*4.6.* •*OH Measurement*

The terephthalic acid (TA) method was employed for determination of hydrogen groups. TA was dissolved in 1.4 mM NaOH solution to obtain a 0.2 mM TA solution. The fluorescence microplate reader (FlexStation3, Molecular Devices, San Francisco, CA, USA) was used to measure the absorbance value of hydroxyl group at an excitation wavelength of 310 nm and an emission wavelength of 425 nm.

#### *4.7. Total Content of NO<sup>2</sup>* <sup>−</sup> *and NO<sup>3</sup>* − *Determination*

The total concentrations of NO<sup>2</sup> <sup>−</sup> and NO<sup>3</sup> − were analyzed with Nitric Oxide (NO) assay kit (Nitrate reductase method, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The reaction between NO<sup>3</sup> <sup>−</sup> and nitrate reductase leads to the formation of NO<sup>2</sup> −.

#### *4.8. Identification of CJAP Plasma Treatment Fragments by Orbitrap HR-LC-MS*/*MS*

The Q. Exactive Orbitrap HR-LC-MS/MS was applied to analyze the obtained CJAP plasma treatment peptide mixtures. An Ultimate 3000 UPLC system equipped with an Accucore aQ column (2.1 × 150 mm, 2.6 µm Thermo Scientific, Waltham, MA, USA) was coupled to the quadrupole electrostatic field orbital trap mass spectrometer (Q Exactive, Thermo Scientific, Waltham, MA, USA) in positive mode. The ESI pattern was used as ion source with a scanning range from 50 to 2400 *m*/*z*. Solvent A and solvent B consisted of an aqueous solution of 0.1% formic acid in water and of 0.1% formic acid in acetonitrile, respectively. A gradient of acetonitrile from 99% solvent A to 99% solvent B was generated for 20 min at a flow rate of 0.2 mL/min. The Xcalibur software (Thermo Scientific, Waltham, MA, USA) was applied for analysis of MS result.

#### *4.9. Quantitative 2-DE and LC-MS Analysis*

The CJAP plasma-treated wheat gliadins were separated by 2-DE as described by Nagib et al. (2010) [43]. First, 200 µL of the gliadin sample (1.25 µg/µL) was mixed with hydration loading buffer containing 8 M urea, 0.2% Bio-Lyte (*w*/*v*), 4% CHAPS (*w*/*v*) and 65 mM dithiothreitol (DTT). Each sample was subjected to IPG prefabricated strips pH 3–10 (7 cm, ReadyStrip, Bio-Rad, Irvine, CA, USA) with 16 h rehydration. IEF was applied using a PROTEAN IEF Cell (Bio-Rad, Irvine, CA, USA) at 17 ◦C with a series of voltages: 250 V and 500 V for desalting, 500–4000 V linear boost and 4000 V for focus. After IEF, IPG prefabricated strips were equilibrated in an equilibration buffer containing 6 M urea, 2% SDS (*w*/*v*), 0.375 M Tris-HCl (pH 8.8) and 20% (*v*/*v*) glycerol including 2% (*w*/*v*) DTT for 15 min, followed by a strip equilibration buffer containing 2.5% iodoacetamide (*w*/*v*) for another 15 min. In the second dimension, SDS-PAGE of a 12% separation gel at 20 mA was applied until blue dye line reached the end of gel.

Three sets of selected protein spots from 2-D gels were excised for trypsin digestion (37 ◦C, 16 h), according to the literature reported by Martinez-Esteso et al. (2016) [44]. The obtained peptide samples from digested protein spots were dissolved (0.1% formic acid, 5% acetonitrile) and loaded into the Q Exactive mass spectrometer equipped with a 75 µm i.d. × 150 mm, packed with an Acclaim PepMap RSLC C18, 2 µm, 100 Å, nanoViper column for sequence identification (Thermo Scientific, Waltham, MA, USA). Solvent A and solvent B consisted of an aqueous solution of 0.1% formic acid in water and of 0.1% formic acid, 80% ACN, respectively. The MS original file was processed and converted using MM File Conversion software to obtain the MGF format file, and the NCBI database and UniProt sapiens database were retrieved using MASCOT.

## *4.10. Qualitative and Quantitative Detection of Carbonyl Groups*

The content of carbonyl groups was measured by 2,4-dinitrophenyl -hydrazine (DNPH) method [24]. First, 50-µL gliadin sample was supplemented with 800 µL of 10 mM DNPH in 2.5 M HCl, with 2.5 M HCl as a control. Two tubes were placed at room temperature for 1 h. The samples were precipitated with 1 mL of 20% trichloroacetic acid (TCA) (*w*/*v*) and left in an ice box for 5 min. After centrifugation (10,000× *g*, 10 min, 4 ◦C), the precipitate was washed with 1 mL of 10% TCA and centrifuged (10,000× *g*, 10 min, 4 ◦C). The precipitates were washed three times with 1 mL ethanol-ethyl acetate (1:1, *v*/*v*). The obtained pellets were dissolved in 500 µL of 6 M guaninide hydrochloride with general vortexing and again centrifuged (10,000× *g*, 10 min, 4 ◦C). Finally, 220 µL of supernatant from each tube was collected and the carbonyl content was calculated based on the absorption at 375 nm.

#### *4.11. Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)*

A commercial ELISA kit (Gliadin, ml058393-2, Shanghai Enzyme-linked Biotechnology Co. Ltd., Shanghai, China) was applied to measure the concentration of prolamin. In the first step, 10 µL of CJAP-treated and untreated samples were added to 40 µL of sample diluent. All samples were then incubated at 37 ◦C for 30 min. The samples were washed five times with a washing solution. The samples were mixed with 50 µL of HRP-Conjugate reagent, incubated again at 37 ◦C for 30 min and then incubated and washed as described above. Thereafter, 50 µL each of reagent A and reagent B were mixed in the dark at 37 ◦C for 10 min. Finally, 50 µL of stop solution was used to terminate reaction. The concentration of prolamin can be calculated by absorption curve measured at a wavelength of 450 nm. Samples without addition of HRP-Conjugate reagents served as a blank control.

#### *4.12. SE-HPLC Analysis*

The gluten proteins were collected with 50 mM of sodium phosphate buffer with 0.5% SDS (*w*/*v*, pH 6.9) from flour of Zhengmai 9023 (*T. aestivum* L.), based on the documentation reported by Li et al. (2017) [45]. A 20-µL portion of supernatant filtered from a nylon membrane (pore size 0.45 µm) was poured into a Waters 1525 binary HPLC pump, fractionated on a Phenomenex Biosep-SEC-s4000 column (20 min, 0.5 mL min−<sup>1</sup> ) and then further tested at 214 nm using a Waters 2998 photodiode array detector (Waters Corp., Milford, CT, USA).

#### *4.13. Statistical Analysis*

The statistical software GraphPad Prism 6.0 (Microsoft Corporation, Redmond, WA, USA) was used for statistical analysis via one-way analysis of variance (ANOVA) followed by Tukey mean-comparison procedure. The 5% significant differences (*p* < 0.05) and 1% highly significant differences (*p* < 0.01) were evaluated using Origin 8.1 (OriginLab Corporation, Northampton, MA, USA).

#### **5. Conclusions**

In conclusions, fragmentation at Pro residues, along with modifications at Phe and Tyr residues of two CD-related peptides—QQPFP and PQPQLPY—could be achieved with the application of CAJP plasma. A variety of RONS such as OH, O3, H2O2, NO<sup>2</sup> <sup>−</sup>, and NO<sup>3</sup> − were observed to increase during plasma processing. In addition, the immunoreactivity of gliadin using the R5 antibody appeared to be reduced because a large amount of recognition epitopes were modified after plasma treatment. Furthermore, the longer the plasma treatment, the greater was the formation of carbonyl and hydroxylation products and, consequently, the smaller was the size of the formed aggregates. The structural changes in the two model peptides, as well as the quantitative changes in gliadin after plasma treatment, favored the preparation of gluten-related product for the celiac disease patients.

*Int. J. Mol. Sci.* **2020**, *21*, 1012

**Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/21/3/1012/ s1. Figure S1. HR-LC chromatogram analysis of CJAP plasma-modified QQPFP and PQPQLPY identified by Orbitrap HR-LC-MS/MS at treatment times of 0 min, 5 min, 10 min, 30 min and 60 min, respectively. (A) QQPFP, (B) PQPQLPY. The black arrow indicates CJAP plasma-modified products of two celiac-toxic peptides. Figure S2. Two model peptides QQPFP and PQPQLPY modified by CJAP plasma. (A) The changes in the HR-LC-MS/MS peak area of QQPFP and PQPQLPY with the different treatment time. (B) The changes in HR-LC-MS/MS peak area of products P1~P4 from QQPFP with the different treatment time. (C) The changes in HR-LC-MS/MS peak area of products P5~P7 from PQPQLPY with different treatment time. Figure S3. Extract ion chromatograms (EIC) selected for certain fragmentation ions were utilized to certify the intensity changes of P3 (A), P4 (B) and P5 (C). Figure S4. SDS–PAGE analysis of the gliadins in wheat with different CJAP plasma treatment times. M: Protein molar mass standard, lane 1: 0 min treatment, lane 2: 5 min treatment, lane 3: 10 min treatment, lane 4: 30 min treatment and lane 5: 60 min treatment.

**Author Contributions:** G.H., G.Y. and F.S. conceived and designed the study. F.S., X.X. and Y.Z. performed all the biological experiments and analyzed the data. F.S., J.D., and M.M. participated in the physics experiments. Y.W. provided some help in the SE-HPLC experiment and its data analysis. D.Q. and X.L. proposed some valuable suggestions to the study. F.S. and X.X. wrote the draft manuscript. G.H. and G.Y. revised and finalized the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** The work was supported by National Genetically Modified New Varieties of Major Projects of China (2016ZX08010004-004) and the National Natural Science Foundation of China (Nos. 31771418, 31570261), and Key Project of Hubei Province (2017AHB041).

**Acknowledgments:** The authors acknowledge the Analytical and Testing Center of Huazhong University of Science and Technology (HUST) for technical assistance in the fragments identification of celiac-toxic peptides by used of Orbitrap HR-LC-MS/MS.

**Conflicts of Interest:** The authors declare that the research was accomplished without any commercial or financial relationships that could be identified as a potential conflict of interest.

#### **Abbreviations**

