Biomarker profiling and reproducibility study of MALDI-MS measurements of Escherichia coli by analysis of variance-principal component analysis

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has proved useful for the characterization of bacteria and the detection of biomarkers. Key challenges for MALDI-MS measurements of bacteria are overcoming the relatively large variability in peak intensities. A soft tool, combining analysis of variance and principal component analysis (ANOVA-PCA) (Harrington, P. D.; Vieira, N. E.; Chen, P.; Espinoza, J.; Nien, J. K.; Romero, R.; Yergey, A. L. Chemom. Intell. Lab. Syst. 2006, 82, 283-293. Harrington, P. D.; Vieira, N. E.; Espinoza, J.; Nien, J. K.; Romero, R.; Yergey, A. L. Anal. Chim. Acta. 2005, 544, 118-127) was applied to investigate the effects of the experimental factors associated with MALDI-MS studies of microorganisms. The variance of the measurements was partitioned with ANOVA and the variance of target factors combined with the residual error was subjected to PCA to provide an easy to understand statistical test. The statistical significance of these factors can be visualized with 95% Hotelling T2 confidence intervals. ANOVA-PCA is useful to facilitate the detection of biomarkers in that it can remove the variance corresponding to other experimental factors from the measurements that might be mistaken for a biomarker. Four strains of Escherichia coli at four different growth ages were used for the study of reproducibility of MALDI-MS measurements. ANOVA-PCA was used to disclose potential biomarker proteins associated with different growth stages.     

Monitoring the growth of a bacteria culture by MALDI-MS of whole cells.

We have probed the time evolution of a growing bacteria culture by extracting samples periodically and performing matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) on whole cells. The mass spectra generated by this method contain tens of peaks in the 3-11-kDa mass range. Cultures of E. coli strain K-12 were grown in two types of containers and at two nutrient concentrations and sampled periodically from 6 to 84 h after inoculation. The relative intensities of several of the stronger peaks vary quite dramatically as a function of time. These temporal characteristics must be taken into account when MALDI-MS is applied to identify bacteria. The results also suggest that MALDI-MS can be used to follow the aging of a bacteria culture.     

Investigation of MALDI-TOF and FT-MS techniques for analysis of Escherichia coli whole cells

Recently, it has been demonstrated that bacteria can be characterized using whole cells and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). However, identification of specific bacterial proteins usually requires analysis of cellular fractions or purified extracts. Here, the first application of Fourier transform mass spectrometry (FTMS) to analysis of bacterial proteins directly from whole cells is reported. It is shown that accurate mass MALDI-FTMS can be used to characterize specific ribosomal proteins directly from Escherichia coli cells. High-accuracy mass measurements and high-resolution isotope profile data confirm posttranslational modifications proposed previously on the basis of low-resolution mass measurements. Seven ribosomal proteins from E. coli whole cells were observed with errors of less than 27 ppm. This was accomplished directly from whole cells without fractionation, concentration, or overt overexpression of characteristic cellular proteins. MALDI-FTMS also provided information regarding E. coli lipids in the low-mass region. Although ions with m/z values below 1000 have been observed by FTMS of whole cells, this represents the first report of detection of ions in the 5000 to 10,000 m/z range by MALDI-FTMS using whole cells.     

一株草苷膦极端抗性菌株的分离和分子鉴定

从极端污染的土壤中 ,分离到一株对草苷膦有极端抗性的细菌菌株HTG7。它能够在以草苷膦为选择压力的限制性培养基上生长 ,其最高耐受能力为 5 0 0mmol/L。采用PCR扩增获得该菌 16SrDNA片段。核苷酸序列分析表明 ,该菌的 16SrDNA核苷酸序列与盐单胞菌属同源性最高 ,在 95 %～ 98%之间。在细菌系统分类学上 ,HTG7归属盐单胞菌属Variabil。     

An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.     

Improvements in protein identification by MALDI-TOF-MS peptide mapping

A new strategy for identifying proteins in sequence data-bases by MALDI-MS peptide mapping is reported. The strategy corrects for systematic deviations of determined peptide molecular masses using information contained in the opened database and thereby renders unnecessary internal spectrum calibration. As a result, data acquisition is simplified and less error prone. Performance of the new strategy is demonstrated by identification of a set of recombinant, human cDNA expression products as well as native proteins isolated from crude mouse brain extracts by 2-D electrophoresis. Using one set of calibration constants for the mass spectrometric analyses, 20 proteins were identified without applying any molecular weight restrictions, which was not possible without data correction. A sequence database search program has been written that performs all necessary calculations automatically, access to which will be provided to the scientific community in the Internet.     

Analysis of whole bacterial cells by flow field-flow fractionation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The purpose of this study is to develop a novel bacterial analysis method by coupling the flow field-flow fractionation (flow FFF) separation technique with detection by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The composition of carrier liquid used for flow FFF was selected based on retention of bacterial cells and compatibility with the MALDI process. The coupling of flow FFF and MALDI-TOF MS was demonstrated for P. putida and E. coli. Fractions of the whole cells were collected after separation by FFF and further analyzed by MALDI-MS. Each fraction, collected over different time intervals, corresponded to different sizes and possibly different growth stages of bacteria. The bacterial analysis by flow FFF/MALDI-TOF MS was completed within 1 h with only preliminary optimization of the process.     

鲜食葡萄贮运期黑斑病害致病病原菌分离鉴定

目的 通过研究鲜食葡萄黑斑病害的致病病原菌分离和鉴定,为鲜食葡萄采后病害防控措施开发提供理论支撑。方法 选用贮藏后的马奶等4个鲜食葡萄品种为研究对象,用组织分离法对引起黑斑病害的病原菌进行分离纯化,在PDA培养基上,观察拟菌株形态以及菌丝和孢子形态特征,采用有伤和无伤活体接种健康果实,并观察其发病特征,应用ITS通用引物对病原菌DNA进行扩增并测序。结果 分离出3种优势致病菌(A,B和C),检出频率分别为75.0%,32.0%和42.11%。有伤和无伤活体接种均具有致病性,且有伤大于无伤。对比《真菌鉴定手册》,初步确定拟菌株A为链格孢霉,拟菌株B为灰葡萄孢霉,拟菌株C为拟茎点霉,采用rDNA-ITS序列分析法证实拟菌株A与KJ489375.1、GU190188.1的序列相似度达到99%,结合两者结果确定主要致病病原菌为链格孢霉属小孢子种。结论 明确引起鲜食葡萄贮运期黑斑病害的主要致病菌为链格孢霉属小孢子种。     

Microorganism identification by matrix-assisted laser/desorption ionization mass spectrometry and model-derived ribosomal protein biomarkers

An improved data analysis method is described for rapid identification of intact microorganisms from MALDI-TOF-MS data. The method makes no use of mass spectral fingerprints. Instead, a microorganism database is automatically generated that contains biomarker masses derived from ribosomal protein sequences and a model of N-terminal Met loss. We quantitatively validate the method via a blind study that seeks to identify microorganisms with known ribosomal protein sequences. We also include in the database microorganisms with incompletely known sets of ribosomal proteins to test the specificity of the method. With an optimal MALDI protocol, and at the 95% confidence level, microorganisms represented in the database with 20 or more biomarkers (i.e., those with complete or nearly completely sequenced genomes) are correctly identified from their spectra 100% of the time, with no incorrect identifications. Microorganisms with seven or less biomarkers (i.e., incompletely sequenced genomes) are either not identified or misidentified. Robustness with respect to variations in sample preparation protocol and mass analysis protocol is demonstrated by collecting data with two different matrixes and under two different ion-mode configurations. Statistical analysis suggests that, even without further improvement, the method described here would successfully scale up to microorganism databases with roughly 1000 microorganisms. The results demonstrate that microorganism identification based on proteome data and modeling can perform as well as methods based on mass spectral fingerprinting.     

16S rRNA基因与16S-23S rRNA转录单元内间隔区序列分析及其在节旋藻和螺旋藻分类鉴定中的应用

测定了节旋藻属3个品系和螺旋藻属1个品系的全长16SrRNA基因基因和16S rRNA转录单元内间隔区序列（ITS）,分析了已知的节旋藻、螺旋藻和相关品系的相应序列的同源性,构建了系统发生树,并评价了这两段DNA序列在节旋藻、螺旋藻种属分类和种质鉴定中的意义。结果表明：（1）16SrRNA基因序列和ITS序列均可用于节旋藻属和螺旋藻属的属间分类,以两序列为基础的系统学分析结果一致;（2）ITS序列变异程度高于16SrRNA序列,适用于节旋藻和螺旋藻属内品系或种质鉴定;（3）节旋藻属可明确界定,16SrRNA基因序列相似性大于98％,ITS序列相 似性大于88％;（4）螺旋藻属某些品系间16SrRNA序列和ITS序列相似性较低,与不同属间的序列相似性程度为同一水平。     

Multiarray sensors with pattern recognition for the detection, classification, and differentiation of bacteria at subspecies and strain levels

This work describes the integration of a fully autonomous electrochemical biosensor with pattern recognition techniques for the detection and classification of bacteria at subspecies and strain level. The system provides a continuous, real-time monitoring of bacteria activity upon exposure to antibiotics. The system utilizes 96-well-type electrodes array (DOX-dissolved oxygen sensor) with principal component analysis (PCA) for rapid and routine classification of different classes of bacteria and related strains. A representative sample of a section of the bacteria kingdom has been analyzed and classified using the proposed DOX-PCA system, including the following: Corynebacterium glutamicum, Micrococcus luteus, Staphylococcus epidermidis, Yersinia ruckeri, Escherichia adecarboxylata, Comamonas acidovorans, Alcaligenes odorans, Bacillus globigii, and three strains of Escherichia coli (K12, SM10, ATCC 25922). The new classification scheme is based on the hypothesis that, under identical experimental conditions, various bacteria consume oxygen at different rates and are affected in different ways by selected antibiotics. Thus, the response of the individual electrode in the array is indirectly altered, compared to that of cells growing on medium, by the addition of the antibiotic. By using three different antibiotics in separate wells, a unique fingerprint can be created for a specific bacterium. With the proposed DOX-PCA system, classification of bacteria was achieved at subspecies and strain level in real time. This study represents a basic research tool that may allow researchers to rapidly detect, quantify, and classify bacteria type at subspecies and strain levels.     

Differentiation of Aeromonas isolated from drinking water distribution systems using matrix-assisted laser desorption/ionization-mass spectrometry

The genus Aeromonas is one of several medically significant genera that have gained prominence due to their evolving taxonomy and controversial role in human diseases. In this study, matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was used to analyze the whole cells of both reference strains and unknown Aeromonas isolates obtained from water distribution systems. A library of over 45 unique m/z signatures was created from 40 strains that are representative of the 17 recognized species of Aeromonas, as well as 3 reference strains from genus Vibrio and 2 reference strains from Plesiomonas shigelloides. The library was used to help speciate 52 isolates of Aeromonas. The environmental isolates were broken up into 2 blind studies. Group 1 contained isolates that had a recognizable phenotypic profile and group 2 contained isolates that had an atypical phenotypic profile. MALDI-MS analysis of the water isolates in group 1 matched the phenotypic identification in all cases. In group 2, the MALDI-MS-based determination confirmed the identity of 18 of the 27 isolates. These results demonstrate that MALDI-MS analysis can rapidly and accurately classify species of the genus Aeromonas, making it a powerful tool especially suited for environmental monitoring and detection of microbial hazards in drinking water.     

Identification of bacterial proteins observed in MALDI TOF mass spectra from whole cells.

Characteristic ions in the MALDI TOF mass spectra from bacterial cells have been associated with four known proteins. The proteins, observed both from cells and in filtered cellular suspensions, were isolated by HPLC and identified on the basis of their mass spectra and their partial amino acid sequence, determined using the Edman method (10-15 residues). The acid resistance proteins HdeA and HdeB give rise to ions near m/z 9735 and 9060 in MALDI TOF mass spectra from cells and from extracts of both Escherichia coli 1090 and Shigella flexneri PHS-1059. However, the proteins associated with proteolytic cleavage by the peptidase Lep, rather than the precursor proteins, were observed, both using cells and from cellular extracts. A cold-shock protein, CspA, was associated with the ion near m/z 7643 from Pseudomonas aeruginosa. Similarly, a cold-acclimation protein, CapB, was identified as the source of the ion near m/z 7684 in P. putida. This last protein was homologous with a known CapB from P. fragi. While these experiments involved the detection of known or homologous proteins from typical bacteria, this same approach could also be applied to the detection of unique proteins or biomarker proteins associated with other bacteria of public health significance.     

Studies of histidine as a suitable isoelectric buffer for tryptic digestion and isoelectric trapping fractionation followed by capillary electrophoresis-mass spectrometry for proteomic analysis

The use of histidine as a protein digestion buffer followed by isoelectric trapping separations using "membrane separated wells for isoelectric focusing and trapping" (MSWIFT) and mass spectrometry (MS) analysis is described. Tryptic digestion of bovine serum albumin (BSA) performed in histidine buffered solutions yields similar amino acid sequence coverage values to those obtained using ammonium bicarbonate buffer. Time course studies suggest that histidine buffers provide faster migration of peptides from the loading compartment compared to digestions prepared in ammonium bicarbonate due to differences in conductivities of the two buffers. In addition, this sample preparation method and MSWIFT separations have been coupled with capillary electrophoresis (CE) and matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS) as an alternative separation approach for proteomic studies. Tryptic peptides of ribosomal proteins in histidine are fractionated using MSWIFT followed by CE-MALDI-MS, which further illustrates the ability to couple fractions from a pI based separation device to CE-MS. Specifically, two-dimensional CE-MS plots provide a direct correlation between the numbers of basic residues within the peptide sequence displayed in charge-state trend lines. Combining MSWIFT and CE-MS provides added information regarding peptide sequence, specifically pI and in-solution charge state. Post-translational modifications can also be identified using this method.     

大肠杆菌热敏肠毒素和耐热肠毒素基因融合及其免疫原性研究

采用基因工程技术将编码大肠杆菌热敏肠毒素亚单位（ＬＴＢ）基因和耐热肠毒素（ＳＴ）基因进行体外重组,得到的融合基因能在大肠杆菌中表达。重组菌株免疫动物后,均能诱发产生抗ＬＴ和ＳＴ抗体。实验结果表明,ＬＴ／ＳＴ融合蛋白不仅保持了ＬＴＢ的免疫原性和与神经节甙酯ＧＭ１的结合能力,而且也赋予本来没有免疫原性的ＳＴ免疫原性,并极大地降低了ＳＴ的生物毒性,为构建理想的致腹泻大肠菌苗奠定了基础。     

C-terminal sequence analysis of peptides and proteins using carboxypeptidases and mass spectrometry after derivatization of Lys and Cys residues

C-Terminal sequence analysis of peptides and proteins using carboxypeptidase digestion in combination with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is convenient for protein and peptide characterization. After a short digestion, a sequence up to 20 residues can be identified, but the total number depends on the individual sequence. Due to the accuracy limits of the MALDI time-of-flight arrangement, the assignment of several residues with close mass values, including Lys/Glx, may remain ambiguous. We have used derivatization of lysine residues by guanidination to overcome the problem of Lys identification. The reaction is rapid and specific and results in full derivatization. In the case of Cys-containing peptides, problems arise from the fact that carboxypeptidases Y and P do not cleave peptides that contain nonderivatized cystine, cysteic acid, or (carboxymethyl)cysteine. Successful identification of Cys residues within the sequence is instead achieved by conversion of Cys to 4-thialaminine by (trimethylamino)-ethylation. The two derivatizations of Lys and Cys side chains provide opportunities for proton attachment and therefore facilitate the analysis by MALDI-MS. This C-terminal sequence analysis method is also useful for large proteins after fragmentation with specific enzymes.     

Identification of sporulated and vegetative bacteria using statistical analysis of fourier transform mid-infrared transmission data

A combined mid-infrared spectroscopic/statistical modeling approach for the discrimination and identification, at the strain level, of both sporulated and vegetative bacterial samples is presented. Transmission mode spectra of bacteria dried on ZnSe windows were collected using a Fourier transform mid-infrared (FT-IR) spectrometer. Five Bacillus bacterial strains (B. atrophaeus 49337, B. globigii Dugway, B. thuringiensis spp. kurstaki 35866, B. subtilis 49760, and B. subtilis 6051) were used to construct a reference spectral library and to parameterize a four-step statistical model for the systematic identification of bacteria. The statistical methods used in this initial feasibility study included principal component analysis (PCA), classification and regression trees (CART), and Mahalanobis distance calculations. Internal cross-validation studies successfully classified 100% of the samples into their correct physiological state (sporulated or vegetative) and identified 67% of the samples correctly as to their bacterial strain. Analysis of thirteen blind samples, which included reference and other bacteria, nonbiological materials, and mixtures of both nonbiological and bacterial samples, yielded comparable accuracy. The primary advantage of this approach is the accurate identification of unknown bacteria, including spores, in a matter of minutes.     

Neutral loss fragmentation pattern based screening for arginine-rich natural products in xenorhabdus and photorhabdus

Although sharing a certain degree of structural uniformity, natural product classes exhibit variable functionalities such as different amino acid or acyl residues. During collision induced dissociation, some natural products exhibit a conserved fragmentation pattern close to the precursor ion. The observed fragments result from a shared set of neutral losses, creating a unique fragmentation pattern, which can be used as a fingerprint for members of these natural product classes. The culture supernatants of 69 strains of the entomopathogenic bacteria Photorhabdus and Xenorhabdus were analyzed by MALDI-MS(2), and a database comprising MS(2) data from each strain was established. This database was scanned for concordant fragmentation patterns of different compounds using a customized software, focusing on relative mass differences of the fragment ions to their precursor ion. A novel group of related natural products comprising 25 different arginine-rich peptides from 16 different strains was identified due to its characteristic neutral loss fragmentation pattern, and the structures of eight compounds were elucidated. Two biosynthesis gene clusters encoding nonribosomal peptide synthetases were identified, emphasizing the possibility to identify a group of structurally and biosynthetically related natural products based on their neutral loss fragmentation pattern.     

一株牙Ping出血症病原菌的分子生物学鉴定

从患出血症养殖牙Ping分离到一株病原菌M4,革兰氏阴性,杆状,有极生和侧生鞭毛,能运动,菌落半透明。进行了常规生理生化和BIOLOG－GN细菌鉴定系统测试。结果表明菌株M4与V.carchariae的表型特征非常相似。为了进一步确定M4的分类学地位,测定了其16SrRNA基因序列,分析了相关细菌相应序列的同源性,构建了系统发生树,结果表明菌株M4和V.carchariae的亲缘关系最近。综合上述结果,菌株M4可鉴定为Vibiro carchariae。     

Affinity capture using vancomycin-bound magnetic nanoparticles for the MALDI-MS analysis of bacteria

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) provides a straightforward means to differentiate microorganism species based on mass spectral fingerprinting. The pathogen cell concentration in an infected sample, however, is generally lower than that capable of being detected directly by MALDI-MS. Furthermore, the presence of proteins or metabolites in biological fluids always causes unavoidable interference for the identification of microorganism species. Vancomycin, which binds to D-Ala-D-Ala moieties on the cell walls of Gram-positive bacteria and, therefore, inhibits peptidoglycan synthesis, is one of the most potent antibiotics. Thus, we have employed vancomycin-modified magnetic nanoparticles as affinity probes to selectively trap Gram-positive pathogens from sample solutions; i.e., these bacteria can be isolated from sample solutions by applying a magnetic field. The isolated cells could then be characterized by MALDI-MS. This approach effectively reduces the interference of protein and metabolite signals in the mass spectra of Gram-positive bacteria because vancomycin has such high specificity for the D-Ala-D-Ala units of the cell walls. The lowest cell concentration we detected for both Staphylococcus saprophyticus and Staphylococcus aureus in a urine sample (3 mL) was approximately 7 x 10(4) cfu/mL.