scholarly journals A novel mechanism of “metal gel-shift” by histidine-rich Ni2+-binding Hpn protein from Helicobacter pylori strain SS1

2016 ◽  
Author(s):  
Rahul Mahadev Shelake ◽  
Yuki Ito ◽  
Junya Masumoto ◽  
Eugene Hayato Morita ◽  
Hidenori Hayashi
Keyword(s):  
Helicobacter Pylori ◽  
Metal Binding ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Metal Species ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Gel Shift ◽  
Tof Ms

AbstractSodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is a universally used method for determining approximate molecular weight (MW) in protein research. Migration of protein that does not correlate with formula MW, termed “gel shifting” appears to be common for histidine-rich proteins but not yet studied in detail. We investigated “gel shifting” in Ni2+-binding histidine-rich Hpn protein cloned from Helicobacter pylori strain SS1. Our data demonstrate two important factors determining “gel shifting” of Hpn, polyacrylamide-gel concentration and metal binding. Higher polyacrylamide-gel concentrations resulted in faster Hpn migration. Irrespective of polyacrylamide-gel concentration, preserved Hpn-Ni2+ complex migrated faster (3-4 kDa) than apo-Hpn, phenomenon termed “metal gel-shift” demonstrating an intimate link between Ni2+ binding and “gel shifting”. To examine this discrepancy, eluted samples from corresponding spots on SDS-gel were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS). The MW of all samples was the same (6945.66±0.34 Da) and identical to formula MW with or without added mass of Ni2+. MALDI-TOF-MS of Ni2+-treated Hpn revealed that monomer bound up to six Ni2+ ions non-cooperatively, and equilibrium between protein-metal species was reliant on Ni2+ availability. This corroborates with gradually increased heterogeneity of apo-Hpn band followed by compact "metal-gel shift" band on SDS-PAGE. In view of presented data metal-binding and “metal-gel shift” models are discussed.

scholarly journals A New Method for Extraction and Analysis of Ricin Samples through MALDI-TOF-MS/MS

Toxins ◽  
2019 ◽  
Vol 11 (4) ◽  
pp. 201 ◽  
Author(s):  
Roberto B. Sousa ◽  
Keila S. C. Lima ◽  
Caleb G. M. Santos ◽  
Tanos C. C. França ◽  
Eugenie Nepovimova ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Sequences ◽  
Intact Protein ◽  
Toxic Activity ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Flight Mass Spectrometry ◽  
Sds Page ◽  
Tof Ms

We report for the first time the efficient use of accelerated solvent extraction (ASE) for extraction of ricin to analytical purposes, followed by the combined use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and MALDI-TOF MS/MS method. That has provided a fast and unambiguous method of ricin identification for in real cases of forensic investigation of suspected samples. Additionally, MALDI-TOF MS was applied to characterize the presence and the toxic activity of ricin in irradiated samples. Samples containing ricin were subjected to ASE, irradiated with different dosages of gamma radiation, and analyzed by MALDI-TOF MS/MS for verification of the intact protein signal. For identification purposes, samples were previously subjected to SDS-PAGE, for purification and separation of the chains, followed by digestion with trypsin, and analysis by MALDI-TOF MS/MS. The results were confirmed by verification of the amino acid sequences of some selected peptides by MALDI-TOF MS/MS. The samples residual toxic activity was evaluated through incubation with a DNA substrate, to simulate the attack by ricin, followed by MALDI-TOF MS/MS analyses.

Electroelution of Intact Proteins from SDS-PAGE Gels and Their Subsequent MALDI-TOF MS Analysis

2006 ◽  
pp. 353-364 ◽  
Author(s):  
Zhentian Lei ◽  
Ajith Anand ◽  
Kirankumar S. Mysore ◽  
Lloyd W. Sumner
Keyword(s):  
Maldi Tof Ms ◽  
Intact Proteins ◽  
Maldi Tof ◽  
Ms Analysis ◽  
Sds Page ◽  
Tof Ms

S1. A modified method for blood culture direct testing using 2% SDS detergent and heat. v1

2021 ◽  
Author(s):  
kwenrich not provided
Keyword(s):  
Blood Culture ◽  
Clinical Laboratory ◽  
Sodium Dodecyl ◽  
Agar Plate ◽  
Drying Methods ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Modified Method ◽  
Flight Mass Spectrometry ◽  
Tof Ms

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can accurately identify bloodstream pathogens directly from positive blood culture bottles without the need to wait for agar plate growth. In this study, 2% sodium dodecyl sulfate (SDS) detergent was assessed to determine its benefit in the removal of interfering cellular components for testing on the Bruker Microflex LT MALDI-TOF MS instrument with the Biotyper® CA system. Additionally, the use of a heat-drying step was evaluated for performance improvement over conventional air-drying of samples on the MALDI steel target plate. The modified method with 2% SDS outperformed the in-house protocol in overall success with percentage scores of 91% and 55% ( respectively). The data results support the potential of applying a simple lysing step to an existing in-house extraction method and the use of modified drying methods. The modified techniques evaluated in this study proved beneficial for identifying most blood culture pathogens encountered in the clinical laboratory, and they can allow for reduced turnaround times and more appropriate antibiotic treatments.

scholarly journals 2155. Accelerated Confirmation of Porin Loss in Carbapenem-Resistant Enterobacterales: A MALDI-TOF Mass Spectrometry-Based Approach

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S731-S731
Author(s):  
Carlos Correa-Martinez ◽  
Evgeny A Idelevich ◽  
Karsten Becker
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Gold Standard ◽  
Sodium Dodecylsulfate ◽  
Resistant Bacteria ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Carbapenem Resistant ◽  
Sds Page ◽  
Tof Ms

Abstract Background The accurate identification of carbapenem resistance mechanisms is decisive for the appropriate selection of antibiotic regimens. Numerous methods can detect carbapenemase-producing carbapenem-resistant bacteria (CPCR). However, non-CPCR (NCPCR) are routinely assumed to display porin loss as a diagnosis of exclusion. No further confirmatory tests are performed since the gold standard (sodium dodecylsulfate polyacrylamide gel electrophoresis, SDS–PAGE) is laborious and time consuming. We propose a test for rapid and easy detection of porin loss by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Methods Clinical meropenem-resistant Enterobacterales strains (10 CPCR, 10 NCPCR) and control strains recommended by EUCAST (5 carbapenemase-producing, one with porin loss, one-negative control) were analyzed. Membrane proteins were extracted by successive centrifugation of bacterial suspensions (McFarland 0.5) and addition of ethanol, formic acid and acetonitrile. MALDI-TOF MS of the protein extracts was performed on a 96-spot target (Bruker Daltonics, Germany). Peaks between 35 and 40 kDa were analyzed for the presence of porins and compared with the bands observed in the SDS–PAGE of the protein extracts. Results Within the molecular weight range of 35–40 kDa, the MALDI-TOF MS-based method revealed peaks in all CPCR isolates corresponding to those observed in the carbapenemase-producing control strains. In contrast, the control strain with porin loss as well as all CNCR isolates showed a lower quantity of peaks in this range. All peaks observed correlated with the bands observed in the SDS–PAGE of the protein extracts at the corresponding molecular weight (Figure 1). Conclusion Yielding results that reliably correspond to the current gold standard, we propose a method for accelerated detection of porin loss as an alternative to the diagnosis of exclusion usually made in routine settings. With a processing time of approximately 20 minutes, the method can be easily implemented in the clinical setting. Applying this MALDI-TOF MS-based approach, valuable information will be provided about a resistance mechanism that otherwise remains unexplained. Disclosures All authors: No reported disclosures.

Molecular characterisation of high molecular weight glutenin allele Glu-B1h encoding 1Bx14+1By15 subunits in bread wheat (Triticum aestivum L.)

2014 ◽  
Vol 65 (3) ◽  
pp. 215 ◽  
Author(s):  
Lele Xiao ◽  
Ke Wang ◽  
Yanlin Liu ◽  
Xingguo Ye ◽  
Wujun Ma ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Liquid Chromatography ◽  
High Molecular Weight ◽  
Bread Wheat ◽  
Sodium Dodecyl ◽  
Reversed Phase ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

In this study, the authentic high molecular weight glutenin (HMW-GS) allele Glu-B1 h encoding for subunits 1Bx14 and 1By15 from German bread wheat cultivars Hanno and Imbros was identified and cross-verified by a suite of established protein analysis technologies, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography, reversed-phase ultra-performance liquid chromatography, and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). The complete encoding sequences were isolated by allele-specific PCR, and consist of 2367 bp for 1Bx14 and 2151 bp for 1By15 and encode 789 and 717 amino acid residues, respectively. The deduced molecular masses of two subunit genes were 82 340.13 Da and 74 736.13 Da, corresponding well to those determined by MALDI-TOF-MS. The presence and authenticity of 1Bx14 and 1By15 subunits were further confirmed by liquid chromatography coupled to tandem mass spectrometry and heterologous expression in E. coli. Comparative analysis demonstrated that 1Bx14 possessed one deletion and 20 single-nucleotide polymorphism variations compared with seven other Glu-B1 x-type HMW-GS genes that mainly resulted from C–T substitutions, whereas compared with five other Glu-B1 y-type HMW-GS genes, 1By15 displayed few variations. Phylogenetic analysis based on the complete coding sequences of the published HMW-GS genes showed that 1Bx14 had a high divergence with other 1Bx subunit genes, whereas 1By15 displayed greater similarity with 1By20. A possible evolutionary route for 1Bx14 gene formation is proposed, which might have resulted from an intra-strand illegitimate recombination event that occurred ~1.32 million years ago.

scholarly journals Further characterization of the subunits of the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) by SDS-PAGE electrophoresis and MALDI-TOF-MS

2011 ◽  
Vol 46 (11) ◽  
pp. 2144-2151 ◽  
Author(s):  
Francisco Adriano O. Carvalho ◽  
José Wilson P. Carvalho ◽  
Patrícia S. Santiago ◽  
Marcel Tabak
Keyword(s):  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Tof Ms ◽  
Page Electrophoresis

scholarly journals Markedly Increased Vitamin B12 Concentrations Attributable to IgG–IgM–Vitamin B12 Immune Complexes

2006 ◽  
Vol 52 (11) ◽  
pp. 2107-2114 ◽  
Author(s):  
Raffick AR Bowen ◽  
Steven K Drake ◽  
Rachna Vanjani ◽  
Edward D Huey ◽  
Jordan Grafman ◽  
...  
Keyword(s):  
Vitamin B12 ◽  
Size Exclusion Chromatography ◽  
Immune Complexes ◽  
Size Exclusion ◽  
Protein G ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Tof Ms

Abstract Background: High serum vitamin B12 concentrations have been reported in patients with hepatic disease, disseminated neoplasia, myeloproliferative disorders, and hypereosinophilic syndromes. We recently discovered an extraordinarily increased vitamin B12 concentration in a patient without these underlying conditions. Methods: Affinity and size-exclusion chromatography, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and ELISA methods were used to determine the cause of the increased vitamin B12 concentrations in this patient’s serum. Results: The protein G column eluates from 2 apparently healthy volunteers and 2 patients with recent vitamin B12 treatment for anemia had vitamin B12 concentrations of <74 pmol/L, whereas the vitamin B12 concentration in the protein G column eluate from the patient was 7380 pmol/L. The elution profile from size-exclusion chromatography of vitamin B12-binding proteins in the patient’s serum revealed an abnormal vitamin-B12-binding protein. SDS–PAGE analysis of the concentrated eluates from the protein G column, under reducing conditions, revealed an additional band with an apparent molecular mass of 76 kDa, which was not present in control column eluates. MALDI-TOF MS identified this band as an IgM heavy chain. By use of a modified ELISA, we determined that the IgM present in the patient’s eluates was associated with the IgG to form IgG-IgM immune complexes. Conclusions: This case demonstrates the unusual circumstance of a patient with markedly increased vitamin B12 concentrations attributed to immune complexes composed of IgG, IgM, and vitamin B12 and illustrates techniques that can be used to identify this occurrence.

Identification of LAB and Fungi in Laru, a Fermentation Starter, by PCR-DGGE, SDS-PAGE, and MALDI-TOF MS

2018 ◽  
Vol 28 (1) ◽  
pp. 32-39 ◽  
Author(s):  
Lenny S. F. Ahmadsah ◽  
Eiseul Kim ◽  
Youn-Sik Jung ◽  
Hae-Yeong Kim
Keyword(s):  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Sds Page ◽  
Fermentation Starter ◽  
Tof Ms

Solution- and Crystal-Phase Covalent Modification of Lysozyme by a Purpose-Designed Organoruthenium Complex. A MALDI-TOF MS Study of its Metal Binding Sites

ChemBioChem ◽  
2003 ◽  
Vol 5 (1) ◽  
pp. 99-109 ◽  
Author(s):  
Michèle Salmain ◽  
Bertrand Caro ◽  
Françoise Le Guen-Robin ◽  
Jean-Claude Blais ◽  
Gérard Jaouen
Keyword(s):  
Metal Binding ◽  
Binding Sites ◽  
Covalent Modification ◽  
Crystal Phase ◽  
Maldi Tof Ms ◽  
Metal Binding Sites ◽  
Maldi Tof ◽  
Tof Ms
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