MALDI-TOF Mass Spectrometry For Identification Research Articles

Evaluation of MALDI-TOF mass spectrometry for identification of environmental yeasts and development of supplementary database

Yeast identification using traditional methods which employ morphological, physiological, and biochemical characteristics can be considered a hard task as it requires experienced microbiologists and a rigorous control in culture conditions that could implicate in different outcomes. Considering clinical or industrial applications, the fast and accurate identification of microorganisms is a crescent demand. Hence, molecular biology approaches has been extensively used and, more recently, protein profiling using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has proved to be an even more efficient tool for taxonomic purposes. Nonetheless, concerning to mass spectrometry, data available for the differentiation of yeast species for industrial purpose is limited and reference databases commercially available comprise almost exclusively clinical microorganisms. In this context, studies focusing on environmental isolates are required to extend the existing databases. The development of a supplementary database and the assessment of a commercial database for taxonomic identifications of environmental yeast are the aims of this study. We challenge MALDI-TOF MS to create protein profiles for 845 yeast strains isolated from grape must and 67.7 % of the strains were successfully identified according to previously available manufacturer database. The remaining 32.3 % strains were not identified due to the absence of a reference spectrum. After matching the correct taxon for these strains by using molecular biology approaches, the spectra concerning the missing species were added in a supplementary database. This new library was able to accurately predict unidentified species at first instance by MALDI-TOF MS, proving it is a powerful tool for the identification of environmental yeasts.

Isolation and characterization of thermostable phycocyanin from Galdieria sulphuraria

Phycocyanin is a highly valuable pigmented protein synthesized by several species of cyanobacteria and red alga. In this study we demonstrate the production of thermostable phycocyanin from the unicellular red alga Galdieria sulphuraria. Phycocyanin was extracted by repeated freeze-thaw cycles and purified in a two-step process using ammonium sulfate fractionation, at 25% and 50% concentrations. Purified phycocyanin exhibited maximum absorbance at 620 nm, and the purity ratio (A620/A280) was found to be greater than 4. The recovery efficiency of phycocyanin from the crude extract was above 80%. In total, approximately 19 milligram pure phycocyanin was obtained from 3 g of wet cell mass of Galdieria sp. Subunits α and β of the protein were separated by SDS-PAGE and analyzed by MALDITOF mass spectrometry for identification, which confirmed that the isolated protein is phycocyanin. The molecular weight of α and β subunits of phycocyanin was found to be 17.6 and 18.4 kDa, respectively.

MALDI‐TOF mass spectrometry identification of filamentous fungi in the clinical laboratory

This study aimed to validate the effectiveness of a standardised procedure for the MALDI-TOF mass spectrometry (MS)-based identification on a large sample of filamentous fungi routinely identified in university hospitals' laboratories. Non-dermatophyte filamentous fungi prospectively isolated in the routine activity of five teaching hospitals in France were first identified by conventional methods in each laboratory and then by MS in one centre. DNA sequence-based identification resolved discrepancies between both methods. In this study, of the 625 analysed filamentous fungi of 58 species, 501 (80%) and 556 (89%) were correctly identified by conventional methods and MS respectively. Compared with the conventional method, MS dramatically enhanced the performance of the identification of the non-Aspergillus filamentous fungi with a 31-61% increase in correct identification rate. In conclusion, this study on a large sample of clinical filamentous fungi taxa demonstrates that species identification is significantly improved by MS compared with the conventional method. The main limitation is that MS identification is possible only if the species is included in the reference spectra library. Nevertheless, for the routine clinical laboratory, MS provides the means to attain markedly accurate results in filamentous fungi identification, which was previously restricted to only a few reference laboratories.

Eficacia de la espectrometría de masas MALDI-TOF en la identificación de bacterias anaerobias

Aim of the studyMALDI-TOF mass spectrometry (MS) is becoming a major resource in the Clinical Microbiology laboratory. Results on some groups of microorganisms are still controversial. We have studied the reliability of MALDI-TOF MS for the identification of anaerobic clinical isolates was studied compared to conventional biochemical methods, with rRNA 16S sequencing being used as a reference when discrepancies arose. Material and methodsA total of 126 anaerobic bacteria clinical isolates were studied by using API20A kits (bioMérieux, Marcy l’Étoile, France) and MALDI-TOF MS (Autoflex II, Bruker Daltonics, Germany), and using the data library BioTyper 2.0 (Bruker Daltonics, Germany). When discrepancies arose, or MALDI-TOF MS was not able to identify any microorganism, rRNA 16S sequencing was used as the reference standard. ResultsThe biochemical method and MALDI-TOF MS agreed in identifying 60.9% of isolates at species level, and 20.3% of isolates at genus level. Among the 48 discrepancies observed, rRNA 16S sequencing supported MALDI-TOF MS identification, at species level, in 32 isolates (66.7%), and in 8 isolates (16.7%) at genus level. rRNA 16S sequencing supported biochemical identification in only two isolates (4.2%) at species level, and in 26 isolates (54.2%) at genus level. The eight isolates for which MALDI-TOF MS did not manage to identify, or the identification obtained was rejected by sequencing, belonged to species that are still not added to the BioTyper II data library. ConclusionsResults obtained in this study show that, overall, MALDI-TOF MS identification of anaerobic bacteria is more reliable than identification obtained by conventional biochemical methods (24% more correct identifications at species level). The number of major errors (incorrect identification at the genus level) is also 2.5-times lower. Moreover, all the major errors obtained by MALDI-TOF MS were due to the absence of some species in the data library. Thus, when data libraries are more complete, reliability differences between both methods will probably be even higher.

Bilan de l’utilisation en routine de la spectrométrie de masse dans un laboratoire hospitalier de microbiologie

Objectives Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories for the identification of microorganisms. The mass fingerprint obtained by analyzing the ribosomal proteins and proteins associated with membranes is compared to spectra and superspectra of database. Microbiological Laboratory of Mulhouse acquired this system 1 year ago. Method The MALDI-TOF MS analyses of all strains were performed on Axima ® (Shimadzu), data analysis with Saramis ® software (Anagnostec) and integration with SirWeb ® software (I2A society). Results Rapidly, laboratory technicians and biologists are able to turn the system after a few days of training. The data prospectively gathered in the present study demonstrated that MALDI-TOF mass spectrometry identification is an efficient method included fastidious bacteria. Exactly 98.8 % of 10,000 isolates were identified in genus and species levels. Use of identification techniques was seldom necessary for a few clinical relevant isolates. The database is in constant evolution. The superspectra contain characteristic signals of genus, species allowing reliable microorganism identifications with high confidence. Conclusion In conclusion, our results show that MALDI-TOF MS is a fast and reliable technique for identification of most bacterial strains routinely isolated in a clinical microbiology. Identification and susceptibility testing with a single cell colony are more often possible. The simple tests, such as Gram staining and oxidase and catalase tests are usually performed in our laboratory.

Association of the POLG1 T(−365)C, ANT1 G(−25)A, and PEO1 G(−605)T polymorphisms with diabetic polyneuropathy in patients with Type 1 diabetes mellitus

Modern proteomic techniques make it possible to identify numerous changes in protein expression in tumor in comparison to normal tissues. Despite the wide application of proteomics in current studies, identification of proteins with stable concentration differences in normal and cancer cells remains rather difficult. The current study was directed to the search of new potential protein colorectal cancer markers using comparative proteomics of protein extracts obtained from primary tumors and adjacent normal tissues. This widespread neoplasm is characterized by lack of evident symptoms at early stages of cancerogenesis. It is highly important to develop fast and sensitive methods of molecular diagnostics. We studied paired cancerous and normal clinical tissue samples from 11 patients with colorectal adenocarcinomas by comparative 2-D PAGE and MALDI-TOF mass-spectrometry identification. Sixteen proteins with stable differential expression were selected and identified, including 13 overexpressed and 3 downregulated proteins. In summary, we describe the discovery overexpression of GPD1 and RRBP1 proteins and lack of expression for HNRNPH1 and SERPINB6 proteins which are new candidate biomarkers of colon cancer.

Presence of hemocyanin with diphenoloxidase activity in deepwater pink shrimp ( Parapenaeus longirostris) post mortem

Polyphenoloxidase and hemocyanin are two proteins which although very similar perform different physiological functions in crustaceans. This paper reports the presence of hemocyanin with diphenoloxidase activity in deepwater pink shrimp ( Parapenaeus longirostris) post mortem. Polyacrylamide gels and specific inhibitors and substrates of mono- and diphenoloxidases were used for purposes of recognition, and MALDI-TOF mass spectrometry for identification. Presumptive polyphenoloxidase was found in an inactive form in cephalothorax following capture, subsequently becoming active during storage. Also in the course of storage, hemocyanin acquired the ability to oxidize diphenols. Ascorbic acid, sodium metabisulphite and tropolone inhibited the prooxidant activity of both presumptive polyphenoloxidase and hemocyanin in the gels. 4-Hexylresorcinol did not avoid the appearance of activity bands in the gel corresponding with hemocyanin, maybe because 4-hexylresorcinol is described as slow-binding inhibitor. The acquired prooxidant activity of hemocyanin following capture is especially important because of the rapid development of melanosis in deepwater pink shrimp during storage.

Tracking cell signaling protein expression and phosphorylation by innovative proteomic solutions.

The most challenging and fruitful biomedical research endeavor of this decade will be the mapping of cell signaling systems and establishing their linkages to normal and disease-related processes. Amongst other things, the Human Genome Sequencing Project has greatly facilitated MALDI-TOF mass spectrometry identification of proteins that have been resolved by standard 2D gel electrophoresis. However, the low abundance of protein kinases and other signal transduction proteins has rendered their analyses particularly problematic without some means of purification and enrichment from cell and tissue lysates. Antibodies have been the most specific affinity probes for tracking target proteins, but their variable quality and high cost preclude their deployment in most discovery-based proteomics studies. Current multi-immunoblotting techniques can permit the probing of a single mini-SDS-PAGE gel with 50 or more antibodies at a time to monitor large changes in the expression and phosphorylation states of signaling proteins. The development of new affinity probes to replace antibodies is necessary to drive large scale proteomics studies. Such affinity probes could include short peptide antibody mimetics (PAM's) and oligonucleotide aptamers that when spotted in 2D array formats (e.g. membrane macroarrays, glass microarrays) or presented on specific beads (e.g. Luminex beads) can capture target proteins for their specific enrichment. The bound target proteins can then be detected using reporter antibodies or other specific probes for their quantitation by high throughput systems. These new proteomics methodologies will accelerate assessment of specific protein expression, post-translational modification, protein-protein interactions and protein-drug interactions to provide a more holistic view of cellular operations and how they might be manipulated under pathological circumstances.

Monitoring of genes that respond to overproduction of an insoluble recombinant protein inEscherichia coli glucose-limited fed-batch fermentations

The cellular response of Escherichia coli to overproduction of the insoluble heterologous protein alpha-glucosidase of Saccharomyces cerevisiae during a glucose-limited fed-batch fermentation was analyzed on the transcriptional and the translational levels. After the induction of the tac-regulated overexpression of the recombinant model protein, a significant but transient increase of the mRNA levels of the heat shock genes lon and dnaK could be observed. The mRNA level of the gene coding for the inclusion body-associated protein IbpB showed the strongest increase and remained at a clearly higher level until the end of the fermentation. By contrast, the mRNA levels of htrA and ppiB were decreased after induction of the alpha-glucosidase overexpression. Analysis of the soluble cytoplasmic protein fraction 3 h after induction revealed increased levels of the chaperones GroEL, DnaK, and Tig and a decrease in the protein levels of the two ribosomal proteins S6 and L9, the peptidylprolyl-cis-trans-isomerase PpiB, and the sigma(38)-dependent protein Dps. Analysis of the aggregated protein fraction revealed a remarkably inhomogeneous composition of the alpha-glucosidase inclusion bodies. N-terminal sequencing and MALDI-TOF mass spectrometry identification showed that most of these spots are fragments of the heterologous alpha-glucosidase. Host stress proteins, like DnaK, GroEL, IbpA, IbpB, and OmpT, have been found to be associated with the alpha-glucosidase protein aggregates.