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。     

基质辅助激光解析电离飞行时间质谱与细菌的快速鉴定

用传统的方法鉴别细菌往往需要较长的时间(≥48h)和复杂的程序,不利于细菌的快速鉴定。基质辅助激光解析电离飞行时间质谱是最近采用的用于细菌检测的生物质谱技术,可以对完整的细菌进行检测。这种技术以激光作为能量来源,将待测细菌的表面成分解析为离子,产生重现性很好的质谱图。将未知细菌质谱图与细菌质谱图库进行比较,可以达到对细菌进行鉴剐的目的。此种质谱技术的应用,对微生物的快速鉴别有重要意义。     

生物质谱技术及其应用

质谱是带电粒子按质荷比大小顺序排列的图谱,最初主要用来测定元素或同位素的原子量,随着科学的发展及高性能质谱仪器的出现,质谱被越来越多地应用生命科学研究的许多领域,以其质辅助激光解吸附飞行时间质谱和电喷雾质谱为代表的现代生物质谱技术,为蛋白质等生物大分子的研究提供了必要的技术手段。本文在简介近年来比较常用的几种生物质谱技术的基础上,概述了生物质谱技术在蛋白质,核酸研究及检测分析等几个方面的初步应用。     

基质辅助激光解析电离飞行时间质谱技术在病原真菌检验中的应用进展

基质辅助激光解析电离飞行时间质谱技术是一种可用于检测大分子物质的“软电离”质谱分析技术,近年来在致病性真菌研究中的作用日显突出,该文对近几年该技术在病原真菌研究中的应用进展作一综述。     

MALDI-TOF MS技术在侵袭性丝状真菌快速鉴定中的应用

目的 探索不同培养基、不同培养时间和不同蛋白质提取方法对侵袭性丝状真菌质谱鉴定准确率的影响,旨在提高基质辅助激光解析电离飞行时间质谱技术鉴定侵袭性丝状真菌的准确率.方法 采用分子生物学方法为金标准,同时运用基质辅助激光解析电离飞行时间质谱技术对所收集临床丝状真菌进行鉴定.根据分子生物学的鉴定结果,去除VITEK-MSv...     

生物质谱技术与蛋白质组学

生物质谱技术是蛋白质组学的支撑技术.详细论述了质谱技术的分类与基本分析原理,重点论述了质谱技术的发展变化,包括基质辅助激光解吸飞行时间质谱技术,电喷雾质谱技术,MALDI-Q-TOF和MAL-DI-TOF-TOF等质谱技术,以及质谱技术在蛋白质组学研究中的应用与未来的发展和挑战.     

质谱流式技术用于单细胞检测

质谱流式技术(mass cytometry)是利用质谱原理对单细胞进行多参数检测的流式技术,能够在单细胞水平实现超过50种标志物的同时测量,显著增强了对细胞生长进程和复杂细胞系统的评估能力。该文简要介绍了质谱流式技术的基本工作原理,并从金属元素标记、质量分析器、高维单细胞数据处理等方面展开论述,阐明设计新型金属元素标签和选择飞行时间质谱的必要性,归纳分析高维单细胞数据的算法并总结各种算法的优点和局限性。     

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。     

基质辅助激光解析电离飞行时间质谱在医学真菌领域的应用进展

基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）是近年来新兴的微生物检测技术,通过核糖体蛋白分析实现对真菌快速、准确鉴定。本文针对MALDI-TOF MS用于致病真菌鉴定、分类、体外抗真菌药物敏感性检测以及临床微生物样本直接检测等方面作一综述。     

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。     

Differential expression of proteomics models of colorectal cancer, colorectal benign disease and healthy controls

Background

Colorectal cancer (CRC) is often diagnosed at a late stage with concomitant poor prognosis. The hypersensitive analytical technique of proteomics can detect molecular changes before the tumor is palpable. The surface-enhanced laser desorption/ionization-time of flight-mass spectra (SELDI-TOF-MS) is a newly-developed technique of evaluating protein separation in recent years. The protein chips have established the expression of tumor protein in the serum specimens and become the newly discovered markers for tumor diagnosis. The objective of this study was to find new markers of the diagnosis among groups of CRC, colorectal benign diseases (CBD) and healthy controls. The assay of SELDI-TOF-MS with analytical technique of protein-chip bioinformatics was used to detect the expression of protein mass peaks in the sera of patients or controls. One hundred serum samples, including 52 cases of colorectal cancer, 27 cases of colorectal benign disease, and 21 cases of healthy controls, were examined by SELDI-TOF-MS with WCX2 protein-chips.

Results

The diagnostic models (I, II and III) were setup by analyzed the data and sieved markers using Ciphergen - Protein-Chip-Software 5.1. These models were combined with 3 protein mass peaks to discriminate CRC, CBD, and healthy controls. The accuracy, the sensitivity and the particularity of cross verification of these models are all highly over 80%.

Conclusions

The SELDI-TOF-MS is a useful tool to help diagnose colorectal cancer, especially during the early stage. However, identification of the significantly differentiated proteins needs further study.     

表面增强激光解吸电离飞行时间质谱技术筛查肺癌血清特异性蛋白质及其临床意义

目的:探讨用表面增强激光解吸电离飞行时间质谱(SELDI-TOF-MS)技术筛查肺癌血清特异性蛋白质的临床意义。方法:应用SELDI-TOF-MS对35例正常对照组、43例治疗前肺癌病人的血清样品进行蛋白质指纹图谱测定,用BioMarker Wizard 3．01及BioMarker Parrern System 5．01分析软件对测得的数据进行处理及建立诊断模型。结果:共检测到251个蛋白质峰,筛选出差异蛋白质峰11个,以质荷比(m/z)分别为M2799_26,M3227_41,M5739_70和M8164_30的4个蛋白质峰为依据组合构建分类决策树模型,分出5个终节点。决策树模型的原始判别总准确率为91．0%(71/78),敏感性为88．4%(38/43),特异性为94．3%(33/35);交叉验证总准确率为85．9%(67/78),敏感性为88．4%(38/43),特异性为82．9%(29/35)。结论:SELDI-TOF-MS在肺癌血清特异性蛋白质的筛选及诊断模型的建立有一定的临床意义。     

SELDI-TOF-MS of saliva: methodology and pre-treatment effects

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.     

Urine profiling by SELDI-TOF/MS: monitoring of the critical steps in sample collection, handling and analysis

The topic of this study is the impact of several pre-analytical and analytical variables on proteomic profiling of human urine by surface enhanced laser desorption/ionization time of flight-mass spectrometry (SELDI-TOF-MS) in healthy subjects. Urine storage at room temperature caused a progressive degradation of proteins, which was prevented by the addition of protease inhibitors only up to 2 h from the collection. The timing of collection over the day had only a minor impact on protein profile, although influencing the intensity of peaks. Repeated freeze/thaw cycles (up to five) did not affect either the number or the intensity of the peaks. A comparison of the protein profile from eight different healthy individuals showed fairly consistent inter-subject similarities, along with between-subject differences, which were markedly dependent on the sex and the type of ProteinChip array used. The addition of a variety of denaturing agents improved the quality of the spectra with all the chips tested (CM10, Q10 and H50), but not with the copper-coated IMAC-30 chip. Finally, SPA matrix allowed to achieve a better performance of SELDI-TOF/MS spectrum, as compared with CHCA, regardless of the ProteinChip array used and even in the low m/z range (2500-10,000). In conclusion, we suggest that a careful choice of a number of pre-analytical and analytical conditions is required to accomplish and define a unifying protocol for the analysis of human urine by SELDI-TOF/MS, in physiological and in pathological states.     

Identification of novel and downregulated biomarkers for alcoholism by surface enhanced laser desorption/ionization-mass spectrometry

Since personal and verbal reporting of alcohol use is not necessarily accurate, objective markers to assess alcohol consumption are required. The currently available markers, however, are limited in sensitivity and specificity for screening of excessive alcohol drinkers. Therefore, searches for novel markers are warranted. Recently, surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) has been successfully used to detect disease-associated proteins in complex biological specimens. We used the ProteinChip SELDI technology to generate comparative protein profiles of the consecutive serum samples obtained during abstinence from a total of 16 chronic alcoholic patients hospitalized for a rehabilitation program. We recognized two peaks (5.9 and 7.8 kDa), both of which had been downregulated on admission, the expression level of which significantly increased after a one-week abstinence. These changes were also seen in nonresponders of gamma-glutamyltransferase. These two proteins were partially purified and subjected to amino acid sequencing. The 5.9 kDa protein was identified as a fragment of fibrinogen alphaE chain and the 7.8 kDa was a fragment of apoprotein A-II. These novel protein fragments may be promising biomarkers for excessive alcohol drinking.     

Proteomic analysis of lymph

This report provides the first proteomic analysis of normal ovine lymph. By establishing the fact that lymph is more than an ultrafiltrate of blood plasma, it documents that the lymph proteome contains an array of proteins that differentiates it from plasma. The protein chip technology, surface-enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS), two-dimensional gel electrophoresis (2-D PAGE) and MS, were employed to examine the protein expression profiles of ovine lymph. Using a weak cation exchange chip surface to assay lymph and plasma samples by SELDI-TOF-MS showed that the analysis of peak maps from lymph contained three protein peaks that were found only in lymph, while analysis of peak maps from plasma samples showed that five protein peaks were found only in plasma. Lymph and plasma samples showed eight peaks that were common to both. There were also more ions present in plasma than in lymph, which is consistent with the 2-D PAGE analysis. MS analysis of a large number of protein spots from 2-D PAGE gels of lymph produced MS/MS sequences for 18 proteins that were identified by searching against a comprehensive protein sequence database. As in plasma, large protein spots of albumin dominated the protein pattern in lymph. Other major proteins identified in 2-D PAGE gels of lymph included, fibrinogen alpha- and beta-chains, immunoglobulin G (IgG) heavy chain, serotransferrin precursor, lactoferrin, and apolipoprotein A-1. Two proteins that were identified and were differentially expressed in lymph were glial fibrillary astrocyte acidic protein and neutrophil cytosol factor-1. By bringing the technologies of proteomics to bear on the analysis of lymph, it is possible to detect proteins in lymph that are quantitatively and qualitatively differentially expressed from those of plasma.     

Comparison of software tools to improve the detection of carcinogen induced changes in the rat liver proteome by analyzing SELDI-TOF-MS spectra

A common animal model of chemical hepatocarcinogenesis was used to demonstrate the potential identification of carcinogenicity related protein signatures/biomarkers. Therefore, an animal study in which rats were treated with the known liver carcinogen N-nitrosomorpholine (NNM) or the corresponding vehicle was evaluated. Histopathological investigation as well as SELDI-TOF-MS analysis was performed. SELDI-TOF-MS is an affinity-based mass spectrometry method in which subsets of proteins from biological samples are selectively adsorbed to a chemically modified surface. The proteins are subsequently analyzed with respect to their mass-charge ratios (m/z) by a time of flight (TOF) mass spectrometry (MS) approach. As data preprocessing of SELDI-TOF-MS spectra is essential, baseline correction, normalization, peak detection, and alignment of raw spectra were performed using either the Ciphergen ProteinChip Software 3.1 or functions implemented in the library PROcess of the BioConductor Project. Baseline correction and normalization algorithms of both tools lead to comparable results, whereas results after peak detection and alignment steps differed. Variability between technical and biological replicates was investigated. A linear mixed model with factors experimental group and time point was applied for each protein peak, taking into account the different correlation structure of technical and biological replicates. Alternatively, only median intensity values of technical replicates were used. Results of both models were similar and correlated well with those of the histopathological evaluation of the study. In conclusion, statistical analyses lead to comparable results, whereas parameter settings for preprocessing proved to be crucial.     

Mitochondrial protein patterns correlating with impaired insulin secretion from INS-1E cells exposed to elevated glucose concentrations

Extended hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and eventually beta-cell apoptosis in individuals with type 2 diabetes mellitus. In an attempt to dissect mechanisms behind the detrimental effects of glucose, we focused on measuring changes in expression patterns of mitochondrial proteins. Impaired GSIS was observed from INS-1E cells cultured for 5 days at 20 or 27 mM glucose compared to cells cultured at 5.5 or 11 mM glucose. After culture, mitochondria were isolated from the INS-1E cells by differential centrifugation. Proteins of the mitochondrial fraction were bound to a strong anionic surface (SAX2) protein array and mass spectra generated by SELDI-TOF-MS. Analysis of the spectra revealed proteins with expression levels that correlated with the glucose concentration of the culture medium. Indeed, such differentially expressed proteins created patterns of protein changes, which correlated with impairment of GSIS. In conclusion, the study reveals the first glucose-induced differentially expressed patterns of beta-cell mitochondrial proteins obtained by SELDI-TOF-MS.     

Comparative SELDI-TOF-MS profiling of low-molecular-mass proteins from Lignosus rhinocerus (Cooke) Ryvarden grown under stirred and static conditions of liquid fermentation

Mushrooms are considered as important source of biologically active compounds which include low-molecular-mass protein/peptides (LMMP). In this study, we attempted to profile the LMMP from Lignosus rhinocerus, a wild medicinal mushroom, grown by static cultures (SC) and in stirred tank reactor (STR). Crude water extract (CWE) and protein fractions were profiled using H50 ProteinChip® arrays and SELDI-TOF-MS. Three protein peaks of 5.8, 6.9 and 9.1 kDa were found to be common to spectra of L. rhinocerus CWE from both culture conditions. Partial protein purification has resulted in detection of more peaks in the spectra of protein fractions. For protein fractions of L. rhinocerus cultured in STR, most peaks were observed in the range of 3-8 kDa whereas some peaks with molecular mass up to 14.3 kDa were noted in spectra of protein fractions from SC. Our results have demonstrated the optimization of profiling method using SELDI-TOF-MS for fungal LMMP.     

Discrimination of Francisella tularensis subspecies using surface enhanced laser desorption ionization mass spectrometry and multivariate data analysis

Francisella tularensis causes the zoonotic disease tularemia, and is considered a potential bioterrorist agent due to its extremely low infection dose and potential for airborne transmission. Presently, F. tularensis is divided into four subspecies; tularensis, holarctica, mediasiatica and novicida. Phenotypic discrimination of the closely related subspecies with traditional methods is difficult and tedious. Furthermore, the results may be vague and they often need to be complemented with virulence tests in animals. Here, we have used surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) to discriminate between the four subspecies of F. tularensis. The method is based on the differential binding of protein subsets to chemically modified surfaces. Bacterial thermolysates were added to anionic, cationic, and copper ion-loaded immobilized metal affinity SELDI chip surfaces. After binding, washing, and SELDI-TOF-MS different protein profiles were obtained. The spectra generated from the different surfaces were then used to characterize each bacterial strain. The results showed that the method was reproducible, with an average intensity variation of 21%, and that the mass precision was good (300-450 ppm). Moreover, in subsequent cluster analysis and principal component analysis (PCA) data for the analyzed Francisella strains grouped according to the recognized subspecies. Partial least squares-discriminant analysis (PLS-DA) of the protein profiles also identified proteins that differed between the strains. Thus, the protein profiling approach based on SELDI-TOF-MS holds great promise for rapid high-resolution phenotypic identification of bacteria.