Absolute quantification of protein NP24 in tomato fruit by liquid chromatography/tandem mass spectrometry using stable isotope-labelled tryptic peptide standard

Ippoushi, Katsunari; Sasanuma, Motoe; Oike, Hideaki; Kobori, Masuko; Maeda‐Yamamoto, Mari

doi:10.1016/j.foodchem.2014.10.008

Cited by 12 publications

(14 citation statements)

References 19 publications

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“…Proteins were extracted using the protocol described by Ippoushi et al ( 2015 ) with slight modifications. Briefly, 100 mg of frozen tissue was homogenized and suspended in 1 mL of 10% (w/v) TCA in 80% (v/v) acetone containing 2% (w/v) DTT.…”

Section: Methodsmentioning

confidence: 99%

Shotgun Proteomics of Tomato Fruits: Evaluation, Optimization and Validation of Sample Preparation Methods and Mass Spectrometric Parameters

et al. 2016

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An optimized protocol was developed for shotgun proteomics of tomato fruit, which is a recalcitrant tissue due to a high percentage of sugars and secondary metabolites. A number of protein extraction and fractionation techniques were examined for optimal protein extraction from tomato fruits followed by peptide separation on nanoLCMS. Of all evaluated extraction agents, buffer saturated phenol was the most efficient. In-gel digestion [SDS-PAGE followed by separation on LCMS (GeLCMS)] of phenol-extracted sample yielded a maximal number of proteins. For in-solution digested samples, fractionation by strong anion exchange chromatography (SAX) also gave similar high proteome coverage. For shotgun proteomic profiling, optimization of mass spectrometry parameters such as automatic gain control targets (5E+05 for MS, 1E+04 for MS/MS); ion injection times (500 ms for MS, 100 ms for MS/MS); resolution of 30,000; signal threshold of 500; top N-value of 20 and fragmentation by collision-induced dissociation yielded the highest number of proteins. Validation of the above protocol in two tomato cultivars demonstrated its reproducibility, consistency, and robustness with a CV of < 10%. The protocol facilitated the detection of five-fold higher number of proteins compared to published reports in tomato fruits. The protocol outlined would be useful for high-throughput proteome analysis from tomato fruits and can be applied to other recalcitrant tissues.

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Section: Methodsmentioning

confidence: 99%

Shotgun Proteomics of Tomato Fruits: Evaluation, Optimization and Validation of Sample Preparation Methods and Mass Spectrometric Parameters

et al. 2016

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“…Sweet pepper and eggplant protein extractions were performed as follows; [8] a whole sweet pepper fruit (39 g, edible part) was symmetrically cut into eight pieces and one piece (4 g) was cut into smaller pieces and ground into a fine powder in liquid nitrogen with a mortar and pestle. The skin (1 g) collected from a sweet pepper fruit and skin (1.2 g) from an eggplant fruit were also ground into a fine powder in liquid nitrogen with a mortar and pestle.…”

Section: Trichloroacetic Acid/acetone Extractionmentioning

confidence: 99%

“…Sweet pepper and eggplant protein extracts obtained using trichloroacetic acid/acetone treatment were digested with trypsin. [8] These extracts were resuspended in 100 μL of 50 mM ammonium bicarbonate and 6 M guanidinium chloride, mixed with 5 μL of 200 mM dithiothreitol in 50 mM ammonium bicarbonate, and boiled for 10 min. For protein alkylation, the mixtures were supplemented with 4 μL of 1 M iodoacetamide in 50 mM ammonium bicarbonate and incubated under shading for 1 h at room temperature.…”

Section: Trypsin Digestionmentioning

confidence: 99%

“…Our assay for NP24 contained in tomato fruit uses GQTWVINAPR (amino acid numbers 54-63, Fig. 1) as an AQUA peptide, [8] and this peptide is generated from PR5-like protein by trypsin digestion in silico; accordingly, the assay can hypothetically be applied to the quantification of PR5-like protein in sweet pepper fruit. However, the osmotin-like protein PR-5 sequence in the region corresponding to the peptide sequence is not known.…”

Section: Lc/mrm Analyses Of Osmotin-like Proteinsmentioning

confidence: 99%

“…We previously quantified NP24 in tomato fruit using the absolute quantification (AQUA) method with a stable isotope-labeled internal standard (SIIS) peptide and liquid chromatography/tandem mass spectrometry (LC/MS/MS). [8] In this study, we determined the applicability of the assay to the quantification of PR5-like protein in sweet pepper fruit and osmotin-like protein PR-5 in eggplant fruit.…”

Section: Introductionmentioning

confidence: 99%

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Quantification method of osmotin-like proteins in sweet pepper and eggplant by liquid chromatography/tandem mass spectrometry based on the aqua technology

Ippoushi

Sasanuma

Oike

et al. 2016

International Journal of Food Properties

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Analysis of A1‐type and A2‐type β‐casein in Maiwa Yak and Pien‐niu milk by HPLC‐high‐resolution MS and tandem MS

Chen

Ren

Wang

et al. 2021

J of Separation Science

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In this study, a peptide-based method employing ultra high performance liquid chromatography electrostatic field orbitrap high-resolution mass spectrometry and triple quadrupole mass spectrometry was developed for quantification of A1type and A2-type β-casein in milk from Yak, cows, and their offspring of crosses, Pien-niu. The specific peptides of A1-type and A2-type β-casein were screened and confirmed by protein software after analysis of high-resolution mass spectrometry. The multiple reaction monitoring method was established based on the qualitative results, and isotope-label peptides were used as internal standards.The linear correlation coefficients of this method were >0.99. The relative standard deviations of repeatability test were 0.2-3.6%. The recovery rate ranged from 93.3 to 114.4% with relative standard deviations <6% at three different spiking levels. The method was applied to analyze 45 milk samples from different species.The results showed that β-casein in Yak and Pien-niu milk was about 30% higher than that in cow milk. Furthermore, the β-casein in the Yak milk only contains A2-type β-casein. A1-type and A2-type β-casein coexist in most samples of Pienniu and cow milk, a few samples contain only one type of β-casein. These results provide further understanding in nutritional value of milk from Yak and Pienniu.

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Absolute quantification of protein NP24 in tomato fruit by liquid chromatography/tandem mass spectrometry using stable isotope-labelled tryptic peptide standard

Cited by 12 publications

References 19 publications

Shotgun Proteomics of Tomato Fruits: Evaluation, Optimization and Validation of Sample Preparation Methods and Mass Spectrometric Parameters

Shotgun Proteomics of Tomato Fruits: Evaluation, Optimization and Validation of Sample Preparation Methods and Mass Spectrometric Parameters

Quantification method of osmotin-like proteins in sweet pepper and eggplant by liquid chromatography/tandem mass spectrometry based on the aqua technology

Analysis of A1‐type and A2‐type β‐casein in Maiwa Yak and Pien‐niu milk by HPLC‐high‐resolution MS and tandem MS

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