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。     

肿瘤蛋白标志物分析技术研究进展

目前,我国的肿瘤发病率为285.91/10万,平均每分钟有6人被诊断为恶性肿瘤,因此肿瘤的早期诊断的重要性显而易见。随着分子生物学、免疫学诊断技术的飞速发展,寻找肿瘤诊断和预后的特异性标志物已成为近期研究的热点。蛋白质是生命活动中多项功能的执行者,可以直接反映基因给予的信息。相比于基因,蛋白质的表达谱更可以直接地反映功能机制。针对蛋白质的生物特性,本文总结了目前临床中筛查肿瘤蛋白标志物分析技术,如双向凝胶电泳、基质辅助激光解吸/电离-飞行时间质谱、表面增强激光解吸/电离-飞行时间质谱、蛋白质芯片技术,并对未来肿瘤蛋白标志物筛查提出新的展望。     

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。     

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．     

评估SELDI-TOF-MS技术检测肾移植术后病人尿液的能力

目的:研究表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术检测肾移植术后病人尿液中蛋白质的能力。方法:根据尿液标本中蛋白质浓度进行稀释处理,采用SELDI-TOF-MS技术,用3种不同芯片(NP20、CM10、IMAC30)与蛋白质浓度的不同组合分别检测肾移植术后病人的尿液。结果:采用NP20/蛋白质未稀释、CM10/蛋白质浓度约0.1g/L、IMAC30/蛋白质浓度约2g/L时,质谱图中蛋白质峰的个数和丰度达到最佳;单一芯片中NP20捕获质荷比为5000～20000的蛋白质能力最佳,而CM10和IMAC30在质荷比为2000～5000时有较大的捕获能力;3种芯片中CM10具有最大的捕获蛋白质的能力。结论:3种芯片检测尿液中蛋白质的能力不同;为了更好地发现疾病特异性标志物,最好多种芯片同时检测,且在各种芯片得到最佳质谱图时的浓度进行检测。     

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。     

乳腺癌蛋白质组学研究

乳腺癌是妇女中最常见的一种癌症,鉴定出与乳腺癌癌变相关的蛋白质以及病情发展过程中蛋白质的变化对揭示乳腺癌变机理及早期诊断是非常重要的。早在蛋白质组学这一概念提出以前,人们已应用2－维凝胶电泳技术（2DE）研究乳腺癌的癌变机理,且随着人类基因序列测序的完成,质谱的应用,以及生物信息学的引入,蛋白质组的研究获得了飞速发展,高通量的蛋白质组研究以及新的技术如激光捕获显微切割（LCM）,表面加强激光解吸／电离飞行时间质谱（SELDI－TOF）,蛋白质阵列,组织阵列等蛋白质组学技术已被用于乳腺癌研究并获得了很快速的发展。乳腺癌蛋白质组学研究已经鉴定了一些具有诊断潜能的生物分子靶标和信号传导因子。介绍了乳腺癌蛋白质组学研究中所使用的最新研究方法和研究进展。     

基质辅助激光解吸电离飞行时间质谱在微生物鉴定中的研究进展

与传统的微生物鉴定技术相比，基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption ionization time-of-flight mass spectrometry, MALDI-TOF MS)是一种准确、可靠和快速的鉴定和分型的技术。本文通过检索近年来国内外相关研究论文，总结最新的研究进展，发现MALDI-TOF MS在临床病原微生物、食源性微生物以及环境微生物等鉴定中有较大的优势，加快了微生物鉴定的进程，同时探索该技术在新领域的最新进展和面临的挑战，以期为我国基质辅助激光解吸电离飞行时间质谱技术的发展提供参考。     

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 Proteomics in Breast Cancer

Background

Proteomic profiling is a rapidly developing technology that may enable early disease screening and diagnosis. Surface-enhanced laser desorption ionization–time of flight mass spectrometry (SELDI-TOF MS) has demonstrated promising results in screening and early detection of many diseases. In particular, it has emerged as a high-throughput tool for detection and differentiation of several cancer types. This review aims to appraise published data on the impact of SELDI-TOF MS in breast cancer.

Methods

A systematic literature search between 1965 and 2009 was conducted using the PubMed, EMBASE, and Cochrane Library databases. Studies covering different aspects of breast cancer proteomic profiling using SELDI-TOF MS technology were critically reviewed by researchers and specialists in the field.

Results

Fourteen key studies involving breast cancer biomarker discovery using SELDI-TOF MS proteomic profiling were identified. The studies differed in their inclusion and exclusion criteria, biologic samples, preparation protocols, arrays used, and analytical settings. Taken together, the numerous studies suggest that SELDI-TOF MS methodology may be used as a fast and robust approach to study the breast cancer proteome and enable the analysis of the correlations between proteomic expression patterns and breast cancer.

Conclusion

SELDI-TOF MS is a promising high-throughput technology with potential applications in breast cancer screening, detection, and prognostication. Further studies are needed to resolve current limitations and facilitate clinical utility.     

Serum protein profile in systemic-onset juvenile idiopathic arthritis differentiates response versus nonresponse to therapy

Systemic-onset juvenile idiopathic arthritis (SJIA) is a disease of unknown etiology with an unpredictable response to treatment. We examined two groups of patients to determine whether there are serum protein profiles reflective of active disease and predictive of response to therapy. The first group (n = 8) responded to conventional therapy. The second group (n = 15) responded to an experimental antibody to the IL-6 receptor (MRA). Paired sera from each patient were analyzed before and after treatment, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Despite the small number of patients, highly significant and consistent differences were observed before and after response to therapy in all patients. Of 282 spectral peaks identified, 23 had mean signal intensities significantly different (P < 0.001) before treatment and after response to treatment. The majority of these differences were observed regardless of whether patients responded to conventional therapy or to MRA. These peaks represent potential biomarkers of active disease. One such peak was identified as serum amyloid A, a known acute-phase reactant in SJIA, validating the SELDI-TOF MS platform as a useful technology in this context. Finally, profiles from serum samples obtained at the time of active disease were compared between the two patient groups. Nine peaks had mean signal intensities significantly different (P < 0.001) between active disease in patients who responded to conventional therapy and in patients who failed to respond, suggesting a possible profile predictive of response. Collectively, these data demonstrate the presence of serum proteomic profiles in SJIA that are reflective of active disease and suggest the feasibility of using the SELDI-TOF MS platform used as a tool for proteomic profiling and discovery of novel biomarkers in autoimmune diseases.     

Modern Tumor Marker Discovery in Urology: Surface Enhanced Laser Desorption and Ionization (SELDI)

During the last two decades, biomarker research has benefited from the introduction of new proteomic analytical techniques. In this article, we review the application of surface enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectroscopy in urologic cancer research. After reviewing the literature from MEDLINE on proteomics and urologic oncology, we found that SELDI-TOF is an emerging proteomic technology in biomarker discovery that allows for rapid and sensitive analysis of complex protein mixtures. SELDI-TOF is a novel proteomic technology that has the potential to contribute further to the understanding and clinical exploitation of new, clinically relevant biomarkers.     

Proteomic changes in hearts of kyphoscoliosis (ky) mutant mice in the absence of structural pathology: implication for the analysis of early human heart disease

Complex molecular changes associated with early stage human heart disease are poorly understood and prevent the development of effective treatments of human cardiac disease. Relatively minor structural changes in early disease may accompany some conditions such as arrhythmias. Our objective was to determine if significant proteomic changes occur in heart tissues in the absence of structural pathology. We used a proteomic "pipeline" based on Ciphergen SELDI-TOF/MS, gel electrophoresis and MALDI-TOF/MS. The kyphoscoliosis (ky) mouse carries a mutation in a putative transglutaminase causing a primary skeletal muscle disease. The ky protein is expressed usually in skeletal and cardiac muscle but its absence from the ky heart causes no structural pathology making it a good model of "occult" heart disease. We discovered 20 statistically validated biomarkers discriminating ky from normal hearts, one cardiac troponin-I was reduced by 40% in ky hearts. A 17% deficit was confirmed subsequently by Western blot. Thus, the proteome of ky hearts was abnormal, giving support to our contention that this SELDI-based analytical approach is capable of making a significant contribution to the analysis of complex proteomic changes in early stage human heart disease.     

Identification of treatment efficacy-related host factors in chronic hepatitis C by ProteinChip serum analysis

Recent development of proteomic array technology, including protein profiling coupling ProteinChip array with surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF/MS), provides a potentially powerful tool for discovery of new biomarkers by comparison of its profiles according to patient phenotypes. We used this approach to identify the host factors associated with treatment response in patients with chronic hepatitis C (CHC) receiving a 48-wk course of pegylated interferon (PEG-IFN) alpha 2b plus ribavirin (RBV). Protein profiles of pretreatment serum samples from 32 patients with genotype 1b and high viral load were conducted by SELDI-TOF/MS by using the three different ProteinChip arrays (CM10, Q10, IMAC30). Proteins showed significantly different peak intensities between sustained virological responders (SVRs), and non-SVRs were identified by chromatography, SDS-PAGE, TOF/MS and tandem mass spectrometry (MS/MS) assay. Eleven peak intensities were significantly different between SVRs and non-SVRs. The three SVR-increased peaks could be identified as two apolipoprotein (Apo) fragments and albumin and, among the eight non-SVR-increased proteins, four peaks identified as two iron-related and two fibrogenesis-related protein fragments, respectively. Multivariate analysis showed that the serum ferritin and three peak intensity values (Apo A1, hemopexin and transferrin) were independent variables associated with SVRs, and the area under the receiver operating characteristic (ROC) curves for SVR prediction by using the Apo A1/hemopexin and hemopexin/transferrin were 0.964 and 0.936. In conclusion, pretreatment serum protein profiling by SELDI-TOF/MS is variable for identification of response-related host factors, which are useful for treatment efficacy prediction in CHC receiving PEG-IFN plus RBV. Our data also may help us understand the mechanism for treatment resistance and development of more effective antiviral therapy targeted toward the modulation of lipogenesis or iron homeostasis in CHC patients.     

Identification of serum proteomic biomarkers for early porcine reproductive and respiratory syndrome (PRRS) infection

ABSTRACT: BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases worldwide. Despite its relevance, serum biomarkers associated with early-onset viral infection, when clinical signs are not detectable and the disease is characterized by a weak anti-viral response and persistent infection, have not yet been identified. Surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI-TOF MS) is a reproducible, accurate, and simple method for the identification of biomarker proteins related to disease in serum. This work describes the SELDI-TOF MS analyses of sera of 60 PRRSV-positive and 60 PRRSV-negative, as measured by PCR, asymptomatic Large White piglets at weaning. Sera with comparable and low content of hemoglobin (< 4.52 ug/mL) were fractionated in 6 different fractions by anion-exchange chromatography and protein profiles in the mass range 1-200 kDa were obtained with the CM10, IMAC30, and H50 surfaces. RESULTS: A total of 200 significant peaks (p < 0.05) were identified in the initial discovery phase of the study and 47 of them were confirmed in the validation phase. The majority of peaks (42) were up-regulated in PRRSV-positive piglets, while 5 were down-regulated. A panel of 14 discriminatory peaks identified in fraction 1 (pH=9), on the surface CM10, and acquired at low focus mass provided a serum protein profile diagnostic pattern that enabled to discriminate between PRRSV-positive and -negative piglets with a sensitivity and specificity of 77% and 73%, respectively. CONCLUSIONS: SELDI-TOF MS profiling of sera from PRRSV-positive and PRRSV-negative asymptomatic piglets provided a proteomic signature with large scale diagnostic potential for early identification of PRRSV infection in weaning piglets. Furthermore, SELDI-TOF protein markers represent a refined phenotype of PRRSV infection that might be useful for whole genome association studies.     

Proteomic analysis of fast and slow muscles from normal and kyphoscoliotic mice using protein arrays, 2-DE and MS

A proteomic strategy based upon the integrated use of SELDI-TOF/MS, 2-DE and MALDI-TOF/MS has been used to identify a panel of fast muscle protein markers: MLC1F, MLC3F, fast troponin C (STNC) and slow muscle markers: MLC1SB and MLC2v. MLC3F, MLC1F and STNC were virtually absent in the physiologically pure slow soleus muscle of kyphoscoliotic mutant mice compared to control BDmice, whereas MLC2v increased threefold. A SELDI-TOF/MS peak at 18,012 Da in spectra from strong anionic exchange protein array fractions of fast vastus muscle was confirmed as STNC by its specific depletion from crude extracts of vastus muscle using an anti-TNC mAb. SELDI-TOF/MS also identified MLC2F phosphorylation in crude muscle extracts after treatment with alkaline phosphatase. High probability protein identifications were achieved by SELDI-TOF/MS PMF based upon the resolution of large peptides formed by partial cleavage and high peptide coverage. When the pI from 2-D gels and molecular weight estimations from SELDI-TOF/MS were entered into the TagIdent algorithm, high probability protein identity predictions were obtained that were confirmed later by PMF. We confirm that SELDI-TOF/MS can be integrated with other proteomics techniques for the efficient analysis of protein expression changes and PTMs associated with physiological changes in skeletal muscle.     

Proteomic analysis of phosphoproteins sensitive to a phosphatidylinositol 3-kinase inhibitor, ZSTK474, by using SELDI-TOF MS

Background  

Phosphoproteins play important roles in a vast series of biological processes. Recent proteomic technologies offer the comprehensive analyses of phosphoproteins. Recently, we demonstrated that surface-enhanced laser desorption/ionization time of flight mass (SELDI-TOF MS) would detect phosphoproteins quantitatively, which was a new application of SELDI-TOF MS.     

Proteomic Approaches to Decipher Mechanisms Underlying Pathogenesis in Multiple Sclerosis Patients

Multiple sclerosis (MS) is a chronic inflammatory demyelinating and neurodegenerative disease of the central nervous system (CNS). The cause of MS is unknown, with no effective therapies available to halt the progressive neurological disability. Development of new and improvement of existing therapeutic strategies therefore require a better understanding of MS pathogenesis, especially during the progressive phase of the disease. This can be achieved through development of biomarkers that can help to identify disease pathophysiology and monitor disease progression. Proteomics is a powerful and promising tool to accelerate biomarker detection and contribute to novel therapeutics. In this review, an overview of how proteomic technology using CNS tissues and biofluids from MS patients has provided important clues to the pathogenesis of MS is provided. Current publications, pitfalls, as well as directions of future research involving proteomic approaches to understand the pathogenesis of MS are discussed.     

Proteomic characterization of inter-alpha inhibitor proteins from human plasma

Inter-alpha inhibitor proteins (IaIp) are a family of structurally related serine protease inhibitors found in relatively high concentrations in human plasma. Recent studies have implicated a role for IaIp in sepsis, and have demonstrated their potential as biomarkers in sepsis and cancer. For characterization of isolated IaI proteins and contaminating proteins during the last steps of the purification process, SELDI-TOF MS and HPLC-ESI-MS/MS were used. After separation by SDS-PAGE or 2-DE, polypeptide bands of 80, 125 and 250 kDa were excised from gels and digested by trypsin. The tryptic peptides were analyzed by both MS methods. The main contamination during the purification process, a band of 80 kDa, contains mainly IaIp heavy chain (HC) H3. HC H1 and H2 were also found in this band. In addition, some vitamin K-dependent clotting factors and inhibitors and other plasma proteins were identified. The 125-kDa band, representing the pre-alpha inhibitor, was found to contain both bikunin and HC H3. The presence of other HC H1, H2 and the recently described HC H4 was also detected by SELDI-TOF MS. The presence of HC H1, H2, and H3 in the 125-kDa band was confirmed by ESI-MS/MS, but not the presence of the H4. Three polypeptides, H1 and H2 together with bikunin, were identified in the 250-kDa band, representing the ITI, by both MS techniques. Once again, the presence of H4 was detected in this band only by SELDI-TOF MS, but the number of corresponding peptides was still not sufficient for final identification of this polypeptide. The importance of the application of proteomic methods for the proper evaluation of therapeutic drugs based on human plasma is discussed.