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 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.

scholarly journals Development of an Optimized MALDI-TOF-MS Method for High-Throughput Identification of High-Molecular-Weight Glutenin Subunits in Wheat

Molecules ◽  
2020 ◽  
Vol 25 (18) ◽  
pp. 4347
Author(s):  
You-Ran Jang ◽  
Kyoungwon Cho ◽  
Se Won Kim ◽  
Susan B. Altenbach ◽  
Sun-Hyung Lim ◽  
...  
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
High Throughput ◽  
Glutenin Subunits ◽  
Maldi Tof Ms ◽  
Molecular Weights ◽  
Maldi Tof ◽  
Rp Hplc ◽  
Group 2 ◽  
Tof Ms

Because high-molecular-weight glutenin subunits (HMW-GS) are important contributors to wheat end-use quality, there is a need for high-throughput identification of HMW-GS in wheat genetic resources and breeding lines. We developed an optimized method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to distinguish individual HMW-GS by considering the effects of the alkylating reagent in protein extraction, solvent components, dissolving volume, and matrix II components. Using the optimized method, 18 of 22 HMW-GS were successfully identified in standard wheat cultivars by differences in molecular weights or by their associations with other tightly linked subunits. Interestingly, 1Bx7 subunits were divided into 1Bx7 group 1 and 1Bx7 group 2 proteins with molecular weights of about 82,400 and 83,000 Da, respectively. Cultivars containing the 1Bx7 group 2 proteins were distinguished from those containing 1Bx7OE using well-known DNA markers. HMW-GS 1Ax2* and 1Bx6 and 1By8 and 1By8*, which are difficult to distinguish due to very similar molecular weights, were easily identified using RP-HPLC. To validate the method, HMW-GS from 38 Korean wheat varieties previously evaluated by SDS-PAGE combined with RP-HPLC were analyzed by MALDI-TOF-MS. The optimized MALDI-TOF-MS method will be a rapid, high-throughput tool for selecting lines containing desirable HMW-GS for breeding efforts.

scholarly journals Endogenous Plasma Peptide Detection and Identification in the Rat by a Combination of Fractionation Methods and Mass Spectrometry

2007 ◽  
Vol 2 ◽  
pp. 117727190700200
Author(s):  
Fabrice Bertile ◽  
Flavie Robert ◽  
Véronique Delval-Dubois ◽  
Sarah Sanglier ◽  
Christine Schaeffer ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Molecular Weight ◽  
Membrane Filtration ◽  
Solid Phase ◽  
Weight Fraction ◽  
Peptide Sequence ◽  
Low Molecular Weight ◽  
Maldi Tof Ms ◽  
Maldi Tof ◽  
Tof Ms

Mass spectrometry-based analyses are essential tools in the field of biomarker research. However, detection and characterization of plasma low abundance and/or low molecular weight peptides is challenged by the presence of highly abundant proteins, salts and lipids. Numerous strategies have already been tested to reduce the complexity of plasma samples. The aim of this study was to enrich the low molecular weight fraction of rat plasma. To this end, we developed and compared simple protocols based on membrane filtration, solid phase extraction, and a combination of both. As assessed by UV absorbance, an albumin depletion >99% was obtained. The multistep fractionation strategy (including reverse phase HPLC) allowed detection, in a reproducible manner (CV < 30%-35%), of more than 450 peaks below 3000 Da by MALDI-TOF/MS. A MALDI-TOF/MS-determined LOD as low as 1 fmol/μL was obtained, thus allowing nanoLC-Chip/MS/MS identification of spiked peptides representing ~10–6% of total proteins, by weight. Signal peptide recovery ranged between 5%-100% according to the spiked peptide considered. Tens of peptide sequence tags from endogenous plasma peptides were also obtained and high confidence identifications of low abundance fibrinopeptide A and B are reported here to show the efficiency of the protocol. It is concluded that the fractionation protocol presented would be of particular interest for future differential (high throughput) analyses of the plasma low molecular weight fraction.

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