Protein Biomarker Identification for the Discrimination of Brucella melitensis Field Isolates From the Brucella melitensis Rev.1 Vaccine Strain by MALDI-TOF MS

Brucella melitensis Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of Brucella isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates, we initially searched for unique marker proteins by in silico comparison of the B. melitensis Rev.1 and 16M proteomes. We found 113 protein sequences of B. melitensis 16M that revealed a homologous sequence in the B. melitensis Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 B. melitensis Rev.1 vaccine and 183 Israeli B. melitensis field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated B. melitensis Rev.1 from the closely related B. melitensis 16M and the Israeli B. melitensis field isolates. In addition, we verified their discriminatory power using a set of B. melitensis strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain B. melitensis isolates on strain level.

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Proteomic Study of Ribosomal Proteins from Escherichia coli, Saccharomyces cerevisiae, Bos taurus, Gallus gallus, and Oncorhynchus tshawytscha: Application in a Teaching Laboratory Setting

American Journal of Undergraduate Research ◽

10.33697/ajur.2014.004 ◽

2014 ◽

Vol 12 (1) ◽

Cited By ~ 1

Author(s):

Yoshihiro Miura ◽

Eric Yeager ◽

James MacKenzie ◽

Kestutis Bendinskas

Keyword(s):

Escherichia Coli ◽

Ribosomal Proteins ◽

Oncorhynchus Tshawytscha ◽

Bos Taurus ◽

Laboratory Setting ◽

Maldi Tof Ms ◽

Teaching Laboratory ◽

Maldi Tof ◽

Proteomic Study ◽

Tof Ms

Ribosomes are central to protein synthesis and our understanding of ribosomes has advanced antibiotics research. The proteomic study of ribosomes presented here utilizes a combination of differential centrifugation and matrix assisted laser desorption/ionization – time of flight mass spectrometry (MALDI-TOF MS) to analyze ribosomes from various species in a teaching laboratory setting. Five biologically varied species were used: Escherichia coli (bacteria), Saccharomyces cerevisiae (yeast), Bos taurus (cow), Gallus gallus (chicken), and Oncorhynchus tshawytscha (Chinook salmon). Samples were lysed, ribosomes were isolated via ultracentrifugation using a discontinuous sucrose gradient and the individual protein subunits were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis. Tryptic digest and MALDI-TOF MS were then conducted on fifteen bands excised from the gel, and the mass spectra of both the whole protein sample and peptides were analyzed. Five out of these fifteen bands were positively identified as various ribosomal proteins, with two uncertain identifications. Additionally, three of the five positively identified proteins that travelled the same distance on the gel were determined to be orthologous. Finally, a class of 14 Biochemistry II students utilized these protocols, identified 3 ribosomal proteins and provided their evaluations of the ultracentrifugation-proteomics teaching laboratory. Key Words: Proteomics, MALDI-TOF MS, ultracentrifugation, ribosomes, teaching laboratory

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Theoretical ribosomal protein mass distribution of Pseudomonas aeruginosa PAO1

10.7287/peerj.preprints.3500 ◽

2018 ◽

Author(s):

Wenfa Ng

Keyword(s):

Pseudomonas Aeruginosa ◽

Ribosomal Protein ◽

Ribosomal Proteins ◽

Small Subunit ◽

Structure And Function ◽

Maldi Tof Ms ◽

Maldi Tof ◽

And Function ◽

Unique Mass ◽

Tof Ms

Ribosomes are the protein synthesis factories of a cell and thus are evolutionary conserved in structure and function. Comprising a large and small subunit, the ribosome is further made up of ribosomal proteins that give structure and function to different parts of the macromolecular complex. Current methods for isolating the ribosome include density gradient ultracentrifugation that separates the ribosome into the large and small subunit. Separation of the various ribosomal proteins that comprise each of the subunit would require a solubilization step followed by the use of sodium dodecyl sulphate and polyacrylamide gel electrophoresis (SDS-PAGE). However, possibility exists for the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to profile the set of ribosomal proteins that could be solubilized from each ribosome subunit. Using ribosomal protein amino acid sequence information from Kyoto Encyclopaedia of Genes and Genomes (KEGG), the molecular weight of each ribosomal protein from Pseudomonas aeruginosa PAO1 was calculated in this report. Obtained results revealed that each ribosomal protein had a unique mass that could be detected by mid-range MALDI-TOF MS instruments. More importantly, the mass of ribosomal proteins constitutes a unique mass fingerprint of each ribosome subunit, which accounts for the different structure and functions of the large and small ribosome subunit. Overall, current mass resolution of MALDI-TOF MS instruments could resolve ribosomal proteins and thus provides a tool for profiling the set of ribosomal proteins that constitute the large and small subunit of the ribosome.

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Brucella melitensis Misidentified as Roseomonas gilardii by MALDI-TOF MS: Experience of a Clinical Microbiology Laboratory

Klimik Dergisi/Klimik Journal ◽

10.5152/kd.2019.24 ◽

2019 ◽

Vol 32 (1) ◽

pp. 105-107

Author(s):

Muharrem Cicek ◽

◽

Asiye Bicakcigil ◽

Bekir Celebi ◽

Burcin Sener ◽

...

Keyword(s):

Clinical Microbiology ◽

Brucella Melitensis ◽

Clinical Microbiology Laboratory ◽

Microbiology Laboratory ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Tof Ms

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Large-scale PRNP genotyping of small ruminants using an automated high-throughput MALDI-TOF MS assay

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638719859043 ◽

2019 ◽

Vol 31 (4) ◽

pp. 629-633

Author(s):

Sergio Migliore ◽

Maurizio Bivona ◽

Enrico Gagliostro ◽

Onofrio Buttitta ◽

Francesca Lo Mascolo ◽

...

Keyword(s):

High Throughput ◽

Large Scale ◽

Genetic Resistance ◽

Small Ruminants ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Automated Processing ◽

Tof Ms

Scrapie resistance or susceptibility in sheep is associated with single nucleotide polymorphisms (SNPs) at codons 136, 154, and 171 of the prion protein gene ( PRNP). In addition, phenylalanine mutation at codon 141 has been recognized as a risk factor for atypical scrapie. In contrast, K222, D146, and S146 alleles confer genetic resistance to classical scrapie in goats. High-throughput genotyping technologies would provide significant benefits in scrapie eradication plans. The ability to resolve oligonucleotides varying in mass by less than a single nucleotide makes MALDI-TOF mass spectrometry (MS) a suitable platform for PRNP genotyping. We evaluated the commercial Myriapod scrapie kit (Diatech Pharmacogenetics), associated with a highly automated processing platform incorporating MALDI-TOF MS technology, to detect SNPs at codons 136, 154, 171, 141, and 222 of small ruminant PRNP. The Myriapod scrapie kit was accredited according to UNI CEI EN ISO/IEC 17025. We present the genotyping results of 10,960 sheep in Sicily and 1,822 goats in Sicily and Calabria (southern Italy) tested during 2017. We found a high frequency (43.9%) of the protective ARR allele in sheep and a promising 12.3% of the resistant K222 variant in goats. This efficient and high-throughput method is suitable for extensive PRNP genotyping, as requested in the European scrapie eradication plan.

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Classification of the Genus Bacillus Based on MALDI-TOF MS Analysis of Ribosomal Proteins Coded inS10andspcOperons

Journal of Agricultural and Food Chemistry ◽

10.1021/jf2004095 ◽

2011 ◽

Vol 59 (10) ◽

pp. 5222-5230 ◽

Cited By ~ 32

Author(s):

Yudai Hotta ◽

Jun Sato ◽

Hiroaki Sato ◽

Akifumi Hosoda ◽

Hiroto Tamura

Keyword(s):

Ribosomal Proteins ◽

Genus Bacillus ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Ms Analysis ◽

Tof Ms

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APPLICATION OF TIME-OF-FLIGHT MASS-SPECTROMETRY FOR DETECTION OF CAUSATIVE AGENT OF BRUCELLOSIS IN BLOOD SAMPLES IN EXPERIMENT

Journal of microbiology epidemiology immunobiology ◽

10.36233/0372-9311-2017-4-9-17 ◽

2017 ◽

pp. 9-17

Author(s):

D. V. Ulshina ◽

D. A. Kovalev ◽

D. G. Ponomarenko ◽

D. V. Rusanova ◽

N. M. Shvetsova ◽

...

Keyword(s):

Mass Spectrometry ◽

Causative Agent ◽

Brucella Melitensis ◽

Time Of Flight ◽

Protein Profiling ◽

Blood Samples ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Tof Ms

Aim.Study the possibility to apply time-of-flight mass-spectrometry for detection of causative agent of brucellosis in blood. Materials and methods. Brucella strains: 5 Brucella melitensis and 21 Brucella abortus. Protein profiling in linear mode on MALD1-TOF mass-spectrometer Microflex «Bruker Daltonics». Results. Technique for disinfection and preparation of blood samples was modified and optimized for MALDI-TOF MS analysis. 120 representative protein profiles of sera extract were obtained that contain brucellosis causative agent. A resulting peak-list (super-spectrum) of the studied protein fraction of blood extract of a conditionally healthy human within the studied group was formed and analyzed. Conclusion. A scheme of brucella detection in blood samples by MALDI-TOF MS is proposed, based on detection of a complex of 15 genus-specific fragments. Signals on mass-spectra of extracts of leukocyte fraction of blood, artificially contaminated with brucellosis causative agents are characterized.

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