蛋白质芯片SELDI-TOF MS技术的研究进展及其在临床中的应用

蛋白质芯片为新一代的蛋白质组研究技术,由美国Ciphergen生物系统公司引进,表面增强激光解吸电离-飞行时间质谱(SELDI-TOFMS)提供一个高通量和高灵敏度的检测平台。投放至今虽短短10来年,但卓越的成果已广为医学科学界重视,尤其在恶性肿瘤的早期诊断、监控和预后研究上。蛋白质是细胞内执行生物功能的最终分子,蛋白质组学研究让人类更深入了解疾病和生命的本源,不断发现的特异性肿瘤标志物更为攻克癌症带来新希望。这里除对表面增强激光解吸电离-飞行时间质谱作较详尽的介绍外,更重点阐述其近年来蛋白质芯片近期的研究进展和在临床中的应用,并就其优劣和发展前景作出评估。     

蛋白质芯片SELDI-TOFMS技术的研究进展及其在临床中的应用

蛋白质芯片为新一代的蛋白质组研究技术,由美国Ciphergen生物系统公司引进,表面增强激光解吸电离-飞行时间质谱(SELDI-TOFMS)提供一个高通量和高灵敏度的检测平台。投放至今虽短短10来年,但卓越的成果已广为医学科学界重视,尤其在恶性肿瘤的早期诊断、监控和预后研究上。蛋白质是细胞内执行生物功能的最终分子,蛋白质组学研究让人类更深入了解疾病和生命的本源,不断发现的特异性肿瘤标志物更为攻克癌症带来新希望。这里除对表面增强激光解吸电离_飞行时间质谱作较详尽的介绍外,更重点阐述其近年来蛋白质芯片近期的研究进展和在临床中的应用,并就其优劣和发展前景作出评估。     

生物质谱结合IMAC亲和提取和磷酸酶水解分析蛋白质磷酸化修饰

蛋白质磷酸化是生物体内非常重要的翻译后修饰方式 ,对磷酸化蛋白质的分析及磷酸化位点的确定有助于理解与其相关的生物功能。基质辅助激光解吸 /电离飞行时间质谱和电喷雾 四极杆 飞行时间质谱这两种生物质谱仪在蛋白质鉴定和翻译后修饰分析中发挥着重要作用。固相金属亲和色谱可选择性亲和提取肽混合物中的磷酸肽 ,结合磷酸酶水解实验和基质辅助激光解吸 /电离飞行时间质谱分析可确定肽混合物中的磷酸肽 ,最后用电喷雾 四极杆 飞行时间串联质谱分析磷酸肽的序列 ,结合数据库检索确定磷酸化位点。     

MALDI-TOF质谱在细菌检测及鉴定中的研究进展

近年来,随着质谱技术的快速发展,软电离方式的出现,基质辅助激光解吸电离飞行时间质谱能够对蛋白质、核酸及脂类等生物大分子进行快速、准确的分析,进而使得其被应用于细菌的检测及鉴定成为可能。本文综述了当前基质辅助激光解吸电离飞行时间质谱在细菌检测及鉴定方面的研究进展。     

采用基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF)鉴定曲霉属Flavi组和Fumigati组的一些种（英文）

一些曲霉是主要的食物腐败菌及人的病原体,特别是免疫系统受损的病人可能导致严重的感染。本研究评价了利用基质辅助激光解吸电离飞行时间质谱对一些临床和环境中重要的Flavi组和Fumigati组曲霉,进行鉴定的可能性,并将结果与形态学及测序结果(ITS区和部分-tubulin和钙调蛋白基因)进行比较分析。通过曲霉中34个Flavi组菌株和30个Fumigati组菌株的质谱分析,来建立基质辅助激光解吸电离飞行时间质谱的数据库。对光谱数据进行聚类分析表明Fumigati组的基质辅助激光解吸电离飞行时间质谱的结果与系统发育结果完全一致;Flavi组的基质辅助激光解吸电离飞行时间质谱方法将A.flavus,A.oryzae,A.sojae和A.parasiticus分开的效果比测序方法好。随后,再选取用于验证数据库的50个菌株中49个(98%)菌株用质谱数据得到正确鉴定。对于分离本研究中曲霉的隐形种,基质辅助激光解吸电离飞行时间质谱方法优于测序方法。这种方法可以用于曲霉临床实验室鉴定的标准方法,因为临床需要快速和稳定的鉴定方法,这对于适当的治疗方案的选择很重要,此方法同样适于环境研究工作。     

基体辅助激光解吸电离质谱法及其应用

基体辅助激光解吸电离质谱是近几年才发展起来的一种新技术,它在生命科学研究中具有广阔的应用前景．对基体辅助激光解吸电离质谱技术的基本原理,运用基体辅助激光解吸电离质谱法研究蛋白质分子量的测定,蛋白质混合物的分离鉴定以及用基体辅助激光解吸电离质谱技术进行超快速的蛋白质序列分析和DNA序列分析的可行性和存在的问题作了介绍和讨论．     

胃癌及癌旁组织定量比较蛋白质组学研究

为寻找胃癌特异的肿瘤标记物,用于胃癌临床诊断及药物治疗靶点的选择,本研究采用荧光差异显示凝胶电泳（DIGE）技术分离并筛选 Cy3、Cy5 及 Cy2 荧光素标记的胃癌及对应癌旁组织差异表达蛋白质,用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）或串联质谱技术进行鉴定并分析。结果共筛选出 33 个差异表达蛋白质点,其中 9 个蛋白质点在胃癌组织中上调,24 个蛋白质点下调。对 22 个蛋白质点采用质谱技术成功鉴定,突变结蛋白、锰超氧化物歧化酶、热休克蛋白 60等在胃癌中高表达,热休克蛋白 27、前列腺素 F 合酶、硒结合蛋白 1、锌指蛋白 160、微管蛋白 α6、真核生物翻译延伸因子 1 α1 等在胃癌组织中低表达,并筛选出 5 个未知蛋白。这些差异表达蛋白可望成为胃癌诊断的特异标记物,并与胃癌的发生、发展及预后等有关,为胃癌的诊断、发生机制的研究提供了新的思路。     

应用IP-2D nano-HPLC-MALDI-TOF-TOF鉴定蛋白质泛素化修饰

近年来,在蛋白质研究中,特别是在蛋白质翻译后修饰(PTM)的研究中,生物质谱技术的应用越来越广泛,与纳升级HPLC的联合应用,使这一技术手段更加有效.针对泛素化在细胞功能调控中发挥关键作用的PTM的特点,将免疫沉淀、2D nano-HPLC和基质辅助激光解吸/电离串联飞行时间质谱(IP-2D nano-HPLC-MALDI-TOF-TOF)有机整合,建立了天然状态下蛋白质泛素化位点的鉴定方法,并应用这一方法确定出K562细胞内具有酪氨酸激酶活性的蛋白c-ABL的泛素化位点.为定性鉴定生理和病理状态下内源性蛋白的泛素化修饰提供了借鉴.     

SELDI-TOF-MS筛选慢性阻塞性肺疾病血清标志蛋白的初步研究

目的:利用表面增强激光解吸电离飞行时间质谱技术(SELDI-TOF-MS)筛选慢性阻塞性肺疾病(COPD)血清特异标志物。方法:应用SELDI-TOF-MS技术检测30例COPD稳定期患者和30例健康对照者血清蛋白指纹图谱,采用Biomarker pattern软件进行分析,建立COPD的诊断模型。结果:COPD患者血清蛋白图谱与对照组相比,在相对分子质量2000-15 000范围内共检测到75个蛋白峰,发现19个有统计学差异的蛋白峰(P0.05)。通过对COPD组与对照组间的数据作进一步分析,经BPS软件分析,建立质荷比(M/Z)3 167、4 645的差异蛋白组成的诊断模型,其诊断敏感度为96.67%,特异度为96.67%。结论:SELDI-TOF-MS技术是一种快速、简单易行、用量少和高通量的分析方法。能直接筛选出COPD血清中特异表达标志物,用特异表达标志物建立的诊断模型能有效区分COPD患者与健康对照者,有望成为COPD诊断的辅助指标。     

异烟肼耐药结核分枝杆菌感染人源巨噬细胞的蛋白质组学研究

利用双向电泳技术,对人源巨噬细胞U937感染异烟肼耐药结核分枝杆菌前后的全细胞蛋白表达图谱进行差异比较和分析,发现其中产生差异的有32个蛋白质斑点,利用基质辅助激光解吸/电离飞行时间质谱技术,对其中5个表达明显上调的蛋白质斑点进行分析鉴定,获得5个明确的肽质量指纹图谱,通过在数据库中进行检索分析,确定这5个蛋白质分别为热休克蛋白105_β、凋亡抑制蛋白-1、磷酸甘油酸变位酶1、组织蛋白酶B、桥粒胶蛋白3.上述发现有助于了解耐药结核分枝杆菌入侵早期导致的巨噬细胞蛋白质组表达变化,为深入研究耐药结核分枝杆菌-宿主相互作用提供了探索方向.     

Proteomic detection of prostate-specific antigen using a serum fractionation procedure: potential implication for new low-abundance cancer biomarkers detection

One of the major obstacles in proteomic analysis of biological fluids is the presence of highly abundant proteins such as albumin and immunoglobulins, which can interfere with the resolution and sensitivity of the proteome profiling techniques used. In this paper, we describe an anion exchange fractionation procedure for serum using denaturating conditions allowing protein-protein interaction disruption before analysis by surface-enhanced laser desorption/ionization and by two-dimensional electrophoresis. This method simplifies the serum proteome into subproteomes and markedly increases resolution and sensitivity without any loss of minor proteins. To confirm the applicability of this method, fractionated serum of a patient with prostate cancer was analyzed for the presence of the prostate-specific antigen (PSA) which is a low-abundance tumor marker protein. The results demonstrate that PSA can be detected by two-dimensional electrophoresis only in serum following fractionation. Hence, this procedure may facilitate the identification of other, so far unknown, tumor markers in patient sera.     

In situ Identification and Localization of IGHA2 in the Breast Tumor Microenvironment by Mass Spectrometry

Modifications in the tumor microenvironment (TME) play a major role in the establishment, progression, and metastasis of cancer. Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique that enables the simultaneous identification and localization of biological compounds within tissues. To detect markers of early TME remodeling in invasive breast cancer, we used MALDI-MSI to compare the molecular profiles of tissues from the breast cancer interface zone, tumor zone, and normal-tissue zone. Using direct-tissue MALDI tandem mass spectrometry (MS/MS), we identified immunoglobulin heavy constant alpha 2 (IGHA2) as a new, zone-specific protein in the breast TME. The zone-specific expression of IGHA2 was verified by immunoblotting and immunohistochemical analysis. IGHA2 expression was consistently positive in tumor cells that were metastatic to regional nodes, with intense expression along the cytoplasmic borders. As a factor related to an increased percentage of nodes with tumor metastasis, IGHA2 expression was upregulated 3.745-fold in cases with an increased number of cancerous nodes (p = 0.0468). Our results provide the first evidence of IGHA2 as a marker of the early process of TME remodeling in invasive breast cancer. Furthermore, IGHA2 may be a novel marker for regional metastases in the lymph nodes of patients with breast cancer.     

Identifying patterns of DNA for tumor diagnosis using capillary electrophoresis-amplified fragment length polymorphism (CE-AFLP) screening

Amplified Fragment Length Polymorphism (AFLP) screening is a genome-wide genotyping strategy that has been widely used in plants and bacteria, but little has been reported concerning its use in humans. We investigated if the AFLP procedure could be coupled with high-throughput capillary electrophoresis (CE) for use in tumor diagnosis and classification. Using CE-AFLP, a series of molecular 'fingerprints' were generated for a set of gastric tumor and normal genomic DNA samples. The CE-AFLP procedure was qualitatively and quantitatively robust, and a variety of clustering tools were used to identify a specific DNA marker 'pattern' of 20 features that classified the tumor and normal samples to reasonable degrees of accuracy (Sensitivity 95%, Specificity 80%). The CE-AFLP-based approach also correctly classified 16 tumor samples, which in a previous study had exhibited no detectable genomic aberrations by comparative genome hybridization (CGH). This is the first reported application of CE-AFLP screening in tumor diagnosis. As the procedure is relatively inexpensive and requires minimal prior sequence knowledge and biological material, we suggest that CE-AFLP-based protocols may represent a promising new approach for DNA-based cancer screening and diagnosis.     

Proteomic analysis of progressive factors in uterine cervical cancer

Human papillomavirus (HPV) infections play a crucial role in the progress of cervical cancer. The high-risk HPV types are frequently associated with the development of malignant lesions. Some of the latest studies have demonstrated that the high-risk HPV 16 and 18 are predominantly detected in the more aggressive cancers. In the present study, we aimed to establish the proteomic profiles and characterization of the tumor related proteins by using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). For proteomic analysis, patients infected by HPV 16 or 18 were included in this study. We compared nuclear protein and cytoplasmic protein, separately by using the subcellular fraction. Differential protein spots between cervical cancer with high-risk HPV, HPV 16 or HPV 18, and HaCaT cell lines were characterized by 2-DE. Those proteins analyzed by peptide mass fingerprinting based on MALDI-TOF MS and database searching were the products of oncogenes or proto-oncogenes, and the others were involved in the regulation of cell cycle, for general genomic stability, telomerase activation, and cell immortalization. However, there was no difference in protein characterization for cervical cancer between HPV 16 and HPV 18 infection. Nonetheless, these data are valuable for the mass identification of differentially expressed proteins involved in human uterine cervical cancer. Moreover, the data has enormous value for establishing the human uterine cervical cancer proteome database that can be used in screening a molecular marker for the further study of human uterine cervical cancer, and also for studying any correlation among the cancers induced by HPV.     

Tumor suppressor gene NGX6 induces changes in protein expression profiles in colon cancer HT-29 cells

Nasopharyngeal carcinoma-associated gene 6 (NGX6; syn. transmembrane protein 8B, TMEM8B) is a recently identified tumor suppressor gene. The underlying mechanisms by which the gene inhibits tumor development are not completely known. To further understand the function of the gene's protein product NGX6, in the present study, we employed two-dimensional difference gel electrophoresis to analyze the protein expression profiles of colon cancer HT-29 cells stably transfected with the gene NGX6. The differentially expressed proteins were selected and identified by matrix-assisted laser desorption/ionization coupled with time-of-flight tandem mass spectrometry. The results showed that 12 proteins were down-regulated and 4 were up-regulated in NGX6-transfected HT-29 cells, compared with vector-transfected HT-29 cells. The MS results were verified by western blot. Bioinformatic analysis showed that these proteins are involved in cell proliferation, metastasis, apoptosis, cytoskeletal structure, metabolism, and signal transduction, suggesting that NGX6 may inhibit colon cancer through the regulation of these biological processes.     

A strategy for high-resolution protein identification in surface-enhanced laser desorption/ionization mass spectrometry: calgranulin A and chaperonin 10 as protein markers for endometrial carcinoma

Surface-enhanced laser desorption/ionization-mass spectrometry (SELDI-MS) has conventionally been practiced on linear time of flight (TOF) which has low mass accuracy and resolution. Here we demonstrate in an examination of both malignant and nonmalignant endometrial tissue homogenates that high mass accuracy and resolution in the MS stage are crucial. Using a commercially available quadrupole/TOF (QqTOF), we were able to resolve two potential cancer markers, subsequently identified off-line as chaperonin 10 and calgranulin A, that differ by 8 Da in mass. Two off-line protein identification protocols were developed: the first was based on size-exclusion chromatography (SEC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), protein extraction, trypsin digestion, and matrix-assisted laser desorption/ionization-tandem MS (MALDI-MS/MS); the second on SEC and shotgun nano-liquid chromatography (nanoLC)-MS/MS. Analyses on a cohort of 44 endometrial homogenates showed 22 out of 23 nonmalignant samples had nondetectable to very low abundance of chaperonin 10 and calgranulin A; 17 of the 21 malignant samples had detectable to abundant levels of both proteins. Immunohistochemical staining of a tissue microarray of 32 samples showed that approximately half of malignant endometrial tissues exhibited positive staining for calgranulin A in the malignant epithelium, while 9 out of 10 benign tissues exhibited negative epithelial staining. In addition, macrophages/granulocytes in malignant as well as nonmalignant tissues showed positive staining. No immunostaining occurred in stroma or myometrium. Calgranulin A, in combination with chaperonin 10 and other proteins, may eventually constitute a panel of markers to permit diagnosis and screening of endometrial cancer.     

Pattern analysis of serum proteome distinguishes renal cell carcinoma from other urologic diseases and healthy persons

Despite having a relatively low incidence, renal cell carcinoma (RCC) is one of the most lethal urologic cancers. For successful treatment including surgery, early detection is essential. Currently there is no screening method such as biomarker assays for early diagnosis of RCC. Surface-enhanced laser desorption/ionization-time of flight mass spectrometry (SELDI-TOF) is a recent technical advance that can be used to identify biomarkers for cancers. In this study, we investigated whether SELDI protein profiling and artificial intelligence analysis of serum could distinguish RCC from healthy persons and other urologic diseases (nonRCC). The SELDI-TOF data was acquired from a total of 36 serum samples with weak cation exchange-2 protein chip arrays and filtered using ProteinChip software. We used a decision tree algorithm c4.5 to classify the three groups of sera. Five proteins were identified with masses of 3900, 4107, 4153, 5352, and 5987 Da. These biomarkers can correctly separate RCC from healthy and nonRCC samples.     

Optimizing microarray-based in situ transcription-translation of proteins for matrix-assisted laser desorption ionization mass spectrometry

The analysis of self-assembled protein microarrays, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, combines two high-throughput platforms for investigation of the proteome. In this article, we describe the fabrication in situ of protein arrays optimized for MALDI characterization. Using the green fluorescent protein (GFP) both as an epitope for immobilization and as a gauge for relative protein expression, we were able to generate amounts of protein on the array slides sufficient for MALDI identification. In addition, expression of N-terminal protein constructs fused to GFP demonstrated mass shifts consistent with that of the full-length protein. We envision this technology to be important for the functional screening of protein interactions.     

Identification of tumor-associated proteins in oral tongue squamous cell carcinoma by proteomics

Oral tongue carcinoma is an aggressive tumor that particularly affects chronic smokers, drinkers and betel squid chewers. Patients often present symptoms at a late stage, and there is a high recurrence rate after treatment. In this article, we report the first proteomic analysis of oral tongue carcinoma to globally search for tumor related proteins. Apart from helping us to understand the molecular pathogenesis of the carcinoma, these proteins may also have potential clinical applications as biomarkers, enabling the tumor to be identified at an early stage in high risk individuals, treatment response to be predicted, and residual or recurrent carcinoma to be detected sooner after treatment. The protein expression profiles of ten oral tongue squamous cell carcinomas and their matched normal mucosal resection margins were examined by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight mass spectroscopy. A number of tumor-associated proteins including heat shock protein (HSP)60, HSP27, alpha B-crystalline, ATP synthase beta, calgranulin B, myosin, tropomyosin and galectin 1 were consistently found to be significantly altered in their expression levels in tongue carcinoma tissues, compared with their paired normal mucosae. The expression profile portrays a global protein alteration that appears specific to oral tongue cancer. The potential of utilizing these tumor related proteins for screening cancer and monitoring recurrence warrants further investigation.     

Molecular classification of liver cirrhosis in a rat model by proteomics and bioinformatics

Liver cirrhosis is a worldwide health problem. Reliable, noninvasive methods for early detection of liver cirrhosis are not available. Using a three-step approach, we classified sera from rats with liver cirrhosis following different treatment insults. The approach consisted of: (i) protein profiling using surface-enhanced laser desorption/ionization (SELDI) technology; (ii) selection of a statistically significant serum biomarker set using machine learning algorithms; and (iii) identification of selected serum biomarkers by peptide sequencing. We generated serum protein profiles from three groups of rats: (i) normal (n=8), (ii) thioacetamide-induced liver cirrhosis (n=22), and (iii) bile duct ligation-induced liver fibrosis (n=5) using a weak cation exchanger surface. Profiling data were further analyzed by a recursive support vector machine algorithm to select a panel of statistically significant biomarkers for class prediction. Sensitivity and specificity of classification using the selected protein marker set were higher than 92%. A consistently down-regulated 3495 Da protein in cirrhosis samples was one of the selected significant biomarkers. This 3495 Da protein was purified on-chip and trypsin digested. Further structural characterization of this biomarkers candidate was done by using cross-platform matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) peptide mass fingerprinting (PMF) and matrix-assisted laser desorption/ionization time of flight/time of flight (MALDI-TOF/TOF) tandem mass spectrometry (MS/MS). Combined data from PMF and MS/MS spectra of two tryptic peptides suggested that this 3495 Da protein shared homology to a histidine-rich glycoprotein. These results demonstrated a novel approach to discovery of new biomarkers for early detection of liver cirrhosis and classification of liver diseases.