MALDI-TOF质谱技术分析与鉴定病原细菌研究

本文通过基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术分析病原细菌的方法进行研究, 阐明影响分析结果的重要因素, 并建立了MALDI-TOF-MS 分析病原细菌的标准方法。对不同属、种和亚种的12株植物病原细菌进行全细胞分析结果表明：MALDI-TOF-MS能快速而准确的区分和鉴定病原细菌, 分析过程简单、灵敏度高。此法在细菌属、种、亚种和菌株水平上, 可快速、准确地区分和鉴定。     

Identification and Cluster Analysis of Streptococcus pyogenes by MALDI-TOF Mass Spectrometry

Background

Whole-cell matrix–assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) has been successfully applied for bacterial identification and typing of many pathogens. The fast and reliable qualities of MALDI-TOF MS make it suitable for clinical diagnostics. MALDI-TOF MS for the identification and cluster analysis of Streptococcus pyogenes, however, has not been reported. The goal of our study was to evaluate this approach for the rapid identification and typing of S. pyogenes.

Methods

65 S. pyogenes isolates were obtained from the hospital. The samples were prepared and MALDI-TOF MS measurements were conducted as previously reported. Identification of unknown spectra was performed via a pattern recognition algorithm with a reference spectra and a dendrogram was constructed using the statistical toolbox in Matlab 7.1 integrated in the MALDI Biotyper 2.0 software.

Results

For identification, 61 of 65 S. pyogenes isolates could be identified correctly by MALDI-TOF MS with BioType 2.0 when compared to biochemical identification (API Strep), with an accuracy of 93.85%. In clustering analysis, 44 of 65 isolates were in accordance with those established by M typing, with a matching rate of 67.69%. When only the M type prevalence in China was considered, 41 of 45 isolates were in agreement with M typing, with a matching rate of 91.1%.

Conclusions

It was here shown that MALDI-TOF MS with Soft Biotype 2.0 and its database could facilitate rapid identification of S. pyogenes. It may present an attractive alternative to traditional biochemical methods of identification. However, for classification, more isolates and advances in the MALDI-TOF MS technology are needed to improve accuracy.     

Multilaboratory Evaluation of the MALDI-TOF Mass Spectrometry System,MicroIDSys Elite,for the Identification of Medically Important Filamentous Fungi

Mycopathologia - With the increasing number of fungal infections and immunocompromised patients, rapid and accurate fungal identification is required in clinical microbiology laboratories. We...     

Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud''s dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex, and C. rugosa complex.     

Viridans Group Streptococci Clinical Isolates: MALDI-TOF Mass Spectrometry versus Gene Sequence-Based Identification

Viridans Group Streptococci (VGS) species-level identification is fundamental for patients management. Matrix-assisted laser desorption ionization—time of flight mass spectrometry (MALDI-TOF MS) has been used for VGS identification but discrimination within the Mitis group resulted difficult. In this study, VGS identifications with two MALDI-TOF instruments, the Biotyper (Bruker) and the VITEK MS (bioMérieux) have been compared to those derived from tuf, soda and rpoB genes sequencing. VGS isolates were clustered and a dendrogram constructed using the Biotyper 3.0 software (Bruker). RpoB gene sequencing resulted the most sensitive and specific molecular method for S. pneumonia identification and was used as reference method. The sensitivity and the specificity of the VITEK MS in S. pneumonia identification were 100%, while the Biotyper resulted less specific (92.4%). In non pneumococcal VGS strains, the group-level correlation between rpoB and the Biotyper was 100%, while the species-level correlation was 61% after database upgrading (than 37% before upgrading). The group-level correlation between rpoB and the VITEK MS was 100%, while the species-level correlation was 36% and increases at 69% if isolates identified as S. mitis/S. oralis are included. The less accurate performance of the VITEK MS in VGS identification within the Mitis group was due to the inability to discriminate between S. mitis and S. oralis. Conversely, the Biotyper, after the release of the upgraded database, was able to discriminate between the two species. In the dendrogram, VGS strains from the same group were grouped into the same cluster and had a good correspondence with the gene-based clustering reported by other authors, thus confirming the validity of the upgraded version of the database. Data from this study demonstrated that MALDI-TOF technique can represent a rapid and cost saving method for VGS identification even within the Mitis group but improvements of spectra database are still recommended.     

Rapid Identification of Gram Negative Bacteria from Blood Culture Broth Using MALDI-TOF Mass Spectrometry

An important role of the clinical microbiology laboratory is to provide rapid identification of bacteria causing bloodstream infection. Traditional identification requires the sub-culture of signaled blood culture broth with identification available only after colonies on solid agar have matured. MALDI-TOF MS is a reliable, rapid method for identification of the majority of clinically relevant bacteria when applied to colonies on solid media. The application of MALDI-TOF MS directly to blood culture broth is an attractive approach as it has potential to accelerate species identification of bacteria and improve clinical management. However, an important problem to overcome is the pre-analysis removal of interfering resins, proteins and hemoglobin contained in blood culture specimens which, if not removed, interfere with the MS spectra and can result in insufficient or low discrimination identification scores. In addition it is necessary to concentrate bacteria to develop spectra of sufficient quality. The presented method describes the concentration, purification, and extraction of Gram negative bacteria allowing for the early identification of bacteria from a signaled blood culture broth.     

Identification by MALDI-TOF Mass Spectrometry of Mercury-resistant Bacteria Associated with the Rhizosphere of an Apple Orchard

In this work, mercury-resistant bacterial strains were isolated from the rhizosphere of an apple orchard, growing in a soil with high levels of mercury (Nuevo San Joaquin, Queretaro State, Mexico). Analysis of the soil in this region by the Cold Vapor Atomic Absortion Spectroscopy method showed that it contained 637 ± 51 mg mercury per kg. Mercury accumulation by fresh apples from this orchard amounted to 15.44 ± 4.33 mg/kg. The bacterial isolates were identified by application of proteomic technique of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). They were found to be strains of Bacillus muralis and Bacillus simplex. All strains showed the ability to catalyze the volatilization of Hg as measured via the nonradioactive X-ray method. In all strains merR and merA genes were detected by polymerase chain reaction. Nucleotide sequence analysis showed that merR from B. simplex was 435 bp in length and that its sequence was similar to merR sequences reported for other bacteria such as Cupriavidus, Ralstonia, Pseudomonas and Serratia. To our knowledge, this is the first report of mercury-resistant Bacillus strains isolated from the rhizosphere of an apple orchard and the first merR gene sequence from such Bacilli.     

MALDI-TOF质谱源后衰变技术鉴定2D胶蛋白点

PMF方法由于具有高灵敏度、高通量和容易自动化等优点,在蛋白质组学鉴定中占有重要的地位。然而,许多样品(比如：小分子蛋白,混合物等)仅仅通过PMF方法不能明确鉴定。在这种情况下,在测定PMF的同一个样品上,选择一个酶解片段峰进行PSD测序,并把这些序列信息输入MS—Tag软件进行搜索,结合PMF方法,表观分子量等电点等参数,能够对胶上的点进行明确的鉴定。本文先用PSD方法对胶上的三个标准蛋白进行鉴定,都得到了非常准确的结果,同时鉴定了胶上的几个未知点。     

一种优化的MALDI-TOF质谱分析多肽C端序列方法

利用基质辅助激光解吸飞行时间 (MALDI TOF)质谱技术 ,测定羧肽酶Y消化蛋白质和多肽 .所产生的缩短肽片段的质量 ,在一张谱图上得到各个不同酶解时间所形成的肽质量梯度 .根据谱图中相邻两肽峰的质量差得到切去氨基酸的信息 ,从而读出C端氨基酸序列 .在pmol水平下对人促肾上腺皮质激素片段 (ACTH 1 3 9) ,人血管紧张肽片段 (angiotensin Ⅰ ,angiotensin Ⅱ )的C端序列进行了测定 .讨论了在不同浓度 ,不同时间 ,不同温度下酶解所得到的序列测定结果 .在优化条件下 ,人ACTH片段得到了C端 2 0个氨基酸残基顺序 ,为目前C端序列分析所得到的最长序列     

A Side by Side Comparison of Bruker Biotyper and VITEK MS: Utility of MALDI-TOF MS Technology for Microorganism Identification in a Public Health Reference Laboratory

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a rapid, highly accurate, and cost-effective method for routine identification of a wide range of microorganisms. We carried out a side by side comparative evaluation of the performance of Bruker Biotyper versus VITEK MS for identification of a large and diverse collection of microorganisms. Most difficult and/or unusual microorganisms, as well as commonly encountered microorganisms were selected, including Gram-positive and negative bacteria, mycobacteria, actinomycetes, yeasts and filamentous fungi. Six hundred forty two strains representing 159 genera and 441 species from clinical specimens previously identified at the Laboratoire de santé publique du Québec (LSPQ) by reference methods were retrospectively chosen for the study. They included 254 Gram-positive bacteria, 167 Gram-negative bacteria, 109 mycobacteria and aerobic actinomycetes and 112 yeasts and moulds. MALDI-TOF MS analyses were performed on both systems according to the manufacturer’s instructions. Of the 642 strains tested, the name of the genus and / or species of 572 strains were referenced in the Bruker database while 406 were present in the VITEK MS IVD database. The Biotyper correctly identified 494 (86.4%) of the strains, while the VITEK MS correctly identified 362 (92.3%) of the strains (excluding 14 mycobacteria that were not tested). Of the 70 strains not present in the Bruker database at the species level, the Biotyper correctly identified 10 (14.3%) to the genus level and 2 (2.9%) to the complex/group level. For 52 (74.2%) strains, we obtained no identification, and an incorrect identification was given for 6 (8.6%) strains. Of the 178 strains not present in the VITEK MS IVD database at the species level (excluding 71 untested mycobacteria and actinomycetes), the VITEK MS correctly identified 12 (6.8%) of the strains each to the genus and to the complex/group level. For 97 (54.5%) strains, no identification was given and for 69 (38.7%) strains, an incorrect identification was obtained. Our study demonstrates that both systems gave a high level (above 85%) of correct identification for a wide range of microorganisms. However, VITEK MS gave more misidentification when the microorganism analysed was not present in the database, compared to Bruker Biotyper. This should be taken into account when this technology is used alone for microorganism identification in a public health laboratory, where isolates received are often difficult to identify and/or unusual microorganisms.     

Subunit Structure of Gas Vesicles: A MALDI-TOF Mass Spectrometry Study

Many aquatic microorganisms use gas vesicles to regulate their depth in the water column. The molecular basis for the novel physical properties of these floatation organelles remains mysterious due to the inapplicability of either solution or single crystal structural methods. In the present study, some folding constraints for the ~7-kDa GvpA building blocks of the vesicles are established via matrix-assisted laser desorption ionization time-of-flight mass spectrometry studies of intact and proteolyzed vesicles from the cyanobacterium Anabaena flos-aquae and the archaea Halobacterium salinarum. The spectra of undigested vesicles show no evidence of posttranslational modification of the GvpA. The extent of carboxypeptidase digestion shows that the alanine rich C-terminal pentapeptide of GvpA is exposed to the surface in both organisms. The bonds that are cleaved by Trypsin and GluC are exclusively in the extended N-terminus of the Anabaena flos-aquae protein and in the extended C-terminus of the Halobacterium salinarum protein. All the potentially cleavable peptide bonds in the central, highly conserved portion of the protein appear to be shielded from protease attack in spite of the fact that some of the corresponding side chains are almost certainly exposed to the aqueous medium.     

蛋白质质谱数据缺失值填补方法的比较与评估

基于质谱数据的蛋白质定量分析一直是目前高通量蛋白质组学的重要研究手段.但是基于现有质谱技术的限制,大规模蛋白质定量过程中往往会产生大量的缺失值,这在一定程度上影响了下游分析的准确性.尽管很多缺失值填补方法被不断提出,但是蛋白质组学领域对于不同情况下缺失值填补方法效力的综合评估仍然缺乏.本研究基于真实数据的分布特征,构建...     

New Diagnostic System for the Identification of Lactose-fermenting Gram-negative Rods

The identification of prompt lactose-fermenting gram-negative rods has generally relied heavily upon colonial morphology coupled with one or more indole, methyl red, Voges-Proskauer, citrate (IMViC) parameters, hydrogen sulfide, and motility. Studies were undertaken to compare diagnoses dependent solely upon the more orthodox criteria to a system for identification based upon hydrogen sulfide, ornithine decarboxylase, and citrate utilization (HOC). The results suggest that the IMViC scheme of identification is neither consistent nor applicable when applied to the current nomenclature of the above group of organisms and should be discarded, whereas the HOC system may prove to be of significant value to clinical microbiologists.     

A Novel Preprocessing Method Using Hilbert Huang Transform for MALDI-TOF and SELDI-TOF Mass Spectrometry Data

Motivation

Mass spectrometry is a high throughput, fast, and accurate method of protein analysis. Using the peaks detected in spectra, we can compare a normal group with a disease group. However, the spectrum is complicated by scale shifting and is also full of noise. Such shifting makes the spectra non-stationary and need to align before comparison. Consequently, the preprocessing of the mass data plays an important role during the analysis process. Noises in mass spectrometry data come in lots of different aspects and frequencies. A powerful data preprocessing method is needed for removing large amount of noises in mass spectrometry data.

Results

Hilbert-Huang Transformation is a non-stationary transformation used in signal processing. We provide a novel algorithm for preprocessing that can deal with MALDI and SELDI spectra. We use the Hilbert-Huang Transformation to decompose the spectrum and filter-out the very high frequencies and very low frequencies signal. We think the noise in mass spectrometry comes from many sources and some of the noises can be removed by analysis of signal frequence domain. Since the protein in the spectrum is expected to be a unique peak, its frequence domain should be in the middle part of frequence domain and will not be removed. The results show that HHT, when used for preprocessing, is generally better than other preprocessing methods. The approach not only is able to detect peaks successfully, but HHT has the advantage of denoising spectra efficiently, especially when the data is complex. The drawback of HHT is that this approach takes much longer for the processing than the wavlet and traditional methods. However, the processing time is still manageable and is worth the wait to obtain high quality data.     

Numerical Taxonomy of Gram-Positive and Catalase-Positive Rods Isolated from Frozen Vegetables

One hundred isolates from peas, beans, and corn were compared with cultures of Corynebacterium, Microbacterium, and Arthrobacter by use of numerical taxonomic procedures. Six groups (clusters), representing 75% of the isolates, resembled Corynebacteriaceae. There was some doubt regarding the genera represented because the groups were not closely related to many of the known strains. The relationship of the different groups to each other, as well as a number of their properties, is presented.     

Identification and Phenotypic Characterization of Sphingomonas wittichii Strain RW1 by Peptide Mass Fingerprinting Using Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

Mass spectrometry is a potentially attractive means of monitoring the survival and efficacy of bioaugmentation agents, such as the dioxin-mineralizing bacterium Sphingomonas wittichii strain RW1. The biotransformation activity of RW1 phenotypes is determined primarily by the presence and concentration of the dioxin dioxygenase, an enzyme initiating the degradation of both dibenzo-p-dioxin and dibenzofuran (DF). We explored the possibility of identifying and characterizing putative cultures of RW1 by peptide mass fingerprinting (PMF) targeting this characteristic phenotypic biomarker. The proteome from cells of RW1—grown on various media in the presence and absence of DF—was partially purified, tryptically digested, and analyzed using matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mascot online database queries allowed statistically significant identification of RW1 in disrupted, digested cells (P < 0.01 to 0.05) and in digested whole-cell extracts (P < 0.00001 to 0.05) containing hundreds of proteins, as determined by two-dimensional gel electrophoresis. Up to 14 peptide ions of the alpha subunit of the dioxin dioxygenase (43% protein coverage) were detected in individual samples. A minimum of 10⁷ DF-grown cells was required to identify dioxin degradation-enabled phenotypes. The technique hinges on the detection of multiple characteristic peptides of a biomarker that can reveal at once the identity and phenotypic properties of the microbial host expressing the protein. The results demonstrate the power of PMF of minimally processed microbial cultures as a sensitive and specific technique for the positive identification and phenotypic characterization of certain microorganisms used in biotechnology and bioremediation.     

Identification and Quantification of Proteoforms by Mass Spectrometry

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post‐translational modifications. In top‐down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top‐down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.