers of the colon, breast, and prostate to Alzheimer's disease, have been studied through serum protein profiling using SELDI-based methods. Although the results from SELDI-based diagnostic studies have generated a great deal of excitement and skepticism alike, the basis of the molecular identities of the features that underpin the diagnostic potential of the mass spectra is still largely unexplored. A detailed investigation has been undertaken to identify the compliment of serum proteins that bind to the commonly used weak cation exchange (WCX-2) SELDI protein chip. Following incubation and washing of a standard serum sample on the WCX-2 sorbent, proteins were harvested, digested with trypsin, fractionated by strong cation exchange liquid chromatography (LC), and subsequently analyzed by microcapillary reversed-phase LC coupled online with an ion-trap mass spectrometer. This analysis resulted in the identification of 383 unique proteins in the WCX-2 serum retentate. Among the proteins identified, 50 (13%) are documented clinical biomarkers with 36 of these (72%) identified from multiple peptides.