Use of SELDI-TOF mass spectrometry for identification of new biomarkers: potential and limitations.

Surface-enhanced laser desorption time of flight mass spectrometry (SELDI-TOF-MS) is an important proteomic technology that is immediately available for the high throughput analysis of complex protein samples. Over the last few years, several studies have demonstrated that comparative protein profiling using SELDI-TOF-MS breaks new ground in diagnostic protein analysis particularly with regard to the identification of novel biomarkers. Importantly, researchers have acquired a better understanding also of the limitations of this technology and various pitfalls in biomarker discovery. Bearing these in mind, great emphasis must be placed on the development of rigorous standards and quality control procedures for the pre-analytical as well as the analytical phase and subsequent bioinformatics applied to analysis of the data. To avoid the risk of false-significant results studies must be designed carefully and control groups accurately selected. In addition, appropriate tools, already established for analysis of highly complex microarray data, need to be applied to protein profiling data. To validate the significance of any candidate biomarker derived from pilot studies in appropriately designed prospective multi-center studies is mandatory; reproducibility of the clinical results must be shown over time and in different diagnostic settings. SELDI-TOF-MS-based studies that are in compliance with these requirements are now required; only a few have been published so far. In the meantime, further evaluation and optimization of both technique and marker validation strategies are called for before MS-based proteomic algorithms can be translated into routine laboratory testing.     

FTICR mass spectrometry in proteomics

Analytical tools that allow rapid screening, low sample consumption and accurate protein identification are of great importance in studies of complex biological samples. Today, mass spectrometry (MS) is a key analytical tools with applications in a wide variety of fields, reaching from the analysis of elemental compositions in various materials to the identification of large protein complexes. One of the fastest growing fields of MS applications is proteomics, or the study of protein expression in an organism. In the traditional proteomic approach, two-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis is applied for the separation and visualization of proteins. In this review, the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry, including up-front multidimensional liquid separations for 'top down' or 'bottom up' proteomic approaches, are presented.     

The application of certified reference materials for clinical mass spectrometry

BackgroundIn the application process of clinical mass spectrometry, there are difficulties such as standardization, low degree of automation, complex instruments, and high requirements for operations, which will affect the accuracy and comparability of results. Reference materials are one of the major approaches to achieve measurement accuracy and metrological comparability.MethodsBased on the problems of reference materials in clinical mass spectrometry, the precautions for the use of reference materials are summarized in the aspects of measurement method validation, calibrator usage, and quality control in this article. Additionally, combined with the previous experience of the author''s laboratory, the operation mode and acceptance criteria of the new calibration solution lot replacement were formulated to ensure the continuous comparability of the measurement system.ConclusionCarefully understand and correctly regulate the use and management of reference materials to ensure the accuracy of test results, thereby improving the comparability and consistency of results between laboratories.     

Automated mass spectrometry: a revolutionary technology for clinical diagnostics.

For various diagnostic analyses and the studies of functional genomics, the use of an accurate and cost-effective analytic platform to analyze large numbers of samples is essential. An automated platform called MassArray (Sequenom, Inc, San Diego, CA), designed for high-throughput diagnostic analyses, has recently been validated. The platform combines miniaturized, two-dimensional chip arrays with proven high-fidelity enzymatic procedures and matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Nanoliter dispensing of samples in high-density formats of 384 or greater results in improved throughput and reduced costs. Automation prevails from the initial assay design through sample processing and data analysis, for the most part eliminating the labor component of assay development and implementation. The MassArray platform is being used in the following areas: (1) molecular diagnosis of genetic disease and infectious agents, (2) pharmacogenomics, (3) paternity and/or identity testing, and (4) agriculture (e.g., marker-assisted breeding). MALDI-TOF mass spectrometry can also be used for analyzing proteins; therefore, genotype/phenotype testing can be performed on a single platform.     

The clinical application of proteomics

BACKGROUND: Proteomics is defined as a scientific approach used to elucidate all protein species within a cell or tissue, and many researchers are taking advantage of proteomic technology to elucidate protein changes between healthy and diseased states. METHODS: The application of proteomic techniques and strategies to the field of medicine is slowly transforming the way biomarker discovery is conducted. However, the complexity of serum is the source of both its promise to clinical applications and its challenge to proteomic analysis. Like any new technology when it is first introduced, proteomics has been touted with much hope and promise. RESULTS AND CONCLUSIONS: We provide a review of the clinical application of proteomics with the emphasis on current practical issues and challenges facing proteomic research.     

Saliva analysis by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS): from sample collection to data analysis.

BACKGROUND: Saliva is one of the most promising and easy-to-collect source of potential biomarkers of oral and systemic disease. We standardized a protocol suitable for pre-analytical treatment and for the analysis of whole normal saliva by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF/MS). METHODS: We evaluated the impact of storage time, freeze/thaw cycles, denaturing agents, glycoproteins depletion, centrifugation, type of matrix and ProteinChip used on the quality of the SELDI protein profile. Moreover, we explored the inter-individual and between-sex differences and the changes in the sample composition over the day. RESULTS: Saliva was qualitatively stable, in the absence of protease inhibitors, for up to 3 h from the collection at room temperature, although the intensity of a number of peaks slightly decreased between 0 and 3 h and the addition of protease inhibitors did not completely revert this trend. The saliva proteome changed during the day and showed relevant between-sex differences. The protein profile remained stable for up to five freeze/thaw cycles. The addition of denaturing solutions and the depletion of glycoproteins improved the quality of the spectra without affecting their reproducibility. CONCLUSIONS: We defined a protocol that improved the quality and the reproducibility of SELDI-TOF/MS analysis, thus potentially supporting the search for putative biomarkers of disease.     

Liquid chromatography tandem mass spectrometry for analysis of steroids in clinical laboratories

Liquid chromatography tandem mass spectrometry is one of the most specific techniques available in clinical laboratories. In the past, immunoassays were the primary methodology for analysis of steroids in biological samples because they are rapid and easy to perform. However, these methods were shown to suffer from the lack of specificity for measuring many of the diagnostically important steroids. LC-MS/MS overcomes many of the limitations of immunoassays, enhances diagnostic utility of the testing, and expands diagnostic capabilities in endocrinology. In addition to the superior quality of the measurements, LC-MS/MS allows high throughput testing using small sample volume with minimal sample preparation, and frees the laboratory from dependence on suppliers of assay specific reagents. LC-MS/MS is being widely employed for routine measurement of steroids, and the methodology plays an important role in the standardization and harmonization of measurements among clinical laboratories.     

串联质谱技术在临床检验中的应用进展

串联质谱技术以其高度的检测灵敏性和特异性 ,在医药领域得到广泛应用。它对样本的需求量小 ,预处理简单 ,大大提高了检测和临床诊断的效率。现对串联质谱技术在临床检验和药物毒理学方面的研究进展作一报道     

用飞行质谱技术筛选结直肠癌患者中特异性生物标志物的临床意义

目的 探讨蛋白质质谱分析方法鉴定结直肠癌生物标志物的临床应用价值。方法 用美国Ciphergen Biosystems公司金属亲和表面芯片(IMAC3)和蛋白芯片仪，检测146例结直肠癌患者，62名正常人，32例结直肠良性疾病患者血清蛋白质的相对含量。结果 结直肠癌患者与正常人和结直肠良性疾病患者血清蛋白质在质荷比为4467000—15122000，其中有10个蛋白质含量差异有显著意义。146例结直肠癌患者中有142例患者被正确检测，62名正常人和32例结直肠良性疾病患者均被正确识别，检测准确率为98．3％(236／240)，灵敏度和特异性分别为97．30%(142／146)和100%(94／94)。结论 该方法灵敏度和特异性高，可快速、准确检测结直肠癌，其临床应用前景广阔。     

蛋白质组学在临床医学中的应用

简述生命科学研究中的两项重要内容:基因组学和蛋白质组学研究的历史现状:虽然基因组学的上游学科为蛋白质组学的发展奠定了基础,但蛋白质组学作为一门独立的学科,其方法学及相关技术在临床应用中有其独特的优势,同时也面临着很大的挑战。     

Monomeric calgranulins measured by SELDI-TOF mass spectrometry and calprotectin measured by ELISA as biomarkers in arthritis

BACKGROUND: SELDI-TOF mass spectrometry (MS) is a high-throughput proteomic approach with potential for identifying novel forms of serum biomarkers of arthritis. METHODS: We used SELDI-TOF MS to analyze serum samples from patients with various forms of inflammatory arthritis. Several protein profiles were collected on different Bio-Rad Laboratories ProteinChip arrays (CM10 and IMAC-Cu(2+)) and were evaluated statistically to select potential biomarkers. RESULTS: SELDI-TOF MS analyses identified several calgranulin proteins [S100A8 (calgranulin A), S100A9 (calgranulin B), S100A9*, and S100A12 (calgranulin C)], serum amyloid A (SAA), SAA des-Arg (SAA-R), and SAA des-Arg/des-Ser (SAA-RS) as biomarkers and confirmed the results with other techniques, such as western blotting, immunoprecipitation, and nano-LC-MS/MS. The S100 proteins were all able to significantly differentiate samples from patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), and ankylosing spondylitis (AS) from those of patients with inflammatory bowel diseases used as an inflammatory control (IC) group, whereas the SAA, SAA-R, and SAA-RS proteins were not, with the exception of AS. The 4 S100 proteins were coproduced in all of the pathologies and were significantly correlated with the plasma calprotectin concentration; however, these S100 proteins were correlated with the SAA peak intensities only in the RA and IC patient groups. In RA, these S100 proteins (except for S100A12) were significantly correlated with the serum concentrations of C-reactive protein, matrix metalloproteinase 3, and anti-cyclic citrullinated peptide and with the Disease Activity Score (DAS(28)). CONCLUSIONS: The SELDI-TOF MS technology is a powerful approach for analyzing the status of monomeric, truncated, or posttranslationally modified forms of arthritis biomarkers, such as the S100A8, S100A9, S100A12, and SAA proteins. The fact that the SELDI-TOF MS data were correlated with results obtained with the classic calprotectin ELISA test supports the reliability of this new proteomic technique.     

胎儿磁共振——磁共振检查的新领域

随着磁共振成像技术的改进,MRI现在可应用于胎儿诊断。MRI提供的胎儿结构信息的深度,意味着它不仅仅是超声有用的辅助诊断手段,史能清晰地显示胎儿结构,避免超声的局限性。目前,MRI在胎儿中枢神经系统异常中的诊断最有价值,在中枢神经系统,在脑室扩大、胼胝体发育不全和后颅窝畸形的诊断中最有优势,此外,在胎儿胸部和其他系统畸形诊断中逐渐显示优势。MRI还能准确测量胎儿器官体积和重量,可以评估胎儿生长受限。一些MRI新技术如弥散加权成像、磁共振波谱和功能成像也逐渐应用于胎儿,可以提高我们对胎儿在子宫内代谢和发育信息的认识。本文将详细介绍胎儿MRI的安全性、技术及各系统临床应用。     

Comparison of 15N analysis by optical emission spectrometry and mass spectrometry for clinical studies during total parenteral nutrition

During total and stable parenteral nutrition, a branched chain amino acid enriched solution containing [15N]leucine was infused into a patient to determine the fate of the nitrogen administered through this formulation. Measurements of 15N isotopic enrichments were performed on the same biological samples (urinary urea, total plasma proteins and albumin) by optical emission spectrometry (OES) and mass spectrometry (MS) to determine if OES with its specific advantages (cost, handling maintenance) constituted even with low enrichments a useful alternative technique to MS considered as the reference method. The average 15N% enrichments are 0.5% for urinary urea, 0.21% for total plasma proteins and 0.14% for albumin. The coefficients of correlation between both series of 15N% enrichment measurements realized by MS and OES vary from 0.999 to 0.998 and 0.988, respectively, for urinary urea, total plasma proteins and albumin. These results show that OES constituted a very useful analytical technique to obtain reliable information in clinical metabolic studies when low 15N enrichments must be determined.     

基于质谱的蛋白质组学诊断所面临的挑战

过去的几年中,基于质谱技术的蛋白质组学分析方法已经成为寻找新的疾病标志物的重要手段之一.由于它具有高灵敏度、高通量、快速以及能和生物信息学技术结合同时快速分析大量的蛋白质或多肽的优点,使之成为临床研究的有力工具.然而,最近的一些研究显示,因为这项技术的重复性和数据统计分析等方面的不足而受到了争议.本文介绍了采用该方法进行标志物筛查时在实验设计、质谱技术以及数据分析等方面面临的一些挑战,探讨其在血清或血浆分析中的应用.     

衍生化串联质谱检测55种酰基肉碱方法的建立及其临床应用

目的 建立衍生化串联质谱检测人体中55种酰基肉碱的方法,考察该方法的影响因素及其在西部地区新生儿遗传代谢性病筛查中的应用和意义,为其有效防治提供科学的依据.方法 利用衍生化串联质谱法分析比较2869例出生3 d至1岁婴幼儿的干血滤纸片.采足跟血于空白采血滤纸上,完全渗透.用已知水平的酰基肉碱同位素内标液萃取干血滤纸片中...