scholarly journals Application of Safirinium N-Hydroxysuccinimide Esters to Derivatization of Peptides for High-Resolution Mass Spectrometry, Tandem Mass Spectrometry, and Fluorescent Labeling of Bacterial Cells

2020 ◽  
Vol 21 (24) ◽  
pp. 9643
Author(s):  
Joanna Fedorowicz ◽  
Magdalena Wierzbicka ◽  
Marek Cebrat ◽  
Paulina Wiśniewska ◽  
Rafał Piątek ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution Mass Spectrometry ◽  
Fluorescent Labeling ◽  
Bacterial Cells ◽  
The Novel ◽  
Sequence Coverage ◽  
E Coli ◽  
High Ionization ◽  
Hydroxysuccinimide Esters ◽  
Amine Groups

Mass spectrometry methods are commonly used in the identification of peptides and biomarkers. Due to a relatively low abundance of proteins in biological samples, there is a need for the development of novel derivatization methods that would improve MS detection limits. Hence, novel fluorescent N–hydroxysuccinimide esters of dihydro-[1,2,4]triazolo[4,3-a]pyridin-2-ium carboxylates (Safirinium P dyes) have been synthesized. The obtained compounds, which incorporate quaternary ammonium salt moieties, easily react with aliphatic amine groups of peptides, both in solution and on the solid support; thus, they can be applied for derivatization as ionization enhancers. Safirinium tagging experiments with ubiquitin hydrolysate revealed that the sequence coverage level was high (ca. 80%), and intensities of signals were enhanced up to 8-fold, which proves the applicability of the proposed tags in the bottom–up approach. The obtained results confirmed that the novel compounds enable the detection of trace amounts of peptides, and fixed positive charge within the tags results in high ionization efficiency. Moreover, Safirinium NHS esters have been utilized as imaging agents for fluorescent labeling and the microscopic visualization of living cells such as E. coli Top10 bacterial strain.

scholarly journals Utilization of Mn-Doped ZnSe/ZnS Core/Shell Quantum Dots for Rapid Detection of Escherichia coli O157:H7 and Methicillin-Resistant Staphylococcus aureus

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Thi-Diem Bui ◽  
Quang-Liem Nguyen ◽  
Thi-Bich Luong ◽  
Van Thuan Le ◽  
Van-Dat Doan
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Quantum Dots ◽  
Fluorescent Labeling ◽  
Core Shell ◽  
Bacterial Cells ◽  
Bacterial Strains ◽  
Methicillin Resistant ◽  
E Coli ◽  
Mn Doped

In this study, Mn-doped ZnSe/ZnS core/shell quantum dots (CSQDs) were synthesized in aqueous solution using polyethylene glycol as a surface stabilizer and successfully applied in the detection of Escherichia coli O157:H7 and methicillin-resistant Staphylococcus aureus (MRSA) for the first time. The CSQDs were conjugated with anti-E. coli antibody and anti-MRSA antibody via protein A supported by 1-ethyl-3-(-3-dimethylaminopropyl)carbodiimide hydrochloride for fluorescent labeling of the intact bacterial cells. The detection was performed for the bacterial strains cultivated in Luria-Bertani liquid medium. The obtained results indicate that E. coli O157:H7 and MRSA can be detected within 30 min at a high sensitivity of 101 CFU/mL. This labeling method based on the highly fluorescent CSQDs may have great potential for use in the food industry to check and prevent outbreaks of foodborne illness.

scholarly journals Old concepts, new molecules and current approaches applied to the bacterial nucleotide signalling field

2016 ◽  
Vol 371 (1707) ◽  
pp. 20150503 ◽  
Author(s):  
Angelika Gründling ◽  
Vincent T. Lee
Keyword(s):  
Mass Spectrometry ◽  
Rapid Identification ◽  
Screening Methods ◽  
Bacterial Cells ◽  
The Novel ◽  
Specific Receptor ◽  
Receptor Proteins ◽  
The Past ◽  
Genome Wide ◽  
Cellular Pathways

Signalling nucleotides are key molecules that help bacteria to rapidly coordinate cellular pathways and adapt to changes in their environment. During the past 10 years, the nucleotide signalling field has seen much excitement, as several new signalling nucleotides have been discovered in both eukaryotic and bacterial cells. The fields have since advanced quickly, aided by the development of important tools such as the synthesis of modified nucleotides, which, combined with sensitive mass spectrometry methods, allowed for the rapid identification of specific receptor proteins along with other novel genome-wide screening methods. In this review, we describe the principle concepts of nucleotide signalling networks and summarize the recent work that led to the discovery of the novel signalling nucleotides. We also highlight current approaches applied to the research in the field as well as resources and methodological advances aiding in a rapid identification of nucleotide-specific receptor proteins. This article is part of the themed issue ‘The new bacteriology’.

scholarly journals Metabolomics Study on Pathogenic and Non-pathogenic E. coli with Closely Related Genomes with a Focus on Yersiniabactin and Its Known and Novel Derivatives

Metabolites ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 221
Author(s):  
Mareike Schulz ◽  
Vasiliki Gaitanoglou ◽  
Olena Mantel ◽  
Yannick Hövelmann ◽  
Florian Hübner ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Liquid Chromatography ◽  
Culture Supernatant ◽  
High Resolution Mass Spectrometry ◽  
Minimum Essential Medium ◽  
E Coli ◽  
Metabolomic Data ◽  
Metabolomics Study ◽  
Resolution Mass

The Escherichia coli (E. coli) strains Nissle 1917 (EcN), 83972 and CFT073 are closely related but differ in their phenotypes and pathogenicity. The aim of this study was to compare the metabolome of these strains based on metabolomic data analysis of bacterial samples using liquid chromatography-high resolution mass spectrometry (LC-HRMS). The strains were cultivated in minimum essential medium at 37 °C for 6 h. The sterilized culture supernatant was analyzed, followed by data processing to create feature lists, and statistical analysis to identify discriminating features in the metabolomes of the three strains. Metabolites were identified using the exact masses, isotope patterns, and fragmentation spectra. The results showed that the metabolome of EcN differs significantly from the metabolomes of E. coli 83972 and CFT073. Based on the analysis, yersiniabactin (Ybt), its metal complexes, and its known structural derivatives escherichelin and ulbactin B were identified as discriminating features; the latter has not been described for E. coli before. Additionally, novel Ytb derivatives were found and tentatively identified by LC-MS/HRMS. All these metabolites were determined in significantly higher levels in the metabolome of EcN compared to E. coli 83972, which may explain a large part of the observed differences of the metabolomes.

scholarly journals Mass Spectrometry–Based Escherichia coli H Antigen/Flagella Typing: Validation and Comparison with Traditional Serotyping

2016 ◽  
Vol 62 (6) ◽  
pp. 839-847 ◽  
Author(s):  
Keding Cheng ◽  
Yi-Min She ◽  
Huixia Chui ◽  
Larissa Domish ◽  
Angela Sloan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Escherichia Coli ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Close Correlation ◽  
Clinical Isolates ◽  
Whole Genome ◽  
Sequence Coverage ◽  
E Coli ◽  
Reference Strains

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.

scholarly journals Accurate Detection of the Four Most Prevalent Carbapenemases in E. coli and K. pneumoniae by High-Resolution Mass Spectrometry

2019 ◽  
Vol 10 ◽  
Author(s):  
Dimard E. Foudraine ◽  
Lennard J. M. Dekker ◽  
Nikolaos Strepis ◽  
Michiel L. Bexkens ◽  
Corné H. W. Klaassen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution ◽  
High Resolution Mass Spectrometry ◽  
E Coli ◽  
Accurate Detection ◽  
High Resolution Mass ◽  
Resolution Mass

scholarly journals The sequence coverage in different methods of mass spectrometry data analysis obtained on model proteins

2017 ◽  
Vol 63 (5) ◽  
pp. 397-404 ◽  
Author(s):  
A.V. Mikurova ◽  
S.E. Novikova ◽  
V.S. Skvortsov ◽  
N.N. Alekseychuk ◽  
A.V. Rybina ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Data Analysis ◽  
Search Engines ◽  
De Novo ◽  
High Resolution Mass Spectrometry ◽  
De Novo Sequencing ◽  
Mass Spectrometry Data ◽  
Sequence Coverage ◽  
Proteomic Data ◽  
A Charge

The aim of this study was to evaluate sequence coverage of five model proteins (CYB5A, SMAD4, RAB27B, FECH, and CXXC1) by means of shotgun proteomic data analysis employing different methods of data treatment including database-dependent search engines (MASCOT and X!Tandem) and de novo sequencing software ((PEAKS, Novor, and PepNovo+). In order to achieve maximal results, multiprotease hydrolysis including enzymes trypsin, LYS-C, ASPN and GluC was performed in solution and using the FASP method. High resolution mass spectrometry was carried out with a Q EXACTIVE HF hybrid mass spectrometer in the positive ionization mode; parent ions with the highest intensity and a charge range from +2 to +6 were fragmented in the HCD mode. 27 experiments were carried out (hydrolysis with each of 5 enzymes in solution, 4 for the FASP protocol, three technical repeats). Using parameters limiting false identification of peptides, the search engines and de novo sequencing software gave similar results. The degree of sequence coverage was not at least 40%, and in the best cases it reached 80-90%. The use of de novo sequencing software resulted in identification of the Y12H amino acid substitution in one model protein (CYB5A).

scholarly journals AI-based identification of grain cultivars via non-target mass spectrometry

2020 ◽  
Author(s):  
Kapil Nichani ◽  
Steffen Uhlig ◽  
Bertrand Colson ◽  
Karina Hettwer ◽  
Kirsten Simon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Resolution Mass Spectrometry ◽  
The Novel ◽  
Food Fraud ◽  
Government Institutions ◽  
Data Independent Acquisition ◽  
Target Mass ◽  
Sample Measurement ◽  
Geographical Traceability

ABSTRACTDetection of food fraud and geographical traceability of ingredients is a continually sought goal for government institutions, producers, and consumers. Herein we explore the use of non-target high-resolution mass spectrometry approaches and demonstrate its utility through a particularly challenging case study – to distinguish wheat and spelt cultivars. By employing a data-independent acquisition (DIA) approach for sample measurement, the spectra are of considerable size and complexity. We utilize artificial intelligence (AI) algorithms (artificial neural networks) to evaluate the extensive proteomic footprint of several wheat and spelt cultivars. The AI model thus obtained is used to classify newer varieties of spelt, processed flour, and bread samples. Additionally, we discuss the validation of such a method coupling DIA and AI approaches. The novel framework for method validation enables calculation of precision parameters for facile comparison of the discriminatory power of the method and in the development of a reliable decision rule.

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