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

Jang, You-Ran; Cho, Kyoungwon; Kim, Se Won; Altenbach, Susan B.; Lim, Sung-Hun; Sim, Jae-Ryeong; Lee, Jong-Yeol

doi:10.3390/molecules25184347

Cited by 12 publications

(17 citation statements)

References 52 publications

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“…MALDI-TOF-MS (Matrix-assisted laser desorption ionization-time of flight mass spectrometry) analysis was carried out with AB Sciex 4800 Plus MALDI TOF analyzer to measure the molecular weight of the recombinant mutI tri-RBD 29,30 . The protein sample was exchanged into water to the concentration of 5 mg/mL, and 0.5 μL of the sample was spotted onto the MALDI target plate, followed by air drying at room temperature.…”

Section: Methodsmentioning

confidence: 99%

Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

Liang

Zhang

Yuan

et al. 2021

Preprint

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The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 is an attractive target for COVID-19 vaccine developments, which naturally exists in a trimeric form. Here, guided by structural and computational analyses, we present a mutation-integrated trimeric form of RBD (mutI tri-RBD) as a broadly protective vaccine candidate, in which three RBDs were individually grafted from three different circulating SARS-CoV-2 strains including the prototype, Beta (B.1.351) and Kappa (B.1.617). The three RBDs were then connected end-to-end and co-assembled to possibly mimic the native trimeric arrangements in the natural S protein trimer. The recombinant expression of the mutI tri-RBD, as well as the homo-tri-RBD where the three RBDs were all truncated from the prototype strain, by mammalian cell exhibited correct folding, strong bio-activities, and high stability. The immunization of both the mutI tri-RBD and homo-tri-RBD plus aluminum adjuvant induced high levels of specific IgG and neutralizing antibodies against the SARS-CoV-2 prototype strain in mice. Notably, regarding to the immune-escape Beta (B.1.351) variant, mutI tri-RBD elicited significantly higher neutralizing antibody titers than homo-tri-RBD. Furthermore, due to harboring the immune-resistant mutations as well as the evolutionarily convergent hotspots, the designed mutI tri-RBD also induced strong broadly neutralizing activities against various SARS-CoV-2 variants, especially the variants partially resistant to homo-tri-RBD. Homo-tri-RBD has been approved by the China National Medical Products Administration to enter clinical trial (No. NCT04869592), and the superior broad neutralization performances against SARS-CoV-2 support the mutI tri-RBD as a more promising vaccine candidate for further clinical developments.

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Section: Methodsmentioning

confidence: 99%

Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

Liang

Zhang

Yuan

et al. 2021

Preprint

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“…The 1Bx7 OE subunit typically exhibits higher abundance resulting from a duplication of the 1Bx7 gene. The 1Bx7 OE and 1Bx7 subunits were also distinguishable by PCR amplification using left and right junction primers between the duplicated segments and long terminal repeat (LTR) retrotransposon borders that gave rise to 1Bx7 OE [5,17]. Therefore, the cultivars presumed to carry 1Bx7 or 1Bx7 OE based on measured RT during RP-UPLC analysis were genotyped for 1Bx7 OE (Figure S4).…”

Section: Identification Of Hmw-gs Compositions In Standard Wheat Cultivarsmentioning

confidence: 99%

“…The extracted genomic DNA was quantified and its quality assessed on a Nan-oDrop spectrophotometer (Thermo Scientific, Waltham, MA, USA), and then it was diluted to 50 ng/µL. PCR analysis to discriminate between 1Bx7 and 1Bx7 OE was performed using the method described by [5,17]. PCR was performed in a reaction volume of 20 µL using 150 ng of genomic DNA, 1.25 U of Go Taq DNA polymerase (Promega, USA), 1× Green Go Taq reaction buffer (containing 1.5 mM MgCl 2 ), 200 µM of dNTP mix (Bioneer, Daejeon, Korea) and 10 pmol each of forward and reverse primers.…”

Section: Genomic Dna Extraction and Pcr Analysismentioning

confidence: 99%

“…Gluten, composed of polymeric glutenin and monomeric gliadin, is the primary factor that produces visco-elasticity in wheat dough and is important for wheat end-use quality [1,2]. Based on their molecular weights and primary structures, glutenins are divided into high-molecular-weight (HMW-GS, MW = 67-100 kDa) and low-molecularweight (LMW-GS, MW = 32-35 kDa) glutenin subunits [3][4][5]. Each Glu-1 locus contains two linked genes that encode x-type and y-type HMW-GS.…”

Section: Introductionmentioning

confidence: 99%

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A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles

et al. 2021

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High-molecular-weight glutenin subunits (HMW-GS) account for only 10% of total wheat storage proteins, but play an important role in the processing quality of wheat flour. Therefore, identifying HMW-GS alleles associated with good end-use quality provides important information for wheat breeders. To rapidly, accurately and reproducibly identify HMW-GS, we established an optimized reversed-phase ultra-performance liquid chromatography (RP-UPLC) method. Separation parameters were optimized using an ACQUITY UPLC Protein BEH C4 column and stepwise ACN gradient, and the separation patterns and retention times (RTs) of 22 subunits were comparatively analyzed in 16 standard wheat cultivars. All HMW-GS proteins were well separated within about 5.5 min, and all analyses were complete within 12 min. We distinguished the 16 subunits based on RT, although three subunits in 1Bx (1Bx7/1Bx7OE and 1Bx17) and three subunits in 1By (1By8*, 1By9 and 1By15) had overlapping RTs; these were differentiated by SDS-PAGE. To distinguish 1Bx7 and 1Bx7OE, which differ in protein abundance, RP-UPLC was combined with PCR analysis of DNA junction markers. The optimized method was successfully applied to determine HMW-GS alleles in a large collection of bread wheat germplasm (1787 lines). This protocol is an appropriate option for selecting lines harboring favorable HMW-GS alleles in wheat breeding.

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“…Gas chromatography-mass spectrometry (GC-MS), solid-phase microextraction (SPME)-GC-MS, high-performance liquid chromatography (HPLC), and liquid chromatography tandem mass spectrometry (LC-MS/MS) are widely used for the detection of volatile oil content [ 2 , 3 , 18 , 19 , 20 , 21 , 22 ]. At present, gas chromatography (GC) is the most widely used for Hou determination [ 2 , 3 ], but it cannot detect Hou in blood.…”

Section: Introductionmentioning

confidence: 99%

Development and Verification of a Precolumn Derivatization LC-MS/MS Method for the Pharmacokinetic Study of Houttuynine of Houttuynia Essential Oil

et al. 2021

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Houttuynia essential oil (HEO) has excellent antiviral, anti-inflammatory, and other pharmacological effects, but the lack of effective analytical methods to quantify HEO in plasma has hindered its better clinical monitoring. Houttuynine (Hou) is one of the main active ingredients and quality control substances of HEO, so the pharmacokinetic study of HEO could be conducted by determining Hou blood concentration. Hou is active and not stable in plasma, which makes its blood concentration difficult to measure. In this work, a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method for Hou determination in rat blood was established that involves Hou being derivatized with 2, 4-dinitrophenylhydrazine to form a stable compound to prevent degradation. Herein, p-Tolualdehyde-2,4-dinitrophenylphenylhydrazone was selected as an internal standard substance and the LC-MS/MS method was evaluated for selectivity, precision, accuracy, calibration limit, matrix effect, recovery, and stability. Good linearity (r2 = 0.998) was reached in the range of 2–2000 ng/mL, and the lower limit of quantification of Hou was determined to be 2 ng/mL. The mean intra-assay accuracy ranged from 77.7% to 115.6%, whereas the intra-assay precision (relative standard deviation, RSD) was below 11.42%. The matrix effect value for Hou in rat plasma was greater than 75%, and for the internal standard (IS) it was 104.56% ± 3.62%. The extraction recovery of Hou were no less than 90%, and for the IS it was 96.50% ± 4.68%. Our method is sensitive and reliable and has been successfully applied to the pharmacokinetic analysis of Hou in rats given HEO via gavage and injection.

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

Cited by 12 publications

References 52 publications

Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

Structure and computation-guided design of a mutation-integrated trimeric RBD candidate vaccine with broad neutralization against SARS-CoV-2

A Rapid, Reliable RP-UPLC Method for Large-Scale Analysis of Wheat HMW-GS Alleles

Development and Verification of a Precolumn Derivatization LC-MS/MS Method for the Pharmacokinetic Study of Houttuynine of Houttuynia Essential Oil

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