Desarrollo y evaluación de una base de datos in house para la identificación rápida de Burkholderia contaminons por EM MALDI-TOF / Development and evaluation of an in-house database for quick identification of Burkholderia contaminans by MALDI-TOF MS

La espectrometría de masas (EM) (matrix assisted laser desorption ionization-time of flight) MALDI-TOF demostró ser una herramienta robusta para la identificación de numerosos grupos taxonómicos. No obstante, presenta limitaciones. Una ventaja clave de la técnica es la flexibilidad para la incorporación de espectros proteicos de microorganismos ausentes en la base de datos comercial. Dada la prevalencia de Burkholderia contaminans en los pacientes fibroquísticos en Argentina, y a que en ellos es crucial el diagnóstico microbiológico rápido y confiable, la EM MALDI-TOF surge como una herramienta estratégica. El objetivo del trabajo fue desarrollar una base de datos adicional con espectros peptídicos de aislamientos de referencia de B. contaminans. La misma demostró ser exitosa para la identificación del 97% de los aislamientos analizados. Por lo cual la EM MALDI-TOF con la base de datos extendida resultó ser una herramienta útil para la identificación y diferenciación de otras especies relacionadas a B. contaminans.

MALDI-TOF (matrix assisted laser desorption ionization-time of flight) mass spectrometry (MS) proved to be a robust tool for the identification of numerous taxonomic groups. However, it has limitations. A key advantage of this technique is the flexibility for the incorporation of protein profiles of microorganisms not included in the commercial database. Due to the prevalence of Burkholderia contaminans in fibrocystic patients in Argentina and the fact that rapid and reliable microbiological diagnosis is crucial in them, MALDI-TOF MS emerges as a strategic tool. The aim of this work was to develop an additional database with peptide spectra of reference isolates of B. contaminans. This database demonstrated to be successful for the identification of 97% of the isolates analyzed. Therefore, MALDI-TOF MS with the extended database was a useful tool for the identification and differentiation of other related species to B. contaminans.

Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry

ABSTRACT Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.

Direct identification of bovine mastitis pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in pre-incubated milk

ABSTRACT The present study aimed to compare two MALDI-TOF identification methods [(a) direct sample identification after pre-incubation; or (b) use of bacteria isolated on pre-culture)] to standard, traditional bench microbiology. A total of 120 quarter milk samples from 40 Holstein lactating cows were screened based on culture-positive results obtained by microbiological culture (reference method) with the following numbers of quarters positive per cow: 4 cows with 1, 8 cows with 2, 12 cows with 3 and 16 cows with 4 infected quarters per cow. For direct identification method, quarter milk samples (n = 120) were skimmed by centrifugation (10,000 × g/10 min) and pre-incubated at 37 ºC for 12 h. After pre-incubation, quarter milk samples were submitted to total bacterial count by flow cytometry and for a preparation protocol for bacterial ribosomal protein extraction followed by MALDI-TOF MS analysis. The direct MALDI-TOF MS identification method compared to microbiological culture correctly identified isolates of coagulase-negative Staphylococci (27.2%), Streptococcus agalactiae (21.8%), Staphylococcus aureus (14.2%), and Streptococcus uberis (5.2%). The pre-incubation protocol of milk samples, associated to the direct identification method by MALDI-TOF MS, did not increase the identification at species level (score >2.0) of pathogens causing subclinical mastitis in comparison to the method without previous incubation.

Concordance analysis between different methodologies used for identification of oral isolates of Candida species / Análisis de concordancia de diferentes metodologías para la identificación de aislamientos orales de especies de Candida

Abstract Background: The yeasts species determination is fundamental not only for an accurate diagnosis but also for establishing a suitable patient treatment. We performed a concordance study of five methodologies for the species identification of oral isolates of Candida in Colombia. Methods: Sixty-seven Candida isolates were tested by; API® 20C-AUX, Vitek®2 Compact, Vitek®MS, Microflex® and a molecular test (panfungal PCR and sequencing). The commercial cost and processing time of the samples was done by graphical analysis. Results: Panfungal PCR differentiated 12 species of Candida, Vitek®MS and Microflex® methods identified 9 species, and API® 20C-AUX and Vitek®2 Compact methods identified 8 species each. Weighted Kappa (wK) showed a high agreement between Panfungal PCR, Vitek®MS, Microflex® and API® 20C-AUX (wK 0.62-0.93). The wK that involved the Vitek®2 Compact method presented moderate or good concordances compared with the other methods (wK 0.56-0.73). Methodologies based on MALDI TOF MS required 4 minutes to generate results and the Microflex® method had the lowest selling price. Conclusion: The methods evaluated showed high concordance in their results, being higher for the molecular methods and the methodologies based on MALDI TOF. The latter are faster and cheaper, presenting as promising alternatives for the routine identification of yeast species of the genus Candida.

Resumen Introducción: La clasificación a nivel de especies de las levaduras del género Candida de origen clínico es fundamental para el diagnóstico y la instauración de un adecuado tratamiento para el paciente. Se realizó un estudio de concordancia de cinco metodologías usadas para la identificación de aislamientos orales de Candida spp en Colombia. Métodos: Sesenta y siete aislamientos de Candida spp fueron identificados a nivel de especie utilizando; API® 20 C AUX‚ Vitek® 2 Compact, MALDI TOF (Vitek® MS y Microflex®) y una prueba molecular, PCR Panfungal y secuenciación. Un análisis del costo comercial y tiempo de procesamiento de las muestras por cada método fue realizado mediante el análisis gráfico de ambas variables. Resultados: La PCR Panfungal y secuenciación diferenció 12 especies de Candida‚ los métodos Vitek® MS y Microflex® identificaron 9 especies y los métodos API® 20 C AUX y Vitek® 2 Compact identificaron 8 especies. El análisis de Kappa ponderado (wK) demostró una concordancia alta entre los métodos PCR Panfungal y secuenciación‚ Vitek® MS‚ Microflex® y API® 20 C AUX‚ concordancias agrupadas en las categorías buena y muy buena (wK 0.62 - 0.93); los Kp que involucraron el método Vitek® 2 Compact presentaron concordancias moderadas o buenas frente a los otros métodos (wK 0.56 - 0.73). Las metodologías basadas en MALDI TOF MS requirieron 4 minutos para generar un resultado y el método Microflex® fue el método que en nuestro medio presentó el menor precio de venta del servicio. Conclusión: Los métodos evaluados presentaron una alta concordancia en sus resultados‚ siendo más alta para los métodos moleculares y las metodologías basadas en MALDI TOF MS; estas últimas son metodologías más rápidas, económicas y precisas, las cuales se presentan como alternativas prometedoras para la identificación rutinaria de especies de levaduras del género Candida.

Displacers improve the selectivity of phosphopeptide enrichment by metal oxide affinity chromatography / Uso de desplazadores para mejorar la selectividad de los fosfopéptidos enriquecidos por cromatografía de afinidad con óxidos de metales

Abstract: Background: A key process in cell regulation is protein phosphorylation, which is catalyzed by protein kinases and phosphatases. However, phosphoproteomics studies are difficult because of the complexity of protein phosphorylation and the number of phosphorylation sites. Methods: We describe an efficient approach analyzing phosphopeptides in single, separated protein by two-dimensional gel electrophoresis. In this method, a titanium oxide (TiO2)-packed NuTip is used as a phosphopeptide trap, together with displacers as lactic acid in the loading buffer to increase the efficiency of the interaction between TiO2 and phosphorylated peptides. Results: The results were obtained from the comparison of mass spectra of proteolytic peptides of proteins with a matrix-assisted laser desorption-ionization-time of flight (MALDI-TOF) instrument. Conclusions: This method has been applied to identifying phosphoproteins involved in the symbiosis Rhizobium etli-Phaseolus vulgaris.

Resumen: Introducción: Un proceso clave en la regulación celular es la fosforilación de proteínas, que se lleva a cabo por cinasas y fosfatasas. Sin embargo, los estudios de fosfoproteómica son difíciles debido a la complejidad de la fosforilación proteica y el número de sitios de fosforilación. Métodos: En el presente trabajo se describe una eficiente estrategia metodológica para analizar fosfopéptidos de proteínas separadas mediante electroforesis bidimensional. En este método, una columna con microesferas de dióxido de titanio (TiO2/NuTip) se utilizó para atrapar los fosfopéptidos en la superficie del TiO2 previamente empacado en una punta. El uso de desplazadores en el buffer de carga, como el ácido láctico, mejoró significativamente la selectividad. Resultados: Los resultados se obtuvieron mediante la comparación de los espectros de masas de péptidos proteolíticos de proteínas analizados utilizando un instrumento de desorción/ionización láser asistida por matriz-tiempo de vuelo (MALDI-TOF). Conclusiones: Este método se ha aplicado para la identificación de fosfoproteínas involucradas en la simbiosis del Rhizobium etli con Phaseolus vulgaris.

Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry for identification of Clostridium species isolated from Saudi Arabia

Abstract The aim of this study was to identify different Clostridium spp. isolated from currency notes from the Ha’il region of Saudi Arabia in September 2014 using MALDI–TOF-MS. Clostridium spp. were identified by Bruker MALDI–TOF-MS and compared with VITEK 2. The confirmation of the presence of different Clostridium spp. was performed by determining the sequence of the 16S ribosomal RNA gene. In this study, 144 Clostridium spp. were isolated. Among these specimens, MALDI–TOF-MS could identify 88.8% (128/144) of the isolates to the species level and 92.3% (133/144) to the genus level, whereas, VITEK 2 identified 77.7% of the (112/144) isolates. The correct identification of the 144 isolates was performed by sequence analysis of the 500 bp 16S rRNA gene. The most common Clostridium spp. identified were Clostridium perfringens (67.36%), Clostridium subterminale (14.58%), Clostridium sordellii (9%) and Clostridium sporogenes (9%). The results of this study demonstrate that MALDI–TOF-MS is a rapid, accurate and user friendly technique for the identification of Clostridium spp. Additionally, MALDI–TOF-MS has advantages over VITEK 2 in the identification of fastidious micro-organisms, such as Clostridium spp. Incorporating this technique into routine microbiology would lead to more successful and rapid identification of pathogenic and difficult to identify micro-organisms.

Proteomic Analysis of Rat Brain Stem with DAI by MALDI-TOF-MS / 法医学杂志

OBJECTIVE@#To establish a diagnostic model for diffuse axonal injury (DAI) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). To screen the proteins or peptides associated with DAI for providing the biomarkers with theoretic foundation.@*METHODS@#Fifteen male Sprague-Dawley rats were randomly divided into DAI group (n = 10) and control group (n = 5). The protein or peptide expression profiles of rat brain stem were detected by MALDI-TOF-MS. ClinProTools 2.2 software was used to find specific peaks, and a diagnostic model was established by the genetic algorithm.@*RESULTS@#There were significant differences in 61 peaks of DAI group (P < 0.05), 9 peaks were down-regulated and 52 up-regulated. The diagnostic model was established based on 5 different peaks. The specificity and sensitivity of cross validation was 96.14% and 95.98%; while the specificity and sensitivity of blind validation showed was 73.33% and 70.00%, respectively.@*CONCLUSION@#A specific and sensitive diagnostic model of DAI can be established by MALDI-TOF-MS to provide a potential value for determining DAI in forensic practice.

Evaluación de la espectrometría de masas: MALDI-TOF MS para la identificación rápida y confiable de levaduras / Evaluation of mass spectrometry: MALDI-TOF MS for fast and reliable yeast identification

La espectrometría de masas, conocida como matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), es una técnica utilizada en la identificación de microorganismos mediante la creación de un espectro basado en el perfil de proteínas, que es único para una especie dada. El objetivo del presente trabajo fue evaluar la identificación de aislamientos clínicos de levaduras por MALDI-TOF MS en un hospital universitario de Argentina y analizar 2 procedimientos para la extracción de proteínas: el recomendado por el fabricante del equipo y una técnica abreviada rápida. Utilizando el primero de estos procedimientos se analizaron 201 aislamientos identificados previamente por métodos convencionales y se obtuvo coincidencia en la identificación a nivel de especie en el 95,38% de los aislamientos analizados. Con 100 de estos aislamientos se utilizó, además, el procedimiento abreviado para la extracción de proteínas; se obtuvo una identificación correcta a nivel de género y especie en el 98,0% de ellos. La espectrometría de masas MALDI-TOF MS demostró ser una técnica rápida, sencilla y precisa para la identificación de levaduras

The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry technique known as MALDI-TOF MS is a tool used for the identification of clinical pathogens by generating a protein spectrum that is unique for a given species. In this study we assessed the identification of clinical yeast isolates by MALDI-TOF MS in a university hospital from Argentina and compared two procedures for protein extraction: a rapid method and a procedure based on the manufacturer's recommendations. A short protein extraction procedure was applied in 100 isolates and the rate of correct identification at genus and species level was 98.0%. In addition, we analyzed 201 isolates, previously identified by conventional methods, using the methodology recommended by the manufacturer and there was 95.38% coincidence in the identification at species level. MALDI TOF MS showed to be a fast, simple and reliable tool for yeast identification

Comparison of MALDI-TOF MS, Housekeeping Gene Sequencing, and 16S rRNA Gene Sequencing for Identification of Aeromonas Clinical Isolates

PURPOSE: The genus Aeromonas is a pathogen that is well known to cause severe clinical illnesses, ranging from gastroenteritis to sepsis. Accurate identification of A. hydrophila, A. caviae, and A. veronii is important for the care of patients. However, species identification remains difficult using conventional methods. The aim of this study was to compare the accuracy of different methods of identifying Aeromonas at the species level: a biochemical method, matrix-assisted laser desorption ionization mass spectrometry-time of flight (MALDI-TOF MS), 16S rRNA sequencing, and housekeeping gene sequencing (gyrB, rpoB). MATERIALS AND METHODS: We analyzed 65 Aeromonas isolates recovered from patients at a university hospital in Korea between 1996 and 2012. The isolates were recovered from frozen states and tested using the following four methods: a conventional biochemical method, 16S rRNA sequencing, housekeeping gene sequencing with phylogenetic analysis, and MALDI-TOF MS. RESULTS: The conventional biochemical method and 16S rRNA sequencing identified Aeromonas at the genus level very accurately, although species level identification was unsatisfactory. MALDI-TOF MS system correctly identified 60 (92.3%) isolates at the species level and an additional four (6.2%) at the genus level. Overall, housekeeping gene sequencing with phylogenetic analysis was found to be the most accurate in identifying Aeromonas at the species level. CONCLUSION: The most accurate method of identification of Aeromonas to species level is by housekeeping gene sequencing, although high cost and technical difficulty hinder its usage in clinical settings. An easy-to-use identification method is needed for clinical laboratories, for which MALDI-TOF MS could be a strong candidate.

Metodología de pre-procesamiento de datos adquiridos por MALDI-MSI en muestras de tejidos / Pre-processing methodology of acquired data by MALDI-MSI in tissue samples

En la última década, la Imagenología por Espectrometría de Masas MALDI (MALDI-MSI) ha demostrado su potencial en el campo de la proteómica. Debido a la alta dimensionalidad de los conjuntos de datos obtenidos en los experimentos de MADI-MSI y las variabilidades inherentes al proceso de adquisición, presentes en los mismos, se hace necesario llevar a cabo una etapa de pre-procesamiento, que reduzca estas distorsiones. La presente investigación propone una metodología de procesamiento de datos de MALDI-MSI sustentada en un conjunto de aplicaciones desarrolladas en MatLab, la Biblioteca Qt4, así como la herramienta de visualización DataCube Explorer. Entre los resultados se pueden destacar la obtención de cambios en las intensidades de los píxeles de las imágenes reconstruidas después de la introducción de ruido, así como el incremento de la Relación Señal-Ruido después de someter los espectros a los métodos de filtrado de Kaiser, Savitzky-Golay y Promedio deslizante, destacándose Kaiser sobre los demás, lo que puede traducirse como una disminución de los niveles de distorsión en los espectros de cada pixel. Se realizó la reconstrucción satisfactoria de la imagen patrón y su visualización con la herramienta DataCube Explorer(AU)

At last decade, MALDI Mass Spectrometry Imaging (MALDI-MSI) has demostrated being a powerful tool in proteomics field. Because of high dimensionality of the acquired datasets in MALDI-MSI experiments and the inherent variabilities to the acquisition process, present in them, it´s necessary carry out a pre-processing stage, which reduces these distortions. This paper proposes a processing methodology of data MALDI-MSI supported in applications developed in MATLAB, Qt4 Library, as well as the visualization tool Datacube Explorer. Among the results obtained, it can be highlighted the changes in the intensities of the pixels of the reconstructed images after introducing noise, as well as the increasing of Signal-Noise Ratio after applying the denoising methods Kaiser, Savitzky-Golay and "sliding average", highlighting Kaiser over the other techniques, which can be interpreted as a decreasing of the distortion levels in each pixel´s spectrum. The reconstruction of example image and its visualization with Datacube Explorer tool were satisfactory(AU)

Utilidad de la espectrometría de masas MALDI-TOF en la identificación de bacterias anaerobias / Utility of MALDI-TOF MS for the identification of anaerobic bacteria

El análisis de espectrometría de masas mediante la metodología hoy conocida como MALDI-TOF MS (Matrix-assited laser desorption/ionization time-of-flight mass spectrometry) se ha convertido en un recurso de referencia para la identificación de microorganismos en microbiología clínica. No obstante, los datos relativos a algunos grupos de microorganismos son todavía controvertidos. El objetivo del presente estudio fue determinar la utilidad del MALDI-TOF MS para la identificación de aislamientos clínicos de bacterias anaerobias. Se analizaron 106 aislamientos de bacterias anaerobias mediante MALDI-TOF MS y por pruebas bioquímicas convencionales. En aquellos casos en los que la identificación por metodología convencional no era aplicable o frente a una discordancia de resultados entre las metodologías citadas, se realizó la secuenciación del gen 16S del ARNr. El método convencional y el MALDI-TOF MS coincidieron a nivel de género y especie en un 95,3 % de los casos considerando la totalidad de los aislamientos estudiados. Al considerar solo el conjunto de los bacilos gram negativos, la coincidencia fue del 91,4 %; entre los bacilos gram positivos, fue del 100 %; los 8 aislados de cocos gram positivos estudiados coincidieron y también hubo coincidencia en el único coco gram negativo incluido. Los datos obtenidos en este estudio demuestran que el MALDI-TOF MS ofrece la posibilidad de llegar a una adecuada identificación de bacterias anaerobias

The analysis by MALDI-TOF MS (Matrix-assited laser desorption/ionization time-of-flight mass spectrometry) has become a reference method for the identification of microorganisms in Clinical Microbiology. However, data on some groups of microorganisms are still controversial. The aim of this study is to determine the utility of MALDI-TOF MS for the identification of clinical isolates of anaerobic bacteria. One-hundred and six anaerobic bacteria isolates were analyzed by MALDI-TOF MS and by conventional biochemical tests. In those cases where identification by conventional methodology was not applicable or in the face of discordance between sequencing methodologies, 16 S rRNA gene sequence analysis was performed. The conventional method and MALDI-TOF MS agreed at genus and species level by 95.3 %. Concordance in gram-negative bacilli was 91.4% and 100% among gram-positive bacilli; there was also concordance both in the 8 isolates studied in gram-positive cocci and in the single gram-negative cocci included. The data obtained in this study demonstrate that MALDI-TOF MS offers the possibility of adequate identification of anaerobic bacteria

Proteomic analysis of the regenerating liver following 2/3 partial hepatectomy in rats

BACKGROUND: Liver regeneration (LR) after 2/3 partial hepatectomy (PH) is one of the most studied models of cell, organ, and tissue regeneration. Although the transcriptional profile analysis of regenerating liver has been carried out by many reserachers, the dynamic protein expression profile during LR has been rarely reported up to date. Therefore, this study aims to detect the global proteomic profile of the regenerating rat liver following 2/3 hepatectomy, thereby gaining some insights into hepatic regeneration mechanism. RESULTS: Protein samples extracted from the sham-operated and the regenerating rat livers at 6, 12, 24, 72, 120 and 168 h after PH were separated by IEF/SDS-PAGE and then analyzed by MALDI-TOF/TOF mass spectrometry. Compared to sham-operated groups, there were totally 220 differentially expressed proteins (including 156 up-regulated, 62 down-regulated, and 2 up/down-regulated ones) identified in the regenerating rat livers, and most of them have not been previously related to liver regeneration. According to the expression pattern analysis combined with gene functional analysis, it showed that lipid and carbohydrate metabolism were enhanced at the early phase of LR and continue throughout the regeneration process. Ingenuity Pathway Analysis indicated that YWHAE protein (one of members of the 14-3-3 protein family) was located at the center of pathway networks at all the timepoints after 2/3 hepatectomy under our experimental conditions, maybe suggesting a central role of this protein in regulating liver regeneration. Additionally, we also revealed the role of Cdc42 (cell division cycle 42) in the termination of LR. CONCLUSIONS: For the first time, our proteomic analysis suggested an important role of YWHAE and pathway mediated by this protein in liver regeneration, which might be helpful in expanding our understanding of LR amd unraveling the mechanisms of LR.

Screening and identification of forensic molecular markers of injury using MALDI-TOF-MS imaging mass spectrometry / 法医学杂志

There are many deficiencies in forensic traumatic molecular markers detected by the techniques of traditional immunohistology and molecular biology, because these markers are isolated and obscure of the mechanism of interaction. The imaging mass spectrometry (IMS) is more suitable for the forensic molecular markers using function of screening, analysis and graphical representation. In this paper, the techniques and the latest research in screening and identification of typical molecular markers by IMS based on matrix-assisted laser adsorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) are reviewed. And its application values in forensic injury are discussed.

Bacterial identification in the diagnostic laboratory: How much is enough.

The major impetus for bacterial identification came after the advent of solid culture media. Morphological appearance of bacterial colonies was often sufficient for their identification in the laboratory. Even in modern times, preliminary identification of most cultivable bacteria is based on such morphological characters. Advances have been made media for the presumptive identifi cation of common organisms encountered in clinical samples. Phenotypic characterisation of bacteria with, physiological tests with a battery of biochemical tests differentiate related bacterial genera as well as confirm their identity. . Each laboratory can select its own method(s) of identification, provided they are based on scientific / epidemiological evidence; clinical laboratory and standards institute (CLSI) is a widely accepted organization and laboratories in many parts of the world follow its recommendations for bacterial identification. Some of the latest advances in identification include Matrix Assisted Laser Desorption Ionization - Time of Flight Mass Spectroscopy (MALDI-TOF) is a state of art facility used for fast and reliable species-specific identification of bacteria including Mycobacteria and fungi including yeasts. However the single most important factor that decides the method of bacterial identification in any laboratory is the cost involved. In the final analysis, selection of tests for bacterial identification should be based on their standardization with proper scientific basis. Considering the cost and lack of easy availability of commercial kits, we have put forward a simplified and rapid method of identification for most commonly encountered bacterial pathogens causing human infection in India

Genotyping and linkage disequilibrium analysis of 67 SNP loci on X chromosome / 法医学杂志

OBJECTIVE@#To screen a panel of SNP loci on X chromosome (X-SNP loci) that is informative in Chinese Han population and evaluate its potential value in forensic identification.@*METHODS@#Sixty-seven candidate X-SNP loci were selected according to the information on dbSNP and HapMap. Genomic DNA extracted from blood samples of 428 unrelated Chinese Han individuals were analyzed through multiplex amplification followed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry, and allele frequencies of the 67 X-SNP loci were calculated. In view of the population data and situation of linkage disequilibrium, X-SNP markers promising in forensic identification were sorted out.@*RESULTS@#Population data of the 67 X-SNP loci were obtained. Except rs12849634, no deviations from Hardy-Weinberg equilibrium could be found. Of the rest 66 X-SNP loci, two loci (rs1229078 and rs1544545) were found to be low informative (minor allele frequency < 0.3). Closely linked alleles were observed at six pairs of X-SNP loci and slightly linked alleles were observed at two pairs of X-SNP loci. Fifty-two X-SNP loci showing independent inheritance and high polymorphisms were finally selected. The markers were promising in forensic identification. CPE in trio and duo cases were 0.999 999 999 96 and 0.999 999 5, respectively. CDP in female and male populations were 0.999 999 999 999 999 999 999 84 and 0.999 999 999 999 999 31, respectively.@*CONCLUSION@#A panel of 52 informative X-SNP loci showing independent inheritance is selected. These markers meet the needs of individual identification and relationship testing in judicially disputed cases.

Human Papilloma Virus Genotyping Assay using Restriction Fragment Mass Polymorphism Analysis, and Its Comparison with Sequencing and Hybrid Capture Assays / 대한진단검사의학회지

BACKGROUND: Infection with human papilloma virus (HPV) is the main cause of cervical cancer, and HPV genotyping is of increasing importance for determining clinical course and management of the disease based on the HPV genotypes. Here, we established a novel matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS) assay, termed restriction fragment mass polymorphism (RFMP) that is suitable for genotyping multiple HPV in an accurate and high-throughput manner. We evaluated the performance of the RFMP assay in HPV genotyping by comparing the results with those of direct or clonal sequencing and hybrid capture (HC) assays. METHODS: The study population consisted of 50 patients with histologically confirmed cervical lesions and a positive test for HPV DNA. HPV genotyping was performed with RFMP, sequencing, and HC assays. The assigned genotypes and risk groups were compared among the methods. RESULTS: Concordance rates in the genotype level between RFMP vs sequencing, sequencing vs HC, and HC vs RFMP were 98% (49/50), 88% (44/50), and 88% (44/50), respectivley. Especially, RFMP and sequencing were 100% concordant when assigned high-risk group was considered identical in 1 case of mixed genotypes identified only in RFMP. The observed discrepancy between HC and the other two methods is due to the assignment of six cases of low, intermediate, or unassigned risk genotypes as high-risk group in HC method. CONCLUSIONS: RFMP, sequencing, and HC assays were highly concordant with each other in HPV genotyping. Compared to sequencing assay, RFMP assay is found to be advantageous in detecting mixed genotype infections. The accuracy and amenability to high-throughput analysis should make the RFMP assay suitable for reliable screening of HPV genotypes in clinical laboratories.

Identification of alpha-2u globulin in the rat preputial gland by MALDI-TOF analysis.

The low molecular mass proteins found in the pheromonal sources such as urine, saliva, glandular secretion etc have been reported as ligand carriers for the processes of chemocommunication in mammals. The preputial gland plays an important role in the production of olfactory signals for pheromonal communication. Thus, in the present study, alpha-2u globulin having molecular mass of 18 kDa has been identified in the preputial gland of Norway rat (Rattus norvegicus) by in-gel trypsin digestion and analyzing the resulting peptides by MALDI-TOF. Since preputial gland is one of the major pheromonal sources in rat, the results suggest that alpha-2u globulin might act as a carrier for hydrophobic odorants of preputial gland.

The amino-terminal domain of human signal transducers and activators of transcription 1: overexpression, purification and characterization.

The dual functional signal transducers and activators of transcription (STAT) proteins are latent cytoplasmic transcription factors that play crucial roles in host defense. Animals that lack these proteins are highly susceptible to microbial and viral infections and chemically induced primary tumours. We have over expressed the amino-terminal domain of human STAT1 (hSTAT1) in Escherichia coli and purified it by affinity chromatography and gel filtration chromatography. The entire process has been monitored by gel electrophoresis. The pure protein has been characterized by mass spectrometry and 2-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Our results indicate that the N-terminus of hSTAT1 exists as a dimer in solution.

Analysis of proteomic components of sera from patients with uremia by two dimensional electrophoresis and matrix assisted laser desorption/ ionization time of flight mass spectrometry / 华中科技大学学报（医学）（英德文版）

The different sera proteomic components between uremia patients and normal subjects were studied through two-dimensional gel electrophoresis technique. Immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis (2DE), silver staining, ImageMaster 2D 5.0 analysis software, matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-TOF-MS) and IPI human database searching were used to separate and identify the proteome of the sera from the patients with uremia. The results showed that satisfactory 2DE patterns of the serum proteins were obtained. Twenty-six protein spots showed significant difference in quantity in uremia patients, and 20 protein spots were identified by MALDI-TOF-TOF-MS. It was concluded that good reproducibility could be obtained by applying immobilized pH gradient 2DE to separate and identify the proteome in serum, which provided the foundation for the further study on uremia toxins pertaining to protein.