Most cited article - PubMed ID 29495560
Proteomic Methods of Detection and Quantification of Protein Toxins
Sample preparation involving the cleavage of proteins into peptides is the first critical step for successful bottom-up proteomics and protein analyses. Time- and labor-intensiveness are among the bottlenecks of the commonly used methods for protein sample preparation. Here, we report a fast online method for postinjection acid cleavage of proteins directly in the mobile phase typically used for LC-MS analyses in proteomics. The chemical cleavage is achieved in 0.1% formic acid within 35 s in a capillary heated to 195 °C installed upstream of the analytical column, enabling the generated peptides to be separated. The peptides generated by the optimized method covered the entire sequence except for one amino acid of trastuzumab used for the method development. The qualitative results are extraordinarily stable, even over a long period of time. Moreover, the method is also suitable for accurate and repeatable quantification. The procedure requires only one manual step, significantly decreasing sample transfer losses. To demonstrate its practical utility, we tested the method for the fast detection of ricin. Ricin can be unambiguously identified from an injection of 10 ng, and the results can be obtained within 7-8 min after receiving a suspicious sample. Because no sophisticated accessories and no additional reagents are needed, the method can be seamlessly transferred to any laboratory for high-throughput proteomic workflows.
In the last two decades, microbiology laboratories have radically changed by the introduction of novel technologies, like Next-Generation Sequencing (NGS) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Nevertheless, emergence of antibiotic-resistant microorganisms represents a global threat of current medicine, being responsible for increasing mortality and health-care direct and indirect costs. In addition, the identification of antibiotic-resistant microorganisms, like OXA-48 carbapenemase-producing Enterobacteriaceae, has been changeling for clinical microbiology laboratories. Even the cost of NGS technology and MALDI-TOF MS equipment is relatively high, both technologies are increasingly used in diagnostic and research protocols. Therefore, the aim of this review is to present applications of these technologies used in clinical microbiology, especially in detection of antibiotic resistance and its surveillance, and to propose a combinatory approach of MALDI-TOF MS and NGS for the investigation of microbial associated infections.
- Keywords
- MLST, NGS, beta-lactamase, carbapenemase, susceptibility testing,
- MeSH
- Anti-Bacterial Agents pharmacology MeSH
- Bacteria classification drug effects genetics isolation & purification MeSH
- Bacterial Infections diagnosis microbiology MeSH
- Drug Resistance, Bacterial * MeSH
- Mass Spectrometry methods MeSH
- Laboratories, Hospital MeSH
- Humans MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
- Names of Substances
- Anti-Bacterial Agents MeSH
Targeted proteomics recently proved to be a technique for the detection and absolute quantification of proteins not easily accessible to classical bottom-up approaches. Due to this, it has been considered as a high fidelity tool to detect potential warfare agents in wide spread kinds of biological and environmental matrices. Clostridium perfringens toxins are considered to be potential biological weapons, especially the epsilon toxin which belongs to a group of the most powerful bacterial toxins. Here, the development of a target mass spectrometry method for the detection of C. perfringens protein toxins (alpha, beta, beta2, epsilon, iota) is described. A high-resolution mass spectrometer with a quadrupole-Orbitrap system operating in target acquisition mode (parallel reaction monitoring) was utilized. Because of the lack of commercial protein toxin standards recombinant toxins were prepared within Escherichia coli. The analysis was performed using proteotypic peptides as the target compounds together with their isotopically labeled synthetic analogues as internal standards. Calibration curves were calculated for each peptide in concentrations ranging from 0.635 to 1101 fmol/μL. Limits of detection and quantification were determined for each peptide in blank matrices.
- Keywords
- Clostridium perfringens, PRM, epsilon toxin, mass spectrometry, protein toxin,
- MeSH
- Bacterial Proteins analysis genetics MeSH
- Bacterial Toxins analysis genetics MeSH
- Chromatography, Liquid MeSH
- Clostridium perfringens * genetics growth & development metabolism MeSH
- Escherichia coli genetics MeSH
- Peptides analysis genetics MeSH
- Proteomics MeSH
- Recombinant Proteins analysis MeSH
- Tandem Mass Spectrometry MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Bacterial Proteins MeSH
- Bacterial Toxins MeSH
- Peptides MeSH
- Recombinant Proteins MeSH
