scholarly journals Genus, Species, and Subspecies Classification of Salmonella Isolates by Proteomics

2021 ◽  
Vol 11 (9) ◽  
pp. 4264
Author(s):  
Shu-Hua Chen ◽  
Christine H. Parker ◽  
Timothy R. Croley ◽  
Melinda A. McFarland

Identification of bacteria by mass spectrometry offers the potential of a high-throughput non-targeted method to determine the presence of Salmonella. While MALDI-TOF mass spectrometry can identify Salmonella at the genus and species level, few studies have reported subtyping beyond the species level due to the diversity and complexity of Salmonella that includes more than 2600 serovars. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) approaches enable profiling of a greater number of proteins over a larger dynamic range and offer the potential to detect small differences between closely related isolates. We evaluate the discriminatory power of bottom-up LC-MS/MS with a collection of nineteen isolates that differ at the genus, species, subspecies, or strain level. Isolates were classified by matching the sequence of identified peptides to reference proteomes translated from genomes with known taxonomic ranks. The degree of proteomic similarity between the tested isolates and reference strains correlated with how closely they were related. All tested Salmonella isolates were easily distinguished from their close relatives, E. coli and Shigella, and readily grouped by species and subspecies. Additionally, each Salmonella isolate most closely matched to its correct serovar. This approach presents a simple and effective proteomic approach to identification of Salmonella genus, species, and subspecies.

2010 ◽  
Vol 76 (11) ◽  
pp. 3637-3644 ◽  
Author(s):  
Rabih E. Jabbour ◽  
Samir V. Deshpande ◽  
Mary Margaret Wade ◽  
Michael F. Stanford ◽  
Charles H. Wick ◽  
...  

ABSTRACT Due to the possibility of a biothreat attack on civilian or military installations, a need exists for technologies that can detect and accurately identify pathogens in a near-real-time approach. One technology potentially capable of meeting these needs is a high-throughput mass spectrometry (MS)-based proteomic approach. This approach utilizes the knowledge of amino acid sequences of peptides derived from the proteolysis of proteins as a basis for reliable bacterial identification. To evaluate this approach, the tryptic digest peptides generated from double-blind biological samples containing either a single bacterium or a mixture of bacteria were analyzed using liquid chromatography-tandem mass spectrometry. Bioinformatic tools that provide bacterial classification were used to evaluate the proteomic approach. Results showed that bacteria in all of the double-blind samples were accurately identified with no false-positive assignment. The MS proteomic approach showed strain-level discrimination for the various bacteria employed. The approach also characterized double-blind bacterial samples to the respective genus, species, and strain levels when the experimental organism was not in the database due to its genome not having been sequenced. One experimental sample did not have its genome sequenced, and the peptide experimental record was added to the virtual bacterial proteome database. A replicate analysis identified the sample to the peptide experimental record stored in the database. The MS proteomic approach proved capable of identifying and classifying organisms within a microbial mixture.


2009 ◽  
Vol 4 (12) ◽  
pp. 1934578X0900401 ◽  
Author(s):  
Lourdes Campaner dos Santos ◽  
Marcelo Aparecido da Silva ◽  
Clenilson Martins Rodrigues ◽  
Virginia Carbone ◽  
Assunta Napolitano ◽  
...  

Liquid chromatography-electrospray ionization multistage ion trap mass spectrometry (LC-ESI-IT-MSn) was used to analyze the secondary metabolites in the methanol extract of the capitulae of Eriocaulon ligulatum. The major components were mono-and diglycosides of flavonoids and naphthopyranones. Eleven compounds, including four new flavonol glycosides, were identified based on their fragmentation patterns in MS experiments and on NMR analysis of the isolated compounds. The described data may contribute to a better understanding of the taxonomic classification of the Eriocaulaceae family.


Author(s):  
Dimard E. Foudraine ◽  
Nikolaos Strepis ◽  
Corné H. W. Klaassen ◽  
Merel N. Raaphorst ◽  
Annelies Verbon ◽  
...  

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in the curbing of drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside modifying enzymes and 16S ribosomal RNA methyltransferases in E. coli and K. pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6’)-Ib, AAC(6’)-Ib-cr, ANT(2”)-I, APH(3’)-VI, ArmA, RmtB, RmtC and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole genome sequencing results. Of the 2460 isolate and resistance mechanism combinations tested, 2416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin and amikacin in only the E. coli and K. pneumoniae isolates (n=191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.


PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e4599 ◽  
Author(s):  
Agnieszka Hareza ◽  
Magda Bakun ◽  
Bianka Świderska ◽  
Małgorzata Dudkiewicz ◽  
Alicja Koscielny ◽  
...  

Many kinases are still ‘orphans,’ which means knowledge about their substrates, and often also about the processes they regulate, is lacking. Here, DIA1/C3orf58, a member of a novel predicted kinase-like family, is shown to be present in the endoplasmic reticulum and to influence trafficking via the secretory pathway. Subsequently, DIA1 is subjected to phosphoproteomics analysis to cast light on its signalling pathways. A liquid chromatography–tandem mass spectrometry proteomic approach with phosphopeptide enrichment is applied to membrane fractions of DIA1-overexpressing and control HEK293T cells, and phosphosites dependent on the presence of DIA1 are elucidated. Most of these phosphosites belonged to CK2- and proline-directed kinase types. In parallel, the proteomics of proteins immunoprecipitated with DIA1 reported its probable interactors. This pilot study provides the basis for deeper studies of DIA1 signalling.


2016 ◽  
Vol 62 (6) ◽  
pp. 839-847 ◽  
Author(s):  
Keding Cheng ◽  
Yi-Min She ◽  
Huixia Chui ◽  
Larissa Domish ◽  
Angela Sloan ◽  
...  

Abstract BACKGROUND Escherichia coli H antigen typing with antisera, a useful method for flagella clinical identification and classification, is a time-consuming process because of the need to induce flagella growth and the occurrence of undetermined strains. We developed an alternative rapid and analytically sensitive mass spectrometry (MS) method, termed MS-based H antigen typing (MS-H), and applied it at the protein sequence level for H antigen typing. We also performed a comparison with traditional serotyping on reference strains and clinical isolates. METHODS On the basis of international guidelines, the analytical selectivity and sensitivity, imprecision, correlation, repeatability, and reproducibility of the MS-H platform was evaluated using reference strains. Comparison of MS-H typing and serotyping was performed using 302 clinical isolates from 5 Canadian provinces, and discrepant results between the 2 platforms were resolved through whole genome sequencing. RESULTS Repeated tests on reference strain EDL933 demonstrated a lower limit of the measuring interval at the subsingle colony (16.97 μg or 1.465 × 107 cells) level and close correlation (r2 > 0.99) between cell culture biomass and sequence coverage. The CV was <10.0% among multiple repeats with 4 reference strains. Intra- and interlaboratory tests demonstrated that the MS-H method was robust and reproducible under various sample preparation and instrumentation conditions. Using discrepancy analysis via whole genome sequencing, performed on isolates with discrepant results, MS-H accurately identified 12.3% more isolates than conventional serotyping. CONCLUSIONS MS-H typing of E. coli is useful for fast and accurate flagella typing and could be very useful during E. coli outbreaks.


2021 ◽  
Vol 105 (18) ◽  
pp. 6819-6833
Author(s):  
Wenbin Wang ◽  
Xinyue Zhou ◽  
Yunong Sang ◽  
Xiaxia Liang ◽  
Jianxin Liu ◽  
...  

Abstract The goal of this work was to identify the target protein and epitope of a previously reported Escherichia coli O157:H7 (ECO157)–specific monoclonal antibody (mAb) 2G12. mAb 2G12 has shown high specificity for the recovery and detection of ECO157. To achieve this goal, the target protein was first separated by two-dimensional gel electrophoresis (2-DE) and located by Western blot (WB). The protein spots were identified to be the outer membrane protein (Omp) C by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF–MS). After that, the target protein was purified by immunoaffinity chromatography (IAC) and subjected to in situ enzymatic cleavage of the vulnerable peptides. Eight eluted peptides of OmpC identified by liquid chromatography–tandem mass spectrometry (LC–MS/MS) were further mapped onto the homologous protein structure of E. coli OmpC (2IXX). The topology of OmpC showed that three peptides had extracellular loops. Epitope mapping with overlapping peptide library and sequence homology analysis revealed that the epitope consisted of a specific peptide, “LGVING,” and an adjacent conservative peptide, “TQTYNATRVGSLG.” Both peptides loop around the overall structure of the epitope. To test the availability of the epitope when ECO157 was grown under different osmolarity, pH, and nutrition levels, the binding efficacy of mAb 2G12 with ECO157 grown in these conditions was evaluated. Results further demonstrated the good stability of this epitope under potential stressful environmental conditions. In summary, this study revealed that mAb 2G12 targeted one specific and one conservative extracellular loop (peptide) of the OmpC present on ECO157, and the epitope was stable and accessible on ECO157 cells grown in different environment. Key points • OmpC is the target of a recently identified ECO157-specific mAb 2G12. • Eight peptides were identified from the OmpC by using LC–MS/MS. • The specificity of mAb 2G12 is mainly determined by the “LGVING” peptide.


1999 ◽  
Vol 37 (7) ◽  
pp. 2255-2261 ◽  
Author(s):  
Val Hall ◽  
G. L. O’Neill ◽  
J. T. Magee ◽  
B. I. Duerden

Identification of Actinomyces spp. by conventional phenotypic methods is notoriously difficult and unreliable. Recently, the application of chemotaxonomic and molecular methods has clarified the taxonomy of the group and has led to the recognition of several new species. A practical and discriminatory identification method is now needed for routine identification of clinical isolates. Amplified 16S ribosomal DNA restriction analysis (ARDRA) was applied to reference strains (n = 27) and clinical isolates (n = 36) of Actinomyces spp. and other gram-positive rods. Clinical strains were identified initially to the species level by conventional biochemical tests. However, given the low degree of confidence in conventional methods, the findings obtained by ARDRA were also compared with those obtained by pyrolysis-mass spectrometry. The ARDRA profiles generated by the combination ofHaeIII and HpaII endonuclease digestion differentiated all reference strains to the species or subspecies level. The profiles correlated well with the findings obtained by pyrolysis-mass spectrometry and by conventional tests and enabled the identification of 31 of 36 clinical isolates to the species level. ARDRA was shown to be a simple, rapid, cost-effective, and highly discriminatory method for routine identification ofActinomyces spp. of clinical origin.


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