Solubility variation of wheat dough proteins: A practical way to track protein behaviors in dough processing

Journal Pre-proofs Solubility Variation of Wheat Dough Proteins: A Practical Way to Track Protein Behaviors in Dough Processing Xiaolong Wang, Rudi Appels, Xiaoke Zhang, Ferenc Bekes, Dean Diepeveen, Wujun Ma, Xinzhong Hu, Shahidul Islam PII: DOI: Reference:

S0308-8146(19)32183-1 https://doi.org/10.1016/j.foodchem.2019.126038 FOCH 126038

To appear in:

Food Chemistry

Received Date: Revised Date: Accepted Date:

23 July 2019 5 November 2019 6 December 2019

Please cite this article as: Wang, X., Appels, R., Zhang, X., Bekes, F., Diepeveen, D., Ma, W., Hu, X., Islam, S., Solubility Variation of Wheat Dough Proteins: A Practical Way to Track Protein Behaviors in Dough Processing, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem.2019.126038

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Solubility Variation of Wheat Dough Proteins: A Practical Way to Track Protein Behaviors in Dough Processing Xiaolong Wanga,b , Rudi Appelsc,*, Xiaoke Zhangd, Ferenc Bekese, Dean Diepeveenb,f, Wujun Mab, Xinzhong Hua and Shahidul Islamb,*

a

College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, 710119,

China b

Australia China Centre for Wheat Improvement, College of Science Health Engineering and Education,

Murdoch University, 90, South Street, Murdoch, WA 6150, Australia c

School of Bio Sciences, University of Melbourne, Parkville, VIC 3010, Australia

d

College of Agronomy, Northwest A & F University, Yangling, Shaanxi 712100, China

e

FBFD PTY LTD, Sydney, Australia

fDepartment

of Primary Industries and Regional Development, Western Australia, 3 Baron-Hay Court,

South Perth, WA 6151, Australia

Corresponding authors: Shahidul Islam; Email: [email protected] Rudi Appels; Email: [email protected]

Running title: Solubility Variation of Wheat Dough Proteins to Track Protein Behaviors

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Abstract: To understand wheat dough protein behavior under dual mixing and thermal treatment, solubility of Mixolab-dough proteins were investigated using nine extraction buffers of different

dissociation

capacities.

Size

exclusion

high

performance

liquid

chromatography (SE-HPLC) and two-dimensional gel electrophoresis (2-DGE) demonstrated that overall changes of protein fractions and dynamic responses of specific proteins during dough processing were well reflected by their solubility variations. After starch pasting, the abundance of 0.5 M NaCl extractable proteins were decreased except for six protein groups including α-amylase inhibitors and superoxide dismutase (SOD). The solubility loss of glutenin proteins at C3 (32 min; 80 ℃) was mainly ascribed to the un-extractable HMW-GSs，LMW-GSs, globulin and triticin, while the extract yield of α-, β-, γ- gliadins and avenin-like proteins (ALPs) increased after starch pasting. Differential responses of dough proteins to extraction systems provides the basis for further exploring wheat protein dynamics in processing. Key Words: 2-DGE, gluten proteins, non-gluten proteins, protein behavior, SE-HPLC.

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1. Introduction Wheat kernel proteins have, classically, been subdivided into glutenins, gliadins, albumins and globulins according to their solubility in water, salt, alcohol and diluted acid or alkali solutions (Osborne, 1924). The three major fractions of wheat grain proteins defined in this way are glutenins, gliadins, albumins/globulins, with each group comprised of suites of individual polypeptides (Juhász, Gell, Békés, & Balázs, 2012). Numerous studies have demonstrated that different protein fractions in the dough matrix are responsible for the rheological properties of wheat dough (Kasarda, Elva, Ellen J-L, Lazo, & Altenbach, 2013). The rheological properties of wheat dough are standard quality assessment measures in the industry (AACC International, 2010). Polymeric glutenins and monomeric gliadins are present in approximately equal amounts in the kernel and together they occupy 80% of the total storage proteins to form the gluten proteins that are essential in the network formation of dough matrix (Broeck, America, Smulders, Bosch, Hamer, Gilissen, et al., 2009). Glutenins are further divided into high molecular weight glutenin subunits (HMW-GSs) and low molecular glutenin subunits (LMW-GSs). Although HMW-GSs only account for 10% of the total protein, they form the network skeleton of gluten polymer, determining 3060% of wheat processing quality (Zhang, Tang, Yan, Zhang, & Zhang, 2009). LMWGSs and gliadins are integrated into gluten polymers via disulfide bonds and hydrogen bonds, and contribute to the viscoelasticity of dough (Figueroa, Maucher, Reule, & Peña, 2009; Gupta, Bekes, & Wrigley, 1991). The gliadin family is comprised of α-, β-, γ- and ω-gliadins, which also play an important role through their contribution to dough 3 / 51

viscosity and extensibility (Fido, Békés, Gras, & Tatham, 1997; Uthayakumaran, Tömösközi, Tatham, Savage, Gianibelli, Stoddard, et al., 2001). Although most of the albumins and globulins (non-gluten proteins) exist in the dough matrix as free monomers, and have limited contribution on dough viscoelasticity, research is increasingly suggesting that some non-gluten proteins such as avenin-like proteins, polymeric albumins and globulins and some enzymes and enzyme inhibitors are also important components of gluten polymers (Békés & Gras, 1992; Debyser, Derdelinckx, & Delcour, 1997; Gupta, Batey, & Macritchie, 1992; Yujuan Zhang, Hu, Islam, She, Peng, Yu, et al., 2018). In order to elucidate the quality and nutritional contribution of specific protein in wheat grain, extensive research focusing on the protein isolation strategy has been carried out (Dupont, Ronald, Rocio, & Vensel, 2005; Vensel, Tanaka, Nick, Wong, Buchanan, & Hurkman, 2010). Alkaline solutions with diluted salt are commonly used in the separation of globulin/albumin. For example, a KCl buffer containing 50 mM Tris-HCl, 100 mM KCl and 5 mM ethylene diamine tetraacetic acid (EDTA) was used to extract albumins and globulins in wheat endosperm (Dupont, Ronald, Rocio, & Vensel, 2005; Paolo, Dale, Christine, Anna Maria, Anna Maria, Birte, et al., 2010). Furthermore, metabolic albumins and globulins were also isolated using a NaCl buffer (10 mM Phosphate, 10 mM NaCl, pH 7.8) (Tasleem-Tahir, Nadaud, Chambon, & Branlard, 2012). Unlike non-gluten proteins (albumins and globulins), the isolation of gluten proteins is more complex. The advanced structure of gluten polymer relies on hydrogen bonds and hydrophobic interactions, and intermolecular disulfide bonds. Therefore, 4 / 51

protein denaturants such as sodium dodecyl sulfate (SDS), acetic acid, ethanol, urea, trichloroacetic acid (TCA) and phenol are widely used to break non-covalent bonds within the gluten polymers during protein extraction, while reducing agents (dithiothreitol (DTT) or 2-mercaptoethanol (2-ME)) or sonication, on the other hand, are applied for the breaking of disulfide bonds (Dupont, Vensel, Tanaka, Hurkman, & Altenbach, 2011; Hurkman & Tanaka, 2004, 2007). Although differences in solubility characteristics have brought unexpected challenges in the separation, quantification and identification of specific proteins in wheat and/or its products, it also enlightened the structure-function research of individual proteins. Component analysis of soluble proteins after partial denaturing or reducing was an important way to explore the structure of gluten polymer in early research. Solubility based component analysis also provided evidence for discovering the polymerization characteristics of HMW-GSs and LMW-GSs, and the distribution of intermolecular disulfide bonds (Kim & Bushuk, 1995; Lawrence & Payne, 1983). Furthermore, the quantification of SDS un-extractable protein polymers (UPP) by SE-HPLC has been generally accepted as a reliable method for wheat processing quality evaluation (Gupta, Khan, & Macritchie, 1993). Therefore, we postulated that dough protein behaviors during processing can be well reflected in their solubility characteristics in a set of extraction buffers with gradient dissociation capacities. The association between protein solubility and dough rheological properties under room temperature has been well studied (Gulia & Khatkar, 2015; Meng & Cai, 2008; 5 / 51

P. Zhang, Jondiko, Tilley, & Awika, 2015). However, under continuous mixing and thermal treatment, dough proteins are subjected to a number of additional molecular interactions with starch, lipids and other ingredients (Blazek & Copeland, 2008; Hadnađev, Dokić, Hadnađev, Pojić, & Torbica, 2014; Jekle, Mühlberger, & Becker, 2016). Research into protein solubility altering dough matrix properties is the subject of our study. We document methods for the separation of non-gluten and gluten proteins, combining SE-HPLC, 2-DGE and tandem mass spectrometry (MS/MS), to analyze the protein solubility of Mixolab-dough samples before and after starch gelatinization. The aims of this study are to understand the solubility characteristics of individual proteins using different extraction buffers, in order to utilize response to extraction methods to track protein behavior during dough processing. 2. Materials and methods 2.1 Materials Grains of Australian Premium White wheat cultivar Westonia were provided by the Department of Primary Industries and Regional Development, Western Australia and ground into flour using the Brabender Quadrumat Junior mill. The dough samples were prepared by a Chopin Mixolab in triplicate, according to the slightly modified method of AACC 54-60.01 (AACC International, 2010). Specifically, the standard ‘‘Chopin+’’ protocol with extended initial mixing stage at 30 ℃ was followed, i.e. initial mixing at 30 ℃ for 16 min (8 min in the original standard method), heating to 90 ℃over 15 min (at a rate of 4 ℃/min), holding at 90 ℃ for 7 min, cooling to 50 ℃ over 10 min 6 / 51

(at a rate of 4 ℃/min) and holding at 50 ℃ for 5 min. The mixing speed was kept constant at 80 rpm. Mixolab-dough samples at 3 min (30 ℃, stable torque within the stability time) and C3/32 min (80 ℃, thermal pasting peak where the starch granules were broken and gelatinized) (Fig. 1) were taken in triplicate, and the flour used for dough preparation was taken as control. The collected dough samples were freeze-dried, ground into powder with motor and pestle and sieved through the screen with 100 mesh. 2.2 SE-HPLC SE-HPLC was applied to quantify protein fractions of dough and flour. Ten milligram dough powder or flour were used for the protein extraction as described previously (Rakszegi, Békés, Láng, Tamás, Shewry, & Bedő, 2005). The buffer containing 0.5% (v/v) SDS-phosphate (pH 6.9) was used initially to separate SDS extractable proteins, thereafter, the insoluble residue was remixed with the same buffer and sonicated for 15 s for separating SDS unextractable protein fractions. All the supernatants were filtered through the 0.45 um polyvinylidene fluoride (PVDF) filter after centrifugation and analyzed by the column of Phenomenex BIOSEP-SEC 4000 in the buffer containing 0.05% (v/v) acetonitrile and 0.05% (v/v) trifluoroacetic acid. The running time of each extract was 10 min with the flow rate at 2 ml/min. Protein fractions were quantified based on their absorption at 214 nm. Six SE-HPLC fractions were obtained from soluble and insoluble extracts (three fractions) according to the two chromatograms. The formulas for quantification of the six protein fractions were reported in our previous study where the soluble and insoluble 7 / 51

fractions of glutenin, gliadin and albumin/globulin were abbreviated as P1s and P1i, P2s and P2i, P3s and P3i, respectively (Wang, Appels, Zhang, Bekes, Torok, Tömösközi, et al., 2017). Detailed formulas for calculating the protein contents are as follows: a) Glutenin (%) = 100×(P1s+P1i)/( P1s+ P2s+ P3s +P1i+ P2i+ P3i) b) Gliadin (%) = 100×(P2s+P2i)/( P1s+ P2s+ P3s +P1i+ P2i+ P3i) c) Albumin/globulin (%) = 100×(P3s+P3i)/( P1s+ P2s+ P3s +P1i+ P2i+ P3i) d) UPP (%) = 100×P1i/(P1i+P1s) e) UG (%) = 100×P2i/(P2i+P2s) f)

UAlb/Glob (%) = 100×P3i/(P3i+P3s)

2.3 Two-dimensional gel electrophoresis Flour and Mixolab-dough proteins were further analysed by 2-DGE (Shahidul, Wujun, Guijun, Liyan, & Rudi, 2011). Non-gluten proteins including most of albumin and globulin were separated by both 0.5 M NaCl (pH 7.0) and 0.1 M KCl (50 mM TrisHCl, 100 mM KCl, 5 mM EDTA, pH 7.8) using the same protocol. Dough power or flour was mixed with the extraction buffer at 350 mg/mL. The mixture was stirred for 5 h at 4 ℃, and the supernatant with dissolved non-gluten proteins was obtained after centrifugation at 12000 g for 20 min. Then the supernatant was mixed with 4 volumes of ice-cold acetone at -20 ℃ to precipitate non-gluten proteins after centrifugation at 12000 g for 20 min. Thereafter, the protein pellet was cleaned by ice-cold acetone 8 / 51

containing 0.07% β-mercaptoethanol and retrieved by centrifugation at 12000 g for 10 min. As listed in Table 1, seven buffers with different levels of extraction strength were used for gluten proteins extraction. Flour or the ground-up Mixolab-dough sample at 3 min or 32 min was mixed with the seven buffers (Table 1) separately at 100mg/mL for the isolation of gluten proteins. Buffer g (0.3% SDS + 15 mM DTT) possessing medium dissociation (buffer containing medium concentration of denaturant and reductant) was applied for the extraction of gluten proteins from the residue left after extracting nongluten proteins with 0.5 M NaCl. Buffer a, which containing much higher concentration of denaturant and reductant (8 M Urea, 4% 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate (CHAPS), 60 mM DTT), was selected for separating all the gluten proteins. Two-dimensional gel electrophoresis of the isolated proteins were carried out following the method published earlier (Wang, Appels, Zhang, Bekes, et al., 2017). Ten milligram of the retrieved non-gluten or gluten protein pellet was dissolved in rehydration buffer (7 M urea, 2 M thiourea, 2% CHAPS, 65 mM DTT, and 2% IPG buffer) and incubated for 5 h at room temperature. After concentration determination of the protein solution using the RC DC protein assay kit (Bio-Rad, Hercules, CA), isoelectric focusing was performed using the Protein IEF cell (Bio-Rad) on 17 cm Bio-Rad IPG strips of 3-10 pH gradient. Finally, the strips were soaked into the rehydration buffer containing 1100 μg protein. Following IEF running, strip equilibration, sodium dodecyl sulfate 9 / 51

polyacrylamide gel electrophoresis (SDS-PAGE), gels staining and analysis of digital gel maps were conducted as described earlier (Wang, Appels, Zhang, Bekes, et al., 2017). 2.4 Protein identification by MS/MS Protein spots which varied significantly on the gels were manually excised for mass spectrometric analysis by the Proteomics International Ltd. Pty at Perth, Australia. Specifically, protein digestion with trypsin, tryptic peptides separation using Ultimate 3000 nano HPLC system, and following spectra analysis with 4000Q TRAP mass spectrometer were performed as described previously (Shahidul, Wujun, Guijun, Liyan, & Rudi, 2011). Specific proteins of interest were identified by Mascot sequencing matching software from Matrix Science. All the searches used the Ludwig NR and the taxonomy was set as Viridiplantae. The detailed settings of the software were referred to earlier study (Shahidul, Wujun, Guijun, Liyan, & Rudi, 2011). Generally, a match was confirmed if at least two digested peptides of one protein were matched to a specific protein entry within the database. 3. Results 3.1 Dough rheological properties in Mixolab process As shown in Fig. 1, the Mixolab curve of Westonia can be separated to five different stages. During the first stage, from 0 min to 5 min, an increase in torque was observed until a maximum was reached at C1/1.6 min and the dough torque stayed ca 1.1 N above until 5 min. From 5 min to C2 (stage 2), torque-value of the dough decreased with 10 / 51

excessive mixing which indicated the weakening of dough protein. A sharp increase in torque-value was observed between C2 and C3 (stage 3) due to the starch gelatinization caused by temperature increase. During this stage, starch granules swell, absorb water and amylose molecules leach out resulting in an increase in the viscosity. Thereafter, the torque-value of dough decreased from C3 to C4 (stage 4) as a result of amylase activity as well as breakdown of other polymer complexes. At the fifth stage, from C4 to C5, the decreasing temperature caused an increase in torque-value as a result of gel formation. According to the dynamic changes of dough torque-value, triplicate dough samples from 3 min (30 ℃ ) and C3/32 min (80 ℃ ) of Mixolab operation, were defined as representing the critical time points for gluten formation and starch gelatinization, respectively, and formed a focus for the protein solubility analysis. 3.2 Solubility profile of protein fractions as revealed by SE-HPLC SE-HPLC analysis was conducted to explore the solubility of different protein fractions. Fig. 2A shows that the overall extractability of total protein decreased after starch gelatinization. Compared to the total protein extractability of dough at 3 min (30 ℃), a 2.3% decrease was observed at C3/32 min (80 ℃ ). The results indicated that the protein extractability was affected by the protein denaturation and polymerization, and also by the interaction between protein and pasting starch (Chen, Deng, Peng, Tian, & Xie, 2010; Wang, Appels, Zhang, Diepeveen, Torok, Tömösközi, et al., 2017). Although SDS buffer sonication performed well in total protein extraction without thermal stress, it could not recover all the proteins after starch gelatinization (Gupta, 11 / 51

Khan, & Macritchie, 1993; Wang, Appels, Zhang, Bekes, et al., 2017). When protein fractions were investigated individually the extractability can change. Glutenin proteins showed significant 8.4% decrease, and albumin/globulin did not show a significant change, between 3 min (30 ℃) and C3/32 min (80 ℃). In contrast, a 2.5 % increase was observed in gliadins between those two time points. The data suggested that the decrease in total protein extractability after heating was mainly contributed by glutenin fraction. In addition, part of albumin/globulin tend to be trapped into the dough matrix at high temperature (80 ℃). In the case of SDS extractable protein fractions, the relative amount of glutenins (Glu%) at C3/32 min (80 ℃) was decreased by 2.4% compared to that of 3 min (30 ℃), and no significant difference was observed on the relative amount of albumin/globulin (Alo/Glob%) (Fig. 2B). However, the relative amount of SDS extractable gliadins (Gli%) increased by 2.3% at C3/32 min (80 ℃) compared to 3 min (30 ℃). The unextractable (only soluble after sonication) portion of different protein fractions were also analysed between the time points (Fig. 2B). The ratio of un-extractable gluten polymers (UPP%) increased by 29.3% (from 33.7% to 62.0%) between 3 min (30 ℃) and C3/32 min (80 ℃). Similar patterns were also observed for the percentage of unextractable gliadins (UG%) and un-extractable albumin/globulin (UAlb/Glob%) between the two time points, where those increased 4.0% (from 11.2% to 15.2%) and 2.5% (from 2.9% to 5.4%), respectively. The findings indicated that the ratio of unextractable proteins increased in all the protein fractions after starch pasting.

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3.3 Solubility profile of protein fractions as revealed by 2-DGE A further detailed investigation on the extractability of protein fractions was carried out following 2-DGE by using nine extraction buffers characterized by different dissociation properties (Table 1). The major differentiating proteins across the two time points were also identified by MS/MS which provided further information of the extractability of protein fractions from Mixolab dough that are described in the following sections. The major change in the Mixolab profile is the increase of viscosity due to starch pasting and in our study we choose the before and after pasting time points for study. 3.3.1 Solubility of albumin/globulin in different extraction buffers The SE-HPLC results showed a small-scale decrease of extractability of albumin/globulin (Fig. 2A) after starch pasting. In order to identify the proteins with differential solubility, NaCl and KCl buffers were used to separate albumin/globulin for the 2-DGE analysis. Considerable differences were observed in the 2-DGE protein profiles of 3 min (30 ℃) and C3/32 min (80 ℃) dough samples (Fig. S1). For example, β-amylase (plate A: Fig. S1), serpins (plate C: Fig. S1), triosephosphate isomerase (plate H: Fig. S1), glyceraldehyde-3-phosphate dehydrogenase and aldose reductase (plate I: Fig. S1) were separated from dough of 3 min (30 ℃ ) by both of the buffers, but they lost their solubility at C3/32 min (80 ℃). Likewise, the solubility of high molecular globulins (plate B: Fig. S1) decreased dramatically at C3/32 min (80 ℃) compared to the 3 min 13 / 51

(30 ℃). In contrast, a higher extractability of α-amylase inhibitors and SOD (plate F: Fig. S1) was observed at C3/32 min (80 ℃), compared to the 3 min (30 ℃). The majority of albumin/globulin showed similar solubility in both of the NaCl and KCl buffers. However, some proteins located at the alkaline end of the gel showed different solubility between NaCl and KCl buffers with an interaction of the time points. For examples, at 3 min (30 ℃ ), similar extractability of low molecular globulins, avein-like b proteins, glucose metabolic enzymes (plate E: Fig. S1), and grain softness protein (GSP) (plate G: Fig. S1) were observed using NaCl and KCl buffers. However, at C3/32 min (80 ℃ ), the solubility of those proteins increased significantly in the buffer NaCl while decreased in the buffer KCl. Generally, both the NaCl and KCl buffers were able to detect albumin/globulin solubility differences during dough processing. However, buffer NaCl showed a better resolution than KCl for the proteins distributed around the neutral pH region (plate I and D: Fig. S1). The MS/MS identification of differentially expressed non-gluten proteins between the two time points had been completed in our previous study (Wang, Appels, Zhang, Bekes, et al., 2017; Wang, Appels, Zhang, Diepeveen, et al., 2017), detailed data can be found in the Fig. S3 and Table S1. 3.3.2 Solubility of gluten proteins in different extraction buffers Another seven extraction buffers were utilized to evaluate the solubility of gluten proteins during Mixolab process by 2-DGE and MS/MS. The typical distribution pattern of gluten proteins is shown in Fig. 3 using protein extracted from 3 min (30 ℃) 14 / 51

dough of Westonia by buffer e (Table 1). The extractability variation of dough gluten proteins in the 2-DGE protein profiles of six buffers (buffer a-f in Table 1) was clearly detected. Differentially extracted proteins (>2 folds; p<0.05) between 3 min and 32 min or between different extraction buffers are presented in Fig. S2. Detailed data for identification of the specific gluten proteins between the two time points or extraction buffers are shown in Fig. S4 and Table S1. HMW-GSs showed greater solubility at 3 min compared to 32 min (Fig. S2, J) in buffer a, c, d and e which can be attributed to either strong ionic denaturant or strong disulfide bond breaker or anionic denaturant of any strength or a combination of two. Decreased solubility of HMW-GS could be explained by denaturation of those proteins or interaction between HWM-GSs and pasting starch induced by high temperature. On the other hand, buffer b and f showed poor extractability of HMW-GSs and no significant difference was observed between two time points. A large decrease in the solubility of β-amylase and serpins were observed with all the buffers at 32 min compared to 3 min (Fig. S2, K+M). In the case of globulin and triticin, their solubility difference between 3 min and 32 min was only detected by buffer a (Fig. S2, L) where a massive extractability decrease was observed at 32 min, but no significant solubility difference was observed with the other four buffers (c, d, e and f) despite of high extractability. Notably, globulin and triticin lost their solubility in buffer b.

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In contrast, α-amylase inhibitors and SOD can be extracted by buffer c, d and e and much higher solubility appeared at 32 min than 3 min (Fig. S2, P). Complex solubility characteristics of 27K proteins and triosephosphate isomerase were observed with the six buffers (Fig. S2, O). Stable solubility between the two time points was observed for 27K proteins and triosephosphate isomerase with buffers a and d although higher abundance was observed with buffer d. In contrast, these proteins exhibited higher solubility in buffer b, c and e at 3 min, while lost solubility at 32 min. In the case of buffer f, 27K proteins were separated only before starch pasting. It was noted that the solubility variation of some of the α-, β-, and γ-gliadins, LMWGSs, ALPs, and enzymes, was not revealed by the six extraction buffers (Fig. S2, N). According to the results from 2-DGE, it is evident that buffers containing SDS not only showed high yield in protein extraction, but also performed well in distinguishing protein solubility characteristics during dough processing. However, as the major constitutes of gluten proteins, solubility changes of group N proteins (Fig. S2, N) were not well reflected by the six buffers mentioned above. So, a sequential extraction strategy using 0.5 M NaCl and buffer g together was applied to magnify the solubility differences during dough processing. Specifically, non-gluten proteins were separated by 0.5 M NaCl at the first step, and thereafter the remaining gluten proteins were further extracted by buffer g. The solubility decrease (>2 folds; p<0.05) of HMW-GSs, βamylase, and serpins, and the solubility increase of γ-gliadins, LMW-GSs, ALPs, globulins, and some enzymes at 32 min were distinctly revealed by this sequential 16 / 51

extraction protocol (Fig. 4 and Table 2). It is also notable that the extract yield increased in γ-gliadins and ALPs after starch pasting as observed in 2-DGE was in consistent with the overall solubility increase of gliadins as revealed by the SE-HPLC analysis. 4. Discussion 4.1 Combination of SE-HPLC and 2-DGE benefits the deep-analysis of dough protein behaviors By using protein extraction buffers with different levels of dissociation capacity, and combining SE-HPLC and 2-DGE, it is possible to establish a response to extractionsystem aimed at tracking protein behavior during dough development, as shown in this study, as well as in our previous researches focusing on the protein behaviors during dough processing (Wang, Appels, Zhang, Bekes, et al., 2017; Wang, Appels, Zhang, Diepeveen, et al., 2017). The overall quantitative fluctuation of different protein fractions, such as albumins/globulins, gliadins and glutenins, was detected by SEHPLC. The 2-DGE analysis was applied to characterize the detailed solubility changes of specific protein at a high resolution. This also allowed us to identify the proteins through mass spectrometric peptide sequencing. Based on the results of SE-HPLC analysis, C3/32 min (80 ℃ ) was identified as a critical point due to the great extractability loss of total protein from the dough matrix (Wang, Appels, Zhang, Bekes, et al., 2017). The 2-DGE analysis and MS/MS identification further proved that the decrease of extract yield was largely due to the solubility loss of HMW-GSs, β-amylase, serpin, 27K protein, SOD and metabolic proteins with higher mass. Furthermore, the 17 / 51

2-DGE results from different extraction buffers have the potential to track protein behaviors that were not uncovered by SE-HPLC. For example, according to the SEHPLC data, the overall solubility of albumin/globulin was decreased after starch pasting, but 2-DGE results demonstrated that peroxidase, globulin-1 and enolase-like proteins (Fig. 4 and Table 2) were released into the buffers with medium dissociation at 32 min. 4.2 Solubility characteristics of non-gluten proteins in dough matrix Most non-gluten proteins were readily separated from Mixolab-dough by the NaCl or KCl buffers before starch pasting since they stay as monomeric proteins in the dough matrix. However, some non-gluten proteins with large molecular weight intended to interact with gluten proteins or pasting starch after thermal treatment causing a decrease in their solubility (Collar & Armero, 2017; Jekle, Mühlberger, & Becker, 2016; Wang, Appels, Zhang, Bekes, et al., 2017). In contrast, non-gluten proteins with low molecular weight, such as α-amylase inhibitors and SOD (plate F: Fig. S1), remained as free monomers during dough processing, and their solubility raised with increasing temperature. Proteins identified as avenin-like b proteins and enzymes involved in glucose metabolism and GSP (plate E and G: Fig. S1) showed different solubility between NaCl and KCl buffers after starch pasting. Compared to 3 min, their solubility increased dramatically at 32 min in buffer NaCl whereas decreased in buffer KCl. This solubility variation might be explained by different pH values of the extraction buffers as reflected in the two-dimensional gel profiles. the isoelectric points of E and G 18 / 51

proteins were both around 8.0 which is close to the pH value of buffer KCl (pH 7.8), so these proteins would tend to aggregate and precipitate in buffer KCl thus causing a lower extraction than in the case of buffer NaCl (pH 7.0). Additionally, salt content in extraction buffers should also be considered as a key factor affecting the solubility of E and G proteins. The results suggested that, after continuous mixing and heating, these two protein groups exhibited much higher solubility in 0.5 M NaCl than that in 0.1 M KCl. 4.3 Solubility characteristics of gluten proteins in dough matrix The solubility characteristics of gluten proteins were more complex than non-gluten proteins. As reflected by the composition of the seven extraction buffers, the type and content of denaturant had a significant impact on the quantitative variation of gluten proteins extraction. HMW-GSs remained extractable by all the buffers before starch pasting, but change in their solubility during dough processing were better reflected by buffers containing anionic denaturant SDS compared to the buffers with ionic or nonionic denaturants. Serpins and β-amylase could be extracted from dough at 3 min by extraction buffers for both the gluten and non-gluten proteins though they lost solubility at 32 min. Previous studies have confirmed that, β-amylase can be polymerised into gluten network via disulfide bonds with LWM-GSs, while serpins easily formed into misfolded polymers due to their inherently unstable structure (Gooptu, Hazes, Chang, Dafforn, Carrell, Read, et al., 2000; Peruffo, Pogna, & Curioni, 1996). Therefore, the solubility decrease of serpins and β-amylase can be interpreted 19 / 51

by their aggregation via covalent bonds or hydrophobic interactions under thermal treatment. Strong anionic denaturant showed high extract yield of 27K proteins and triosephosphate isomerase, but their solubility differences were well exhibited by buffers containing low concentration of denaturant or NaCl (Fig. S2, O, Fig. S1). Furthermore, the sequential extraction results of dough protein at 3 min and 32 min indicated that nearly no 27K proteins and triosephosphate isomerase were extracted from the residue by buffer g after separation of salt soluble proteins using 0.5 M NaCl (Fig. 4). This observation meant that these two protein groups mainly stayed in the dough matrix as free monomers before starch pasting, but after heating, they became bound within the dough matrix with medium energy through hydrogen bonds, hydrophobic interactions or entanglements which can only be separated by buffers with strong denaturant (Julia, Birgitta, Andreas, Beatrice, & Vilgis, 2014; Lagrain, Thewissen, Brijs, & Delcour, 2008). The protein group N, including α-, β-, and γ-gliadins, LMW-GSs, ALPs, and some enzymes, accounted for a large proportion of dough proteins. However, no significant change in extractability was observed using the first six buffers although considerable extract yields were obtained. Variation in group N protein solubility characteristics during dough processing were successfully revealed by a sequential extraction strategy. Separating salt soluble proteins by 0.5 M NaCl first appeared to reduce the masking effects from monomeric non-gluten proteins and gluten proteins bound in dough matrix with low strength and thus amplified the solubility differences of gluten proteins bound

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in dough matrix with medium to high strength as shown by buffer g (Dupont, Ronald, Rocio, & Vensel, 2005; Hurkman & Tanaka, 2007). Nearly all the gluten proteins were extracted by buffer a (8 M urea, 4% CHAPS, 60 mM DTT) and no significant solubility differences were found between the two time points. Thus, buffer containing high concentration of denaturant and reduction agent was showed its potential to be used as a control for separating maximum gluten proteins indiscriminatingly at any stage of dough processing.

21 / 51

5. Conclusion The response-to-extraction system in this study provides a practical way for expanding our knowledge of dough protein dynamics during processing. The dynamic changes in binding strength of different protein fractions during dough processing were well reflected by their solubility variation in different extraction buffers. A response-toextraction system for tracking dough protein behavior under dual mixing and thermal constraint was established by selecting a set of suitable buffers with gradient dissociation capacity and combining the subsequent analysis by SE-HPLC, 2-DGE and MS/MS. To be specific, the overall solubility fluctuation for different protein fractions was quantified by SE-HPLC initially. Further identification of individual proteins by 2-DGE, indicated that the non-gluten proteins were separated by salt buffer (0.5 M NaCl, pH 7.0) firstly, and then from the residue, the gluten proteins bound in dough matrix with medium energy were isolated by buffer with medium dissociation (buffer g: 0.3% SDS, 15 mM DTT, pH 6.9). The strong extraction buffer (buffer a: 8 M Urea, 4% CHAPS, 60 mM DTT, pH 7.5) which was applied to extract the total protein during dough development was established as a control in protein behavior analysis. The application of the more defined extraction buffers for proteins in this paper are suitable for the analysis and investigation of health related attributes of foods such as gluten sensitivity. In addition, the extraction buffers provide more resolution of protein changes during the processing of wheat flour and the innovation of new products. Acknowledgements 22 / 51

This work was supported by the National Natural Science Foundation of China, (Grant No.: 31801460), Key Research and Development Program of Shaanxi (Grant No.: 2019NY-145), Fundamental Research Funds for the Central Universities (Grant No.: GK201803072), and Australia China Centre for Wheat Improvement (ACCWI), Murdoch University, Australia. References AACC International (2010). AACC International. Approved Methods of Analysis, 11th Ed. Methods 5460.01. Determination of Rheological Behavior as a Function of Mixing and Temperature Increase in Wheat Flour and Whole Wheat Meal by Mixolab. Final approval May 28, 2010. AACC International, St. Paul, MN. Békés, F., & Gras, P. W. (1992). Demonstration of the 2-gram mixograph as a research tool. Cereal Chemistry, 69, 229-230. Blazek, J., & Copeland, L. (2008). Pasting and swelling properties of wheat flour and starch in relation to amylose content. Carbohydrate Polymers, 71(3), 380-387. Broeck, H. C. V. D., America, A. H. P., Smulders, M. J. M., Bosch, D., Hamer, R. J., Gilissen, L. J. W. J., & Meer, I. M. V. D. (2009). A modified extraction protocol enables detection and quantification of celiac disease-related gluten proteins from wheat. Journal of Chromatography B, 877(10), 975-982. Chen, J., Deng, Z., Peng, W. U., Tian, J. C., & Xie, Q. G. (2010). Effect of Gluten on Pasting Properties of Wheat Starch. Agricultural Sciences in China, 9(12), 0-1844. Collar, C., & Armero, E. (2017). Impact of heat moisture treatment and hydration level on physicochemical and viscoelastic properties of doughs from wheat-barley composite flours. European Food Research & Technology(5), 1-12. Debyser, W., Derdelinckx, G., & Delcour, J. A. (1997). Arabinoxylan solubilization and inhibition of the barley malt xylanolytic system by wheat during mashing with wheat wholemeal adjunct: Evidence for a new class of enzyme inhibitors in wheat. Journal of the American Society of Brewing Chemists, 55(4), 153-156. Dupont, F. M., Ronald, C., Rocio, L., & Vensel, W. H. (2005). Sequential extraction and quantitative recovery of gliadins, glutenins, and other proteins from small samples of wheat flour. Journal of Agricultural and Food Chemistry, 53(5), 1575-1584. Dupont, F. M., Vensel, W. H., Tanaka, C. K., Hurkman, W. J., & Altenbach, S. B. (2011). Deciphering the complexities of the wheat flour proteome using quantitative two-dimensional electrophoresis, three proteases and tandem mass spectrometry. Proteome Science, 9(1), 129. Fido, R. J., Békés, F., Gras, P. W., & Tatham, A. S. (1997). Effects of α-, β-, γ- and ω-Gliadins on the Dough Mixing Properties of Wheat Flour . Journal of Cereal Science, 26(3), 271-277. 23 / 51

Figueroa, J. D. C., Maucher, T., Reule, W., & Peña, R. J. (2009). Influence of low molecular weight glutenins on viscoelastic properties of intact wheat kernel and relation to functional properties of wheat dough. Cereal Chemistry, 86(4), 372-375. Gooptu, B., ., Hazes, B., ., Chang, W. S., Dafforn, T. R., Carrell, R. W., Read, R. J., & Lomas, D. A. (2000). Inactive conformation of the serpin alpha(1)-antichymotrypsin indicates two-stage insertion of the reactive loop: implications for inhibitory function and conformational disease. Proceedings of the National Academy of Sciences of the United States of America, 97(1), 6772. Gulia, N., & Khatkar, B. S. (2015). Quantitative and Qualitative Assessment of Wheat Gluten Proteins and their Contribution to Instant Noodle Quality. International Journal of Food Properties, 18(8), 1648-1663. Gupta, R., B., Batey, I., L., & Macritchie. (1992). Relationships between protein composition and functional properties of wheat flours. Cereal Chemistry, 69(2), 125-131. Gupta, R. B., Bekes, F., & Wrigley, C. W. (1991). Prediction of physical dough properties from glutenin subunit composition in bread wheats: Correlation studies. Cereal Chemistry, 68(4), 328-333. Gupta, R. B., Khan, K., & Macritchie, F. (1993). Biochemical basis of flour properties in bread wheats. I. Effects of variation in the quantity and size distribution of polymeric protein. Journal of Cereal Science, 18(1), 23–41. Hadnađev, T. R. D., Dokić, L. P., Hadnađev, M. S., Pojić, M. M., & Torbica, A. M. (2014). Rheological and Breadmaking Properties of Wheat Flours Supplemented with Octenyl Succinic Anhydride-Modified Waxy Maize Starches. Food & Bioprocess Technology, 7(1), 235-247. Hurkman, W. J., & Tanaka, C. K. (2004). Improved methods for separation of wheat endosperm proteins and analysis by two-dimensional gel electrophoresis. Journal of Cereal Science, 40(3), 295-299. Hurkman, W. J., & Tanaka, C. K. (2007). Extraction of wheat endosperm proteins for proteome analysis. Journal of Chromatography B Analytical Technologies in the Biomedical & Life Sciences, 849(1), 344-350. Jekle, M., Mühlberger, K., & Becker, T. (2016). Starch–gluten interactions during gelatinization and its functionality in dough like model systems. Food Hydrocolloids, 54, 196-201. Juhász, A., Gell, G., Békés, F., & Balázs, E. (2012). The epitopes in wheat proteins for defining toxic units relevant to human health. Functional & Integrative Genomics, 12(4), 585-598. Julia, M., Birgitta, S., Andreas, B., Beatrice, C. P., & Vilgis, T. A. (2014). Effect of heat treatment on.wheat dough rheology and wheat protein solubility. Food science and technology international = Ciencia y tecnología de los alimentos internacional, 20(5), 341-351. Kasarda, D. D., Elva, A., Ellen J-L, L., Lazo, G. R., & Altenbach, S. B. (2013). Farinin: characterization of a novel wheat endosperm protein belonging to the prolamin superfamily. Journal of Agricultural and Food Chemistry, 61(10), 2407-2417. Kim, H. R., & Bushuk, W. (1995). Changes in some physicochemical properties of flour proteins due to partial reduction with dithiothreitol. Cereal Chemistry, 72(5), 450-456. Lagrain, B., Thewissen, B. G., Brijs, K., & Delcour, J. A. (2008). Mechanism of gliadin–glutenin crosslinking during hydrothermal treatment. Food Chemistry, 107(2), 753-760. Lawrence, G. J., & Payne, P. I. (1983). Detection by Gel Electrophoresis of Oligomers Formed by the Association of High-Molecular-Weight Glutenin Protein Subunits of Wheat Endosperm. Journal of Experimental Botany, 34(140), 254-267. 24 / 51

Meng, X. G., & Cai, S. X. (2008). Association Between Glutenin Alleles and Lanzhou Alkaline Stretched Noodle Quality of Northwest China Spring Wheats. II. Relationship with the Variations at the Glu-1 Loci. Cereal Research Communications, 36(1), 107-115. Osborne, T. B. (1924). The Vegetable Proteins. London: Longmans Green and Co. Paolo, L., Dale, S., Christine, F., Anna Maria, D. L., Anna Maria, M., Birte, S., Domenico, L., & Stefania, M. (2010). Comparative proteome analysis of metabolic proteins from seeds of durum wheat (cv. Svevo) subjected to heat stress. Proteomics, 10(12), 2359-2368. Peruffo, A. D. B., Pogna, N. E., & Curioni, C. (1996). Evidence for the presence of disulfide bonds between beta-amylase and low molecular weight glutenin subunits. In

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International Gluten Workshop, Gluten 96, (pp. 45-54). Rakszegi, M., Békés, F., Láng, L., Tamás, L., Shewry, P. R., & Bedő, Z. (2005). Technological quality of transgenic wheat expressing an increased amount of a HMW glutenin subunit. Journal of Cereal Science, 42(1), 15-23. Shahidul, I., Wujun, M., Guijun, Y., Liyan, G., & Rudi, A. (2011). Differential recovery of lupin proteins from the gluten matrix in lupin-wheat bread as revealed by mass spectrometry and twodimensional electrophoresis. Journal of Agricultural and Food Chemistry, 59(12), 6696-6704. Tasleem-Tahir, A., Nadaud, I., Chambon, C., & Branlard, G. (2012). Expression profiling of starchy endosperm metabolic proteins at 21 stages of wheat grain development. Journal of Proteome Research, 11(5), 2754-2773. Uthayakumaran, S., Tömösközi, S., Tatham, A. S., Savage, A. W. J., Gianibelli, M. C., Stoddard, F. L., & Bekes, F. (2001). Effects of Gliadin Fractions on Functional Properties of Wheat Dough Depending on Molecular Size and Hydrophobicity. Cereal Chemistry, 78(78), 138-141. Vensel, W. H., Tanaka, C. K., Nick, C., Wong, J. H., Buchanan, B. B., & Hurkman, W. J. (2010). Developmental changes in the metabolic protein profiles of wheat endosperm. Proteomics, 5(6), 1594-1611. Wang, X., Appels, R., Zhang, X., Bekes, F., Torok, K., Tömösközi, S., Diepeveen, D., Ma, W., & Islam, S. (2017). Protein-transitions in and out of the dough matrix in wheat flour mixing. Food Chemistry, 217, 542-551. Wang, X., Appels, R., Zhang, X., Diepeveen, D., Torok, K., Tömösközi, S., Bekes, F., Ma, W., Sharp, P., & Islam, S. (2017). Protein interactions during flour mixing using wheat flour with altered starch. Food Chemistry, 231, 247-257. Zhang, P., Jondiko, T. O., Tilley, M., & Awika, J. M. (2015). Effect of high molecular weight glutenin subunit composition in common wheat on dough properties and steamed bread quality. Journal of the Science of Food and Agriculture, 94(13), 2801-2806. Zhang, Y., Hu, X., Islam, S., She, M., Peng, Y., Yu, Z., Wylie, S., Juhasz, A., Dowla, M., Yang, R., Zhang, J., Wang, X., Dell, B., Chen, X., Nevo, E., Sun, D., & Ma, W. (2018). New insights into the evolution of wheat avenin-like proteins in wild emmer wheat (Triticum dicoccoides). Proceedings of the National Academy of Sciences, 115(52), 13312-13317. Zhang, Y., Tang, J., Yan, J., Zhang, Y., & Zhang, Y. (2009). The gluten protein and interactions between components determine mixograph properties in an F recombinant inbred linepopulation in bread wheat. Journal of Cereal Science, 50(2), 219-226.

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Dough temperature (℃)

100

Torque (Nm)

90

3.0

C5

80

2.5

Torque (Nm)

Temperature (℃)

Figures and tables

C3 (32 min)

70

2.0

60 50

C1 3 min

40

1.5

C4

1.0

30 20

0.5

10

C2

0 0

10

Time (min)

20

0.0 30

40

50

Fig. 1. Mixolab curve of Westonia showing sampling points: 3min (30 ℃ ) and C3/32min (80 ℃). The Y axis on the left hand side monitors the temperature changes within the dough during mixing and the Y axis on the right-hand side is the torque required to maintain the dough mixing action (standard units of torque, Newton/meter, Nm).

26 / 51

Glutenin

1.15

Protein abundance (ratio to Westonia flour)

Protein abundance (ratio to Westonia flour)

A

1.10 1.05 1.00 0.95 0.90

0

3

Gliadin

1.15 1.10 1.05 1.00 0.95 0.90

32

0

1.20

Albumin/Globulin

1.15 1.10 1.05 1.00 0.95 0.90

0

3

0

32

40

70 60 50 40 30 20 10 0

0

UPP%

0

3

Time (min)

32

43 42 41 40 39

3 32 Time (min)

Protein content (%)

41

18 16 14 12 10 8 6 4 2 0

18

Gli%

44

0

32

UG%

0

3 32 Time (min)

Alb/Glob%

17 16 15 14

3 32 Time (min)

0

6 Protein content (%)

42

39

Protein content (%)

Protein content (%)

43

3 Time (min)

45

Glu%

44

Protein content (%)

Protein content (%)

45

32

Total protein

1.12 1.10 1.08 1.06 1.04 1.02 1.00 0.98 0.96 0.94

Time (min)

B

3 Time (min)

Protein abundance (ratio to Westonia flour)

Protein abundance (ratio to Westonia flour)

Time (min)

3 32 Time (min)

UAlb/Glob%

5 4 3 2 1 0

0

3

32

Time (min)

Fig. 2. Relative amounts (A) and amount change (B) of different protein fractions during Mixolab dough preparation as identified by SE-HPLC. In group A, Y-axis indicates the relative abundance of the extractable proteins with Westonia flour as a control (abundance is 1.0), and the relative amount of the respective protein group was calculated based on the total peak area of proteins within the group as determined by SE-HPLC system. In group B, Y-axis indicates content of different protein fractions in the dough extracts. 27 / 51

Fig. 3. Two-dimensional gel profile showing typical distribution pattern of gluten proteins extracted from 3 min (30 ℃ ) dough of Westonia by buffer e (Table 1). 28 / 51

Different regions marked on the gel profile represented different protein groups. Specifically, J: HMW-GS; K: β-amylase; L: globulin and triticin; M: Serpins; N: α/βand γ-gliadins, LMW-GSs, ALPs and some enzymes; O: 27K proteins and Triosephosphate isomerase; P: α-amylase inhibitors and SOD. A higher resolution of plates J, K, L, M, N, O, and P were showed in Fig. S2.

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30 / 51

Fig. 4. Two-dimensional gel electrophoresis profile of gluten protein extracted from Westonia flour and dough by buffer g based on the sequential extraction protocol. The marked protein spots showing significant quantitative differences (P<0.05, 2+ folds) between 3 min and 32 min. At 32 min, solubility decrease (p<0.05, 2+ folds) in HMW-GSs, β-amylase, serpins, as well as the extractability increase in γ-gliadins, LMW-GSs, ALPs, globulins, and some enzymes were distinctly revealed by this sequential extraction protocol. The data of identification and abundance variations of the specific proteins between 3 min and 32 min can be found in the Table 2.

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Table 1. Formula and function of the solutions used for separating gluten proteins form Mixolab dough and flour of Westonia

Urea CHAPS Acetic

a

(M)

(%)

8

4

acid (M)

SDS Triton X-100 DTT Sonication Tris-HCl PMSF (%)

4

c

2

d e g

1.5

(mM) (s)

(mM)

A

pH

7.5

2

33 30

Function

(mM) (mM)

60

b

f

(%)

EDT

1.7

17

1+6 5+7

50

6.9

3+6

2

50

6.9

3

0.3

50

6.9

4

3.8

2

6.9

4+7

0.25 0.3

15

Buffer g was only used for the sequential extraction strategy, i.e extracting gluten proteins form the residue left after separating non-gluten proteins with 0.5 M NaCl. The numbers list in the “Function” column indicates the dissociation properties of each buffer, their explanation as following: 1: Strong ionic denaturant; 2: Weak ionic denaturant; 3: Strong anionic denaturant; 4: Weak anionic denaturant; 5: Nonionic denaturant; 6: Strong disulfide bond breaker; 7: Weak disulfide bond breaker.

32 / 51

Table 2. Identification of differential expressed proteins between 3 and 32 min as reflected by the sequential extraction protocol using buffer g. Spot ID

Accession

Protein

Mw (KDa)/PI Value

Matched peptides

Average volume ratio of flour, 3 min and 32 min

Proteins with decreased abundance at 32 min compared to 3 min 2

gi/475523854

Beta-amylase

61.08/5

12(6)

1:0.7:0

3

gi/3334120

Beta-amylase

56.58/5.24

3(3)

1:0.98:0

4

gi/474451266

Beta-amylase

58.71/5.34

11(8)

1:0.83:0.02

7

gi/379060943

Serpin-N3.2

42.97/5.18

11(10)

1:0.56:0

8

gi/75313847

Serpin-Z2A

43.28/5.46

7(5)

1:0.3:0

89

gi/30793446

27K protein, partial

25.14/6.35

2(1)

1:0.21:0

63

gi/262205150

High-molecular-weight glutenin Dx2

88.88/5.82

3(3)

1:0.59:0

71

gi/171027813

Triticin

64.84/6.37

9(7)

1:0.63:0.06

101

gi/474246751

TRIUR3_30124 12S seed storage globulin 1

63.83/6.62

4(1)

1:0.9:0

109

gi/253783729

Glyceraldehyde-3-phosphate dehydrogenase

38.39/7.09

4(3)

1:0.1:0.02

144

NS

1:0.36:0

145

gi/169666929

GluB3-6

44.61/8.91

5(2)

1:0.05:0

168

gi/601292771

Alpha-gliadin protein

34.26/8.24

2(1)

1:0.07:0

170

gi/586499463

Gamma-gliadin

34.55/8.73

5(2)

1:0.15:0

171

gi/586499463

Gamma-gliadin

34.55/8.73

8(6)

1:0.29:0

182

gi|421932514

gli-2 K7X0R6_WHEAT Alpha-gliadin

32.72/8.58

1(1)

1:0.32:0

F15

gi/474072546

Aspartic proteinase oryzasin-1

54.21/6.67

4(0)

1:0.11:0

F16

NS

1:0.2:0

Proteins with increased abundance at 32 min compared to 3 min 79

gi/573955321

probable disease resistance protein RXW24L-like

112.02/7.99

1(1)

1:0.13:0.53

93

gi/54778521

0.19 dimeric alpha-amylase inhibitor, partial

13.31/7.45

5 (2)

1:0:2.19

94

gi/54778521

Dimeric alpha-amylase inhibitor

15.18/5.58

15(10)

1:0:1.49

33 / 51

Average volume ratio

Spot ID

Accession

Protein

Mw (KDa)/PI Value

Matched peptides

95

gi/21713

Alpha-amylase/trypsin inhibitor CM3

18.21/7.44

11(7)

1:0:1.39

96

gi/54778521

0.19 dimeric alpha-amylase inhibitor, partial

13.31/7.45

31(19)

1:0:0.59

152

gi/390979705

Globulin-3A

66.29/8.48

8(0)

1:0:0.35

153

NS

of flour, 3 min and 32 min

1:0:0.97

178

gi/281335544

gliadin/avenin-like seed protein

11.32/8.03

7(0)

1:0:0.07

179

gi/281335544

gliadin/avenin-like seed protein

11.32/8.03

7(0)

1:0:3.42

183

gi|390979705

Globulin-3A

66.29/8.48

28(2)

1:0:0.32

103

gi/195957140

Major allergen CM16

15.77/4.86

22(0)

1:0::4.32

111

gi/227344090

Low molecular weight glutenin

34.83/9.06

1(0)

1:0:0.41

123

gi/140169817

HMW-GS 1Dy12.3

70.00/8.05

5(1)

1:0.52:2.5

124

gi/21779

Glutenin, high molecular weight subunit 12-SwissProt

70.82/7.64

1(0)

1:1.13:87.74

125

gi/568874288

Enolase-like

48.30/6.19

12(5)

0:0:1

126

gi/568874288

Enolase-like

48.30/6.19

14(7)

0:0:1

127

gi/568874289

Enolase-like

48.30/6.19

8(2)

1:0:1.32

128

gi/121101

Gamma-gliadin

37.10/7.62

2(2)

1:0:0.6

129

gi/121101

Gamma-gliadin

37.10/7.62

2(2)

1:0.14:2.02

130

gi/121101

Gamma-gliadin

37.10/7.62

2(2)

1:0:0.67

135

gi/22001285

Peroxidase

38.87/7.62

6(0)

1:0:1.43

137

gi/148906038

Putative uncharacterized protein

41.02/8.62

1(1)

1:0.09:1.37

138

gi/42562301

U-box domain-containing protein 6

85.27/6.02

2(2)

1:0.11:1.28

139

gi/121101

Gamma-gliadin

37.10/7.62

2(1)

1:0:0.9

140

gi/110341795

Globulin 1

24.54/8.57

2(1)

1:0:1.41

141

gi/110341795

Globulin 1

24.54/8.57

2(1)

1:0:0.97

142

gi/110341795

Globulin 1

24.54/8.57

2(2)

1:0:1.11

34 / 51

Average volume ratio

Spot ID

Accession

Protein

Mw (KDa)/PI Value

Matched peptides

143

gi/110341795

Globulin 1

24.54/8.57

4(1)

1:0:1.35

159

gi/363992662

Gamma gliadin

30.48/8.96

1(1)

1:0.09:0.41

160

gi/22001285

Peroxidase

38.87/7.62

4(0)

1:0.21:0.86

161

gi/22001285

Peroxidase

38.87/7.62

5(0)

1:0.05:0.71

164

gi/475567072

Fructose-bisphosphate aldolase

38.79/6.85

5(0)

1:0.11:1.28

of flour, 3 min and 32 min

Protein abundance of flour sample was set as 1 in the comparison. Two-dimensional gel profiles for these proteins were showed in Fig. 4. NS indicates no significant match in the database.

35 / 51

Supplementary figures and tables 32 min (80℃)

3 min (30℃)

Flour （0 min）

A

B I E

C G

H

D F

KCl

KCl

NaCl

NaCl

KCl

NaCl

Fig. S1. Two dimentional gel electrophoresis profile of dough and flour proteins of Westonia extracted by NaCl and KCl, respectively. Proteins spots presented in the marked regions demonstrateing differntially expressed proteins (p<0.05, 2+ folds) between the two time points. For the gels of 3 min (30℃) and 32 min (80℃) using the same extraction buffer, dotted lines on one gel indicate that the extractable proteins in the enclosed regions decreased comppred to the gel from another timepoint. A: βamylase; B: Globulin; C: Serpins; D: Superoxide dismutise (SOD) and 27 K protein; E: Globulin, avenin-like b protein, enzymes involved into glucose metabolism; F: αamylase inhibitors, SOD; G: Grain softness protein (GSP); H: Triosephosphate isomerase. I: Glyceraldehyde-3-phosphate dehydrogenase and aldose reductase. More data for the identification of the differential proteins between the two time points was persented in the Fig. S3 and Table S1. 36 / 51

J

K+M

37 / 51

L

a

b

c

d

e

f 38 / 51

N

39 / 51

40 / 51

Fig. S2. Comparison of different extraction buffers for separating gluten proteins from the dough of Westonia. The enclosed protein spots on the profile indicating significant quantitative differences (P<0.05, 2+ folds) between 3 min and 32 min (horizontal plates) or among idfferent extraction buffers (vertical plates). The coordinates of different protien fractions on the gel profile were illustraged in Fig. 3. J: HMW-GS; K: β-amylase; L: globulin and triticin; M: Serpins; N: α/β- and γ-gliadins, LMW-GSs, ALPs and some enzymes; O: 27K proteins and triosephosphate isomerase; P: α-amylase inhibitors and SOD. The lowcase letters (a, b, c, d, e and f) shown next to the figures represent different extraction buffers listed in Table 1. Detailed data of the identification of these specific proteins were presented in Fig. S4 and Table S1.

41 / 51

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32 min (0.5 M NaCl) 3 min (0.5 M NaCl)

Fig. S3 Two-dimensional gel profiles of non-gluten proteins extracted from Westonia dough at 3 min and 32 min by 0.5 M NaCl. The number on the spots indicated their identification by mass spectrometry as listed in the Table S1. The solubility of non-gluten proteins in NaCl and KCl buffer were showed in Fig. S1.

43 / 51

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3 min (buffer a)

Fig. S4 Two-dimensional gel profile of gluten proteins extracted from Westonia dough at 3 min by buffer g (see Table 1). The number on the spots indicated their identification by mass spectrometry as listed in the Table S1. The solubility comparison of gluten proteins in different extraction buffers were showed in Fig. S2. Table S1. List of the identified dough proteins with different solubility between 3 min and 32 min as revealed by different extraction buffers Spot ID

Accession

Protein

Species

Score/ coverage%

Theoretical Mw (KDa) /PI Value

Matched peptides

1 2 3 4 5 6 7

gi/731910779 gi/475523854 gi/3334120 gi/474451266 gi/390979705 gi/20259685 gi/379060943

Heat shock protein 70 Beta-amylase Beta-amylase Beta-amylase Globulin-3A Beta-D-glucan exohydrolase Serpin-N3.2

Ornithogalum saundersiae Triticum aestivum Triticum aestivum Triticum urartu Triticum aestivum Triticum aestivum Triticum aestivum

425/12 903/27 334/8 952/28 319/12 357/8 976/43

71.01/5.11 61.08/5 56.58/5.24 58.71/5.34 66.28/8.48 67.26/6.86 42.97/5.18

5(4) 12(6) 3(3) 11(8) 5(4) 6(4) 11(10)

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Spot ID

Accession

Protein

Species

Score/ coverage%

Theoretical Mw (KDa) /PI Value

Matched peptides

9 11

gi/75313847 gi/474323981

Serpin-Z2A Globulin-1 S allele

Triticum aestivum Triticum urartu

1102/32 594/22

43.28/5.46 55.30/7.77

10(10) 9(5)

12 13

gi/474323981 gi/390979705

Globulin-1 S allele Globulin-3A

Triticum urartu Triticum aestivum

545/20 371/10

55.30/7.77 66.28/8.48

8(4) 5(5)

14 15

gi/224589266 gi/511779049

Serpin 1 BiP3 ER molecular chaperone

Triticum aestivum Triticum aestivum

206/7 984/19

43.09/5.44 73.10/5.11

3(3) 11(8)

16 19

gi/253783729 gi|253783729

Glyceraldehyde-3-phosphate dehydrogenase Glyceraldehyde-3-phosphate dehydrogenase

Triticum aestivum Triticum aestivum

689/24 207/14

38.39/7.09 36.63/7.07

8(7) 3(1)

20 22

gi/253783729 gi/113595

Glyceraldehyde-3-phosphate dehydrogenase Aldose reductase

Triticum aestivum Hordeum vulgare

589/23 93/13

38.39/7.09 35.78/6.51

7(6) 4(1)

24 25

gi/390979705 gi/390979705

Globulin-3A Globulin-3A

Triticum aestivum Triticum aestivum

570/20 555/20

66.28/8.48 66.28/8.48

9(5) 9(5)

26 33

gi/390979705 gi/343455543

Globulin-3A SOD Superoxide dismutase

Triticum aestivum Triticum aestivum

499/15 73/6

66.28/8.49 25.33/7.17

7(5) 2(2)

34

gi|290350670

Triticum aestivum

132/9

25.08/5.9

5(3)

35

gi/474329936

Uncharacterized protein/88% ident with27K protein, partial Avenin-3

Triticum urartu

102/22

22.20/6.35

3(1)

36 37 38 39 40 43

gi/227809455 gi/21713 gi/110341795 gi/669027789 gi/380875808 gi/475531176

Dimeric alpha-amylase inhibitor Alpha-amylase/trypsin inhibitor CM3 Globulin 1 Histone H2B Superoxide dismutase [Cu-Zn] F775_09613 Lactoylglutathione lyase

Triticum dicoccoides Triticum aestivum Triticum aestivum Triticum aestivum Deschampsia antarctica Aegilops tauschii

49/9 57/8 127/25 110/17 139/28 224/29

15.09/4.96 18.21/7.44 24.53/8.57 14.75/9.94 15.12/5.7 32.55/5.43

2(0) 2(1) 3(1) 2(1) 5(1) 9(1)

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Spot ID

Accession

Protein

Species

Score/ coverage%

Theoretical Mw (KDa) /PI Value

Matched peptides

44 46

gi/475531176 gi/380875808

F775_09613 Lactoylglutathione lyase Superoxide dismutase [Cu-Zn]

Aegilops tauschii Triticum aestivum

184/23 420/42

32.55/5.43 15.13/5.7

7(1) 13(6)

47 48

gi/226897529 gi/259017810

SOD1 Superoxide dismutase [Cu-Zn] DHAR Dehydroascorbate reductase

Triticum aestivum Triticum aestivum

481/42 370/29

15.13/5.71 23.34/5.88

29(18) 5(3)

49 50

gi/123975 gi/123975

Endogenous alpha-amylase/subtilisin inhibitor Endogenous alpha-amylase/subtilisin inhibitor

Triticum aestivum Triticum aestivum

335/46 202/30

19.62/6.77 19.62/6.77

7(2) 9(2)

51 52

gi/122022 gi/54778521

Histone H2B 0.19 dimeric alpha-amylase inhibitor, partial

Triticum aestivum Aegilops tauschii

246/43 244/29

16.47/10 13.31/7.45

7(0) 16(6)

53 54

gi/123975 gi/284178233

Endogenous alpha-amylase/subtilisin inhibitor Xylanase inhibitor protein

Triticum aestivum Triticum aestivum

704/62 435/29

19.62/6.77 33.29/8.66

36(11) 8(4)

55 56

gi/62465516 gi/30793446

Class II chitinase 27K protein, partial

Triticum aestivum Triticum aestivum

123/14 116/8

28.24/8.66 22.76/6.06

2(1) 2(1)

57

gi/30793446

27K protein, partial

Triticum aestivum

79/7

25.08/6.35

2(0)

58

gi/669027442

Triticum aestivum

573/60

16.82/5.83

18(7)

63 64

gi/262205150 gi/170743

Chromosome 3B, genomic scaffold, cultivar Chinese Spring High-molecular-weight glutenin Dx2 HMW glutenin subunit Ax2

Triticum aestivum Triticum aestivum

282/4 1194/20

88.88/5.82 88.42/6.15

3(3) 11(9)

65 66 67 68 69 70

gi/260401175 gi/260401173 gi/16148

Gamma gliadin Gamma gliadin 1-aminocyclopropane-1-carboxylate synthase NS Triticin Globulin-1 S

Triticum aestivum Triticum aestivum Arabidopsis thaliana

81/4 78/4 31/1

35.01/8.9 35.97/7.59 55.50/7.2

1(1) 1(1) 1(1)

Triticum aestivum Triticum urartu

55/1 194/18

64.84/6.37 55.30/7.77

1(1) 10(2)

gi/171027813 gi/474323981

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Spot ID

Accession

Protein

Species

Score/ coverage%

Theoretical Mw (KDa) /PI Value

Matched peptides

71 72

gi/171027813

Triticin NS

Triticum aestivum

826/17

64.84/6.37

9(7)

73 74

gi/485474709 gi/601292771

LMW-i1 Low molecular weight glutenin subunit Alpha-gliadin protein

Triticum aestivum Triticum aestivum

51/2 118/5

36.9/9.02 34.26/8.24

1(0) 1(1)

75 76

gi/23451222 gi/601292771

alpha/beta-gliadin, partial Alpha-gliadin protein

Triticum aestivum Triticum aestivum

62/10 92/5

13.80/7.94 34.26/8.24

1(1) 1(1)

77 78

gi/475542024 gi/260401173

Globulin-1 S Gamma gliadin

Aegilops tauschii Triticum aestivum

111/6 65/4

51.56/6.91 35.97/7.59

2(2) 1(1)

79

gi/573955321

Oryza brachyantha

63/0

112.02/7.99

1(1)

80 81

gi/601292771 gi/21783

probable disease resistance protein RXW24Llike Alpha-gliadin protein Glutenin, low molecular weight subunit

Triticum aestivum Triticum aestivum

74/5 37/1

34.26/8.24 41/9.04

1(1) 1(1)

82

gi/474186084

Alpha/beta-gliadin MM1

Triticum urartu

82/4

23.54/7.77

1(1)

83 84 85 86

gi/23451222 gi/30793446 gi/30793446 gi/11124572

Alpha/beta-gliadin 27K protein, partial 27K protein, partial Triosephosphate isomerase

Triticum aestivum Triticum aestivum Triticum aestivum Triticum aestivum

50 160/8 142/8 308/18

36.51/6.73 22.76/6.06 22.76/6.06 26.82/5.38

1(1) 2(2) 2(1) 4(3)

87 94 95 96 97 99

gi/11124572 gi/54778521 gi/21713 gi/54778521 gi/54778521 gi/75107149

Triosephosphate isomerase Dimeric alpha-amylase inhibitor Alpha-amylase/trypsin inhibitor CM3 0.19 dimeric alpha-amylase inhibitor, partial 0.19 dimeric alpha-amylase inhibitor, partial Chymotrypsin inhibitor WCI

Triticum aestivum Aegilops tauschii Triticum aestivum Aegilops tauschii Aegilops tauschii Triticum aestivum

271/17 357/29 340/50 716/51 152/29 148/16

26.82/5.57 15.18/5.58 18.21/7.44 13.31/7.45 13.31/7.45 12.9/7.42

4(2) 15(10) 11(7) 31(19) 8(4) 4(2)

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Spot ID

Accession

Protein

Species

Score/ coverage%

Theoretical Mw (KDa) /PI Value

Matched peptides

113 121

gi/260600233 gi/387812326

Avenin-like b7 HMW-GS 1Sy18* subunit

Triticum aestivum Aegilops speltoides

64/3 281/25

32.33/7.83 70.67/8.05

1(1) 15(5)

122 123

gi/140169817 gi/140169817

HMW-GS 1Dy12.3 HMW-GS 1Dy12.3

Triticum aestivum Triticum aestivum

64/14 141/16

70.00/8.05 70.00/8.05

4(0) 5(1)

124

gi/21779

Triticum aestivum

21/3

70.82/7.64

1(0)

131 132

gi/110341796

Glutenin, high molecular weight subunit 12SwissProt HMW-GS 1By8 NS

Triticum aestivum

77/11

77.27/8.64

5(1)

155 156

gi/110341801 gi/110341801

Globulin 1 Globulin 1

Triticum aestivum Triticum aestivum

167/25 185/30

24.54/8.05 24.54/8.05

7(3) 9(4)

157 158

gi/110341790 gi/110341795

Globulin 1 Globulin 1

Triticum aestivum Triticum aestivum

204/25 296/33

24.98/8.72 24.54/8.57

6(4) 15(7)

160

gi/22001285

Peroxidase

Triticum aestivum

88/15

38.87/7.62

4(0)

161 175 176 E56

gi/22001285 gi/122232330 gi/338817622 gi|1086237|S4 8186 gi|1086237|S4 8186 gi|60652218|gb |AAX33226.1

Peroxidase Avenin-like b1 Avenin-like b2 Grain softness protein

Triticum aestivum Triticum aestivum Triticum aestivum Triticum aestivum

109/14 59/11 71/11 78/27

38.87/7.62 32.71/8.08 32.49/7.82 11.32/8.03

5(0) 5(0) 6(0) 7(0)

Grain Softness Protein 1

Triticum aestivum

109/14

18.18/8.33

6(2)

Grain softness protein-1B2, partial

Triticum aestivum

130/16

15.99/7.45

3(1)

E57 E58

NS indicates no significant match in the database 49 / 51

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

1. Binding strength of dough protein is showed by its solubility in a set of buffers.

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2. Starch pasting causes overall solubility loss of total protein in dough matrix. 3. Gliadins and some metabolic proteins are released from dough matrix after heating. 4. A response to extraction-system is developed for tracking protein behavior.

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