1 1 Quantitative proteomics analysis of barley-based liquid feed and

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Omics Technologies Applied to Agriculture and Food

Quantitative proteomics analysis of barley-based liquid feed and the effect of protease inhibitors and NADPHdependent thioredoxin reductase/thioredoxin (NTR/Trx) system Abida Sultan, Birgit Andersen, Jesper Bjerg Christensen, Hanne Damgaard Poulsen, Birte Svensson, and Christine Finnie J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01708 • Publication Date (Web): 16 May 2019 Downloaded from http://pubs.acs.org on May 16, 2019

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Journal of Agricultural and Food Chemistry

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Quantitative proteomics analysis of barley-based liquid feed and the effect of protease

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inhibitors and NADPH-dependent thioredoxin reductase/thioredoxin (NTR/Trx) system

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Abida Sultana, §*, Birgit Andersenb,†, Jesper Bjerg Christensenc, Hanne Damgaard Poulsenc, Birte

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Svenssona, Christine Finnieb

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a. Enzyme and Protein Chemistry, Department of Biotechnology and Biomedicine,

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Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs. Lyngby,

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Denmark.

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b. Agricultural and Environmental Proteomics, Department of Systems Biology,

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Technical University of Denmark, Søltofts Plads, Building 224, DK-2800 Kgs.

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Lyngby, Denmark.

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c. Department of Animal Science, Animal nutrition and physiology, Aarhus University,

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Blichers Allé 20, Building S20, DK-8830 Tjele, Denmark.

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§

Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet, Building 220,

DK-2800 Kgs. Lyngby, Denmark. †

Section of Plant and Soil Science, Department of Plant & Environmental Sciences, University of Copenhagen,

Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark.

1 ACS Paragon Plus Environment


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Abstract

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Liquid feeding strategies have been devised with the aim of enhancing grain nutrient

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availability for livestock. It is characterized by a steeping/soaking period that softens the

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grains and initiates mobilization of seed storage reserves. The present study uses 2D gel-based

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proteomics to investigate the role of proteolysis and reduction by thioredoxins over a 48 h of

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steeping period by monitoring protein abundance dynamics in barley-based liquid feed samples

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supplemented with either protease inhibitors or NADPH-dependent thioredoxin

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reductase/thioredoxin (NTR/Trx). Several full-length storage proteins were only identified in

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the water-extractable fraction of feed containing protease inhibitors, illustrating significant

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inhibition of proteolytic activities arising during the steeping period. Application of functional

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NTR/Trx to liquid feed reductively increased solubility of known and potentially new Trx-

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target proteins, e.g. outer membrane protein X, and their susceptibility to proteolysis. Thus

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NTR/Trx system exhibits important potential as a feed additive to enhance nutrient

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digestibility in monogastric animals.

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Keywords: Barley proteome, mass spectrometry, NADPH-dependent thioredoxin

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reductase/thioredoxin system, protease inhibitors

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Introduction

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Monogastric animals are unable to efficiently utilize cereal grain most nutrients including

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nitrogen from grain proteins. Thus, in the past years several strategies have been devised with

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the aim of enhancing nutrient availability with application of proteins/enzymes, essential

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amino acids and minerals. Moreover, instead of dry feeding systems, there has been an

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increase in the use of liquid feeding systems, which is based on cereals soaked in water during

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an incubation period prior to delivery to the livestock 1–3. It is suggested that the steeping

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period softens the grains and mobilizes nutrient components, thereby increasing their

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digestibility. During fermentation, the abundance of lactic acid bacteria will increase, as well

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as the production of organic acids (e.g. lactic acid and butyric acid), which can reduce the

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growth of pathogenic bacteria, and promote gut health and livestock growth performance 4.

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High moisture airtight storage of barley resulted in increased protein solubility compared to

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dry storage (13% humidity) 5, which increased the digestibility of protein in pigs (Ton Nu,

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unpublished results). A review of different feeding studies revealed that liquid feed diets

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generally provided an increased apparent ileal digestibility of proteins, improved weight gain

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and feed conversion ratio 2,3. Proteins constitute an average of 1012% of the grain dry weight,

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of which 80% are storage proteins and the remainder are metabolically active proteins 6. The

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major storage proteins in the barley endosperm are the hordeins, which are deficient in some

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amino acids that are essential to monogastric animals, in particular lysine, methionine and

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threonine. Therefore, barley-based feed is supplemented with other sources of proteins, such as

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legumes or fish proteins 7.

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Seed germination commences upon imbibition and involves activation and de novo synthesis

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of hydrolytic enzymes, such as amylases, glucanases, limit dextrinases and proteases, in the

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aleurone layer that can degrade cell walls and starch, and mobilize reserve storage proteins,



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carbohydrates accumulated in the endosperm to sustain the growing embryo. The protein

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reserves are mobilized by proteases released from the aleurone layer as well as by pre-formed

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proteases already present in the endosperm 8,9. Proteases play a vital role in plant physiology

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by controlling synthesis, turnover and function of proteins. While indispensable to the plant,

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proteases can be potentially detrimental when overexpressed, e.g. cause premature sprouting

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10 ,

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involves modulation of their activities by protease inhibitors, which are widely distributed in

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various tissues of the grain. Plant protease inhibitors are presumed to be a tool of defense

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against proteolytic enzymes of the invading microorganisms and insect pests, as well as in the

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animal gut 8,11,12. Christensen et al. (2014) applied barley recombinant endoprotease B2 to

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soaked and buffered ground barley (pH 4.3) and reported an increase in protein solubility, due

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to increased proteolysis. However, it should be noted that quite high enzyme concentration had

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to be used (9 U/g barley flour) in order to exceed the inhibitory effect of the endogenous

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protease inhibitors 13.

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Upon imbibition and germination, seed proteins transition from the oxidized disulfide form (in dry

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seeds) to more reduced state (sulfhydryl -SH) by action of the cytosolic reductase thioredoxin h

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(Trx) 14. Thioredoxin h is the barley´s own enzyme and promotes mobilization of storage proteins in

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the endosperm via reduction of intramolecular disulfide bonds, altering the protein structure leading

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to loss of biological activity (if any), increased solubility and decreased resistance to proteolysis 15–

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17.

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reductases (NTR) using NADPH. In plants, this tripartite system is highly complex with the

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presence of a diverse collection of Trx isozymes showing distinct temporal- and organelle-specific

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expression. Thioredoxins have also been reported to influence the activity of various proteases (e.g.

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thiocalsin) and amylolytic enzymes (e.g. α-amylase) by reduction of disulfide bonds either directly

hence their activities need to be closely and correctly regulated. A means of regulation

For most organisms, Trxs are reduced enzymatically by NADPH-dependent thioredoxin


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or indirectly by inactivation of specific inhibitor proteins 16,18–21. Reductively inactivating inhibitors

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of proteases and amylases or activating specific proteases all aim toward breakdown of protein and

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starch reserves during germination. Proteomics approaches have enabled identification of a wide

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range of Trx target proteins 21–25. Germinating grains from transgenic barley with overexpression of

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Trx in the endosperm resulted in an increase in protein solubility 21,23. Thioredoxins have also been

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utilized for modification of the solubility of proteins in wheat endosperm 23. Thus, the natural

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NTR/Trx system presents potential for application in the feed industry by facilitating the

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mobilization of seed storage reserves.

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There is a lack of knowledge on susceptibility of dietary proteins and effects of supplemental

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additives/enzymes on the proteolytic activities occurring in feed during the steeping/soaking

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period. In view of this, the aim of the present study was to profile the proteomes of water-

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extractable and urea-extractable protein fractions in barley-based liquid feed over 48 h

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steeping, and monitor protein abundance dynamics associated with application of protease

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inhibitors and functional NTR/Trx system to liquid feed.

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Materials and Methods

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Materials

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Barley grains of cultivar Keops were obtained from Sejet Plant Breeding, Horsens, Denmark (9° 50'

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51.32'' E, 55° 51' 29.27'' N, 34 m). Roche protease inhibitor cocktail were purchased from Merck

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(Darmstadt, Germany). IPG strips, 2D-gels, ampholytes and Destreak reagent were purchased

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from GE Healthcare Life Sciences (Brøndby, Denmark). Trypsin (sequencing grade) was

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purchased from Promega (Stockholm, Sweden). NuPage Bis-Tris gels and protein ladder Mark12



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were purchased from Fisher Scientific (Roskilde, Denmark). Unless specified all the chemicals

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were purchased from Sigma-Aldrich (Copenhagen, Denmark).

106 107

Liquid feed preparation and protein extraction

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Barley-based liquid feed was prepared from mature barley grains (cultivar Keops) harvested in

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2009. Briefly, 100 mg of flour (roller mill 2 mm) was mixed with 275 μL water supplemented

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with either 17 μL of (a) protease inhibitor cocktail (Roche, 25x stock solution, inhibiting serine

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and cysteine proteases) and pepstatin A dissolved in 5% DMSO (Sigma Aldrich, against

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aspartic proteases), (b) 5% DMSO, (c) NTR/Trx (0.03 mg NTR, 0.06 mg Trx and 0.06 mg

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NADPH), or (d) water. Two glass beads were added to aid homogenization and the samples

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were incubated for 48 h at 20°C under continuous agitation. Aliquots were withdrawn at 2 and

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48 h. Constant incubation temperature of 20°C was chosen, as it has been found that optimal

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steeping of liquid feed can be achieved at that temperature 2. Samples were centrifuged (20,000

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x g, 30 min, 4°C) and the supernatants (water-extractable fraction) were collected. The pellets

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were freeze-dried, ground using pestle and mortar and resuspended in 1 mL extraction buffer

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(7 M urea, 2 M thiourea, 2% CHAPS, 0.5% ampholytes pH range 4−7 (GE Healthcare), 200

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mM Destreak reagent (bis (2-hydroxyethyl) disulfide, GE Healthcare), 2% glycerol, 5%

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isopropanol) containing protease inhibitor cocktail (Roche) for 1 h at 4°C under agitation.

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After centrifugation (20,000 x g, 30 min, 4°C), the supernatants (urea-extractable protein

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fraction) were collected and quantified as described below. For each treatment, four replicates

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were prepared.

125 126

Protein content determination and SDS-PAGE


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The protein content of the supernatants (water-extractable and urea-extractable fractions) were

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determined using the amido black method with bovine serum albumin as standard 26.

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Moreover, 10 μL of water-extractable and urea-extractable protein fractions were analyzed by

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SDS-PAGE using 4−12% Bis Tris NuPAGE gels (Novex system, Invitrogen) according to the

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manufacturer’s instructions. The gels were stained with colloidal Coomassie Blue 27 and a broad-

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range molecular mass protein ladder (Mark 12TM, Invitrogen) was used.

133 134

2D-gel electrophoresis of protein extracts of liquid feed

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For 2-DE, 250 μg protein was precipitated overnight at - 20°C in 4 volumes of acetone,

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centrifuged (4,000 x g, 10 min, 4°C), and the pellet dissolved in 350 μL rehydration buffer (7

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M urea, 2 M thiourea, 2% CHAPS, 0.5% ampholytes pH 4−7, 200 mM destreak reagent, trace

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of bromophenol blue). The samples were applied to 18 cm pH 4−7 IPG strips (GE Healthcare)

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for isoelectric focusing (EttanTM IPGphor, GE Healthcare). After rehydration (12 h, 50

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mA/strip, 20°C), isoelectric focusing was performed to reach a total of 50 kVh (4 h at 150 V, 5

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h at 300 V, 1 h at 500 V, 1 h at 1000 V, gradient to 8000 V, hold at 8000 V until a total of 50

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kVh). The strips were equilibrated in 5 mL equilibration buffer (6 M urea, 2% SDS, 30%

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glycerol, 50 mM Tris HCl, pH 8.8, 0.01% bromophenol blue) supplemented with 1%

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dithiothreitol and 2.5% iodoacetamide in first and second equilibration step each for 15 min.

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SDS-PAGE (12−14%, 18 cm × 24 cm, GE Healthcare) was performed on a Multiphor II (GE

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Healthcare) according to the manufacturer’s recommendations using Mark 12 as protein

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ladder. 2-DE was performed in quadruplicate (four biological replicates of each treatment,

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Supplementary Fig. S1) and the gels were stained with colloidal Coomassie Blue 27.

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Image analysis 7 ACS Paragon Plus Environment


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The gel images were scanned (grey scale, 16 Bit) and analyzed by the image analysis software

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Progenesis Samespots v4.0, (nonlinear Dynamics, Newcastle upon Tyne, UK). Gel images

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were warped, matched and aligned to a selected reference gel by automated calculation of

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alignment vectors after fifteen manually assigned landmark vectors. Images of biological

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replicates for each of the treatments were grouped to obtain the average volume of each spot.

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The spot volumes were normalized across different gel images automatically by the software.

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A threshold of 1.5−fold spot volume ratio change, ANOVA p < 0.05, and a false discovery rate

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of 5% (q < 0.05) was chosen as criterion to identify differentially abundant protein spots.

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Power analysis was applied to determine the sample size required to confidently accept the

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outcome of a statistical test (recommended value 80%) 28. The experimental setup with four

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replicate gels had sufficient statistical power (power > 0.8). Principal component analysis

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(PCA) and cluster analysis (dendrogram) were performed to analyze the protein spot patterns

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among gels and abundance profiles of protein spots fulfilling the above criteria.

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In-gel digestion and mass spectrometry

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Spots were excised manually and subjected to in-gel tryptic digestion with minor modifications

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as described below 29. Gel pieces were washed (100 μL 40% ethanol, 10 min), shrunk (50 μL

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100% acetonitrile (ACN)) and soaked in 2 μL 12.5 ng/μL trypsin (Promega, porcine

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sequencing grade) in 25 mM ammonium bicarbonate on ice for 45 min, followed by

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rehydration in 10 μL 25 mM ammonium bicarbonate at 37°C overnight. Tryptic peptides (1

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μL) were loaded onto the AnchorChipTM target plate (Bruker-Daltonics, Bremen, Germany),

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covered by 1 μL matrix solution (0.5 μg/μL α-cyano-4-hydroxycinnamic acid in 90% ACN,

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0.1% trifluoroacetic acid (TFA) and washed in 0.5% TFA. Spectra (MS and MS/MS) were

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acquired by Ultraflex II MALDI-TOF-TOF mass spectrometer (Bruker-Daltonics) using Flex


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Control v3.0 and processed by Flex Analysis v3.0 (Bruker-Daltonics). Peptide mass maps were

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acquired in positive ion reflector mode with minimum 1000 laser shots per spectrum. Tandem

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MS/MS data were acquired with an average of 3000 laser shots for each spectrum. Spectra

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were externally calibrated using a tryptic digest of β-lactoglobulin (5 pmol/μL). Filtering of

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spectra was performed for known trypsin autolysis products (m/z 842.5090, 1045.5637 and

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2211.1040) and keratin peaks. The MS and MS/MS spectra were analyzed using Biotools v3.1

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(Bruker-Daltonics, Bremen, Germany). MASCOT 2.0 (http://www.matrixscience.com) was

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used for database searches in the NCBInr for green plants (1749148 entries), DFCI (Dana-

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Farber Cancer Institute) barley gene index Release 12.0 (http://compbio.dfci.harvard.edu/tgi)

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and MIPS/IBIS (Munich information center for protein sequence - Institute of Bioinformatics

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and Systems Biology) barley genome (79220 entries). Search parameters were monoisotopic

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peptide mass accuracy of 80 ppm; fragment mass accuracy to ± 0.7 Da; a maximum of one

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missed cleavage; carbamidomethylation of cysteine and partial oxidation of methionine,

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signal-to-noise threshold ratio of 1:6. Probability-based MOWSE scores above the calculated

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threshold value (p < 0.05) with a minimum of two matched peptides were considered for

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protein identification. Protein sequences were also assessed for the presence of cysteine

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residues using CYS_REC release 2 (http://linux1.softberry.com/berry.phtml).

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RESULTS

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Protein quantification and SDS-PAGE of liquid feed

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The water-extractable and urea-extractable proteins were obtained from liquid feed during 2 or

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48 h steeping, and supplemented with either protease inhibitors in 5% DMSO or 5% DMSO.

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The pH of the liquid feed was constant between 5.5−6.0 throughout the steeping period (data

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not shown). Due to relatively short steeping period and a stable pH, we did not expect major



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changes in the populating microbiota and proteolytic activities. The amount of water-

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extractable proteins remained relatively steady throughout the 48 h steeping period, Fig. 1A.

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Significant differences, however, were found in the urea-extractable protein fractions between

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samples as well as over time showing an increase in the amount of protein (ANOVA, p < 0.05,

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Fig. 1B). The amount of urea-extractable proteins at both 2 and 48 h were lower in feed

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supplemented with protease inhibitors in DMSO and DMSO compared to feed added water

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(Fig. 1B). Collectively, the total amount of proteins increased for all the samples during 48 h,

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with no major differences between the samples. Similar changes in the amount of water-

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extractable and urea-extractable protein fractions were observed in SDS-PAGE, when loading

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equal volumes of protein extracts (Fig. 1C-D). Distinct high molecular weight protein bands

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were present and/or more intense in feed with protease inhibitors in DMSO at 2 and 48 h

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compared to feed with water or DMSO. For example, the protein band of ca. 40 kDa present in

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feed with protease inhibitors at 48 h is absent or less intense in feed added water and DMSO

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(Fig. 1C, fold change 1.67, densitometric values shown in Supplementary Fig. S1). Similarly,

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low molecular weight protein bands were absent and/or of low intensity in feed with protease

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inhibitors in DMSO compared to the other two samples (indicated with arrows in Fig. 1C-D,

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Supplementary Fig. S1). The observed changes in band intensities in both the water- and urea-

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extractable protein fractions suggests that application of protease inhibitors to some extent

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inhibits proteolytic activities occurring during the 48 h of steeping.

218 219

Proteome analysis of liquid feed supplemented with protease inhibitors

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2D-gel electrophoresis in the pH 4−7 range was performed for four biological replicates from

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each condition. The water-extractable and urea-extractable protein fractions were analyzed

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separately to enrich specific proteins elicited by application of protease inhibitors in DMSO to


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liquid feed. This separate analysis also minimizes co-extraction of storage proteins, which

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would otherwise dominate the 2-DE profile and mask lower abundance proteins in the water-

225

extractable fraction. As expected, the 2-DE patterns of the water-extractable (Fig. 2A and

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Supplementary Fig. S2) and urea-extractable (Fig. 2B and Supplementary Fig. S3) protein

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fractions were distinctively different. Fewer but larger spots were detected on gels with urea-

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extractable protein fraction. Clear differences were observed between feed with protease

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inhibitors in DMSO and feed added water or DMSO at both 2 and 48 h of incubation, with

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absence and/or decrease in spot intensity of several low molecular weight proteins in extracts

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of feed with protease inhibitor in DMSO (Fig. 2). In addition, differences were found between

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the feed samples over time, with decrease in spot intensity of several high molecular weight

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proteins and appearance of additional spots with lower molecular weight proteins over time

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(Fig. 2 and Supplementary Fig. S2 and S3). Gel images were analyzed with the specified

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criteria under image analysis. Principal component analysis (PCA) of complex proteomic

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datasets with many variables can effectively visualize these variables and their

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connections/clustering of co-regulated proteins that would otherwise go unnoticed. Principal

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component analysis essentially compares the abundancy patterns of all the protein spots across all

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samples, using the variation of expression patterns to group individual samples. While there are

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certain limitations of gel-based proteomics, such as gel-to-gel and spot migration variability, it

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is nevertheless, an easy visualization method for mapping differences in protein abundance, as

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well as degradation, isoforms and/or PTMs.

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The biological replicates from each treatment clustered together, demonstrating acceptable

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reproducibility of 2-DE patterns (Fig. 3). Thirty-six percent of the variance could be explained

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by principal component 1 (PC1), whereas 18% of the variance was explained by PC2 (Fig.

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3A). The largest difference was observed between feed with protease inhibitors in DMSO (2



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and 48 h) positioned in the positive quadrant of PC1 (negative PC2) and feed with water (2 and

248

48 h) in the negative quadrant of PC1 (positive PC2). Thus feed with protease inhibitors in

249

DMSO was inversely correlated with feed with water. Differences were also observed between

250

feed added protease inhibitors in DMSO (2 and 48 h) and DMSO (48 h). By contrast, feed with

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protease inhibitors at 2 and 48 h clustered closely together, while feed added water and DMSO (2 and

252

48 h) also clustered together (Fig. 3A), indicating similarity in protein abundance profiles between

253

the samples. A total of 57 protein spots showed differential abundance in the water-extractable

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proteome of feed with protease inhibitors in DMSO after 2 and 48 h of incubation compared to

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feed added water (fold change > 1.5, p < 0.05, Fig. 4A and C). These spots were excised,

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digested with trypsin and analyzed by mass spectrometry. Thirty-nine of the 57 spots were

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confidently identified, where the volume of 32 (14 and 18 at 2 and 48 h) protein spots

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increased and 7 decreased in intensity. Protein identifications are summarized in Table 1 and

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their appearances on the gel images are labeled in Fig. 4A. The majority of the identified

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proteins that increased in abundance were typically involved in primary metabolism such as

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glycolysis, and those arising upon imbibition, including carbohydrate and protein degrading

262

enzymes, and others involved in defense-/stress-related mechanisms.

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In the urea-extractable proteome, the greatest change was also observed for feed supplemented

264

with protease inhibitors in DMSO at 48 h (Fig. 3B). The PCA plot shows that PC1 (26% of the

265

variance) separates the samples according to steeping period with the 2 h samples in the

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positive quadrant and the 48 h samples in the negative. Moreover, there is an overlap between

267

feed added water and DMSO at 48 h incubation, suggesting similar protein abundance profiles.

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Comparative analysis detected 39 spots (fold change > 1.5, p < 0.05) that differed in relative

269

intensity in response to protease inhibitors in DMSO compared to feed added water (Fig. 4B

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and D), of which 18 were confidently identified by MS (Table 2 and Fig. 4B). The 18 protein


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spots that increased in relative abundance were identified as defense/stress-related and storage

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proteins, similar to those found in the water-extractable proteome. Identification of proteins

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that are typically absent and/or degraded (differential molecular weight) suggests that protease

274

inhibitors are to some degree an effective means of inhibiting the proteolytic activities in the

275

liquid feed.

276 277

Proteome analysis of liquid feed supplemented with NTR/Trx system

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Protein quantification analysis revealed no differences in the total amount of water-extractable

279

proteins between feed supplemented with NTR/Trx system and feed added water only. However,

280

the amount of urea-extractable proteins at 48 h were lower in feed with NTR/Trx compared to feed

281

added water (p < 0.05, histograms in Fig. 5), demonstrating the efficiency of NTR/Trx system and

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increased partition of proteins in the water-extractable fraction within the 48 h of steeping period. 2-

283

DE was performed to map and identify the protein changes in liquid feed supplemented with the

284

NTR/Trx system. Representative 2D-gels of the water-extractable and urea-extractable proteomes

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of liquid feed with NTR/Trx system (2 and 48 h) resolved in the pH 4−7 are shown in Fig. 5. The

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water-extractable proteome of liquid feed containing NTR/Trx were highly resolved and crowded in

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both number and intensity of multiple protein spots not observed in the other proteome profiles.

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Thus illustrating that application of NTR/Trx reductively alters the solubility of barley proteins and

289

promotes their mobilization. Comparative analysis enabled detection of 50 spots changing in

290

abundance after 48 h in feed supplemented with NTR/Trx when compared to the control (feed with

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water only). Of these, 32 spots which increased in intensity were confidently identified by MS (Fig.

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6A and C). The majority of the identified proteins were found to be enzyme inhibitors and stress-

293

related enzymes (Table 3). In the urea-extractable proteome of liquid feed supplemented with



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NTR/Trx at 48 h, a total of 27 spots differed significantly in relative intensity of which 24 spots

295

were confidently identified by MS (Fig. 6B, Table 4).

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DISCUSSION

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Profiling the protein dynamics of liquid barley feed over a 48 hour steeping period via

299

application of protease inhibitors

300

Protein reserves are mobilized by proteases released from the aluerone layer, as well as the

301

pre-formed proteases present in the endosperm during imbibition and germination 9. Thus,

302

their inhibition would result in reduced protein solubility/degradation. The aim of this study

303

was to map the proteome of liquid feed and monitor protein changes over a 48 h of steeping

304

period with application of protease inhibitors. The majority of the identified proteins that

305

differed in abundance upon supplementation of protease inhibitors were typically defense-

306

/stress-related proteins, imbibition-related enzymes and proteins involved in the primary

307

metabolism. Several glycolytic and TCA cycle enzymes were found in spots that increased in

308

abundance in water-extractable proteome of feed with protease inhibitors, including fructose-

309

bisphosphate aldolase (FPA, spot 2442), triose-phosphate isomerase (TPI, spots 3391, 3367

310

and 3399), phosphoglycerate kinase (spots 2502), enolase (ENO, spots 2118 and 2099), and

311

cytosolic NADP-malic enzyme (spot 1795) and NADP-dependent malate dehydrogenase

312

(MDH, spot 2704). Glycolytic enzymes in the mature seeds have been suggested to be present

313

in preparation for the rapidly increasing respiratory rate upon imbibition, to provide energy for

314

commencement of germination 30. Several proteins including ENO and TPI were found in more

315

than one spot with differing pI and/or molecular size (Fig. 4A), suggesting that the proteins

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were post-translationally modified (PTM), degraded or found as sequence-related isoforms.

317

Isoelectric heterogeneity is commonly found in 2-DE and has been ascribed to PTMs (e.g.


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glycosylation and phosphorylation) or artifacts arising during processing, e.g. carbamylation

319

by urea displayed as string of spots 31. Moreover, discrepancies in the theoretical and measured

320

molecular weights suggest that several of the identified proteins have been degraded over the

321

48 h of steeping period, e.g. spot 2442 (FPA), 2705 (protein z-type serpin), 3845 (late

322

embryogenesis abundant protein), 3885 (globulin) and 4318 (nucleoporin like protein).

323

Seed imbibition is known to be accompanied with resumption of metabolic activities and

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mobilization of carbohydrate and protein reserves of the endosperm in order to support the

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growth of the new plantlet 32.

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A group of proteins identified in spots with increased intensities in feed with protease

327

inhibitors include proteins that are bound/associated with starch granules (e.g. granule bound

328

starch synthase and α-amylase inhibitor) and a collection of storage proteins (e.g. hordeins,

329

globulin-like proteins, ferritin-like protein and Z-type serpins), thus illustrating the occurring

330

modulation of the protein matrix and embedded starch granules and increased availability of

331

proteins and minerals/iron. Moreover, β-amylase (spot 1942, +8.7 fold change), starch

332

debranching enzyme, were also identified in the water-extractable proteome with protease

333

inhibitors. In mature barley grains, β-amylase occurs both in free and bound form in the

334

starchy endosperm. The bound β-amylase has been proposed to be associated with either the

335

endosperm protein matrix and/or periphery of the embedded starch granules 33. Multiple

336

proteoforms of β-amylase with different pIs have been identified in barley, all of which are

337

products of proteolysis of the C-terminal region. Identification of the full-length β-amylase

338

indicates that application of protease inhibitors to some extend hampers some of the

339

proteolytic activities taking place during the steeping period.

340

Another group of the proteins identified in feed with protease inhibitors include functions,

341

such as defense-related proteins (i.e. chloroform/methanol (CM) soluble proteins α-



Page 16 of 47

342

amylase/trypsin inhibitor CMb (spot 4042), two isoforms of the subtilisin-chymotrypsin

343

inhibitors CI-IB and CI-IC (spot 4405 and 4392) and proteins related to maintenance of

344

oxidative and desiccation stress (i.e. protein disulfide-isomerases (PDIs, spot 1862 and 1866),

345

short-chain dehydrogenase/reductases (spot 2947), enoyl-(acyl-carrier protein) reductase (spot

346

2811), glutathione S-transferase DHAR1 (spot 1767), 27K protein (spot 3379), late

347

embryogenesis abundant protein (spot 3845), cold-regulated protein (spot 3780 and 3759) and

348

glyoxalase (spot 3926)). Imbibition entails an increase in hydrogen peroxide content, which is

349

due to resumption of the metabolic activities in seeds, thus it is not surprising to identify

350

proteins with desiccation protective roles 34–36. In addition, several members of the heat shock

351

protein (HSP) family were identified (HSP70 in spots 3460, 3239, 3255 and 3240), which are

352

known to exhibit chaperone activity, protection from irreversible aggregation, and have a

353

protective role in desiccation tolerance 37,38. Conversely, HSP70 was found in spot 2370 (52

354

kDa, pI 9.8) with 5.3 fold decrease in abundance. Noticeable, the identified protein had a lower

355

measured molecular mass and shifted pI, indicating the occurring mobilization of seed proteins

356

during steeping. Overall, on the basis of sequence coverage and molecular mass, the protein

357

identifications suggest that application of protease inhibitors to liquid feed, in addition to the

358

endogenous protease inhibitors already present, can hamper proteolysis taking place during the

359

48 h steeping period of feed.

360

Minor changes were observed in the urea-extractable proteome in feed with protease inhibitors

361

over a 48 h steeping period, compared to the water-extractable proteome (Fig. 4B). The

362

majority of the spots that were identified in high abundance in the urea-extractable proteome

363

with protease inhibitors were similar to those found in the water-extractable proteome, such as

364

storage proteins (B hordein, spots 1780 and 1825, embryo globulin, spots 2316 and 2460, and

365

seed maturation protein, spot 1766), glycolytic enzymes (TPI, spots 1839 and 2082, and ENO,


Page 17 of 47


366

spot 2070) and a collection of defense-related proteins (the dimeric form of α-amylase

367

inhibitor BDAI-1, spots 2572 and 2584). Serpin protein Z was identified in multiple spots

368

(1460, 1551, 1573, 1604, 1611 and 1719). In the mature grains, serpin protein Z is present in

369

both free and thiol-bound forms, which are released during germination. Serpins have been

370

proposed to function as storage proteins, due to their high lysine content similar to other

371

storage proteins, and their synthesis and deposition during seed development 39. Moreover, the

372

plant serpins have been shown to provide protection against insects by irreversible inhibition

373

of proteases 40. However, the biological function of these proteins still remains unknown, due

374

to the fact that no target endogenous proteases have yet been found in plants. Endogenous

375

protease inhibitors play important roles against proteolysis during germination and/or

376

pathogenesis. Identification of proteins that are typically absent and/or degraded suggests that

377

protease inhibitors can be an effective means of inhibiting proteolytic activities in liquid feed.

378

More importantly, these findings highlight the major reorganization of seed storage reserves

379

taking place during 48 h of steeping period, which could be attributed to activation of pre-

380

formed proteases and/or de novo synthesis of proteases during imbibition.

381 382

The NTR/Trx system reductively alters the solubility of barley proteins

383

Increasing the solubility of proteins is of great interest for livestock feed. Differential proteomics

384

analysis was performed to study the protein changes elicited upon application of the NTR/Trx

385

system to liquid feed. The water-extractable protein profile of liquid feed containing the

386

NTR/Trx system was clearly different compared to control (feed with water only) gels showing

387

spots increasing both in numbers and intensity (Fig. 5). A bulk group of enzyme inhibitors were

388

identified in the water-extractable fraction of feed with NTR/Trx, including α-amylase/trypsin

389

inhibitor CMa (spot 1459), CMd (spot 1266), α-amylase inhibitor BDAI-1 (spots 1554 and 1596)



Page 18 of 47

390

and BMAI-1 (spots 1405, 1406 and 1408) and trypsin/α-amylase inhibitor pUP38 (spot 1486).

391

Protease inhibitors identified included serpins (spots 705, 714, 747, 1444, 731) and subtilisin-

392

chymotrypsin inhibitor CI-1B (spot 1691 and 1683). These inhibitor proteins are well known

393

and recognized targets of Trxs and have been previously reported in cereals 23,25,41. Identification

394

of these small disulfide proteins in the water-extractable proteome of liquid feed supports the

395

view that application of functional NTR/Trx system indirectly increases protein solubility by

396

reducing intra-molecular disulfide bonds of these proteins, thus enhancing their partition in the

397

water-extractable fraction

398

by facilitating mobilization of seed reserves by reductively enhancing solubility of storage

399

proteins and thereby their susceptibility to proteolysis, or by reductively inactivating proteins

400

that inhibit specific amylases and proteases

401

reductively activate enzymes participating during germination, such as β-amylases identified in

402

spots 505 and 541 (5 cysteines). Other known Trx target proteins include glucose-ribitol

403

dehydrogenases (spot 918, 5 Cys) and fructose-1,6-bisphosphate aldolase (spot 755 and 798, 7

404

Cys). Spot 763 was identified as the reversibly glycosylated polypeptide containing up to 9 Cys

405

(3 probable SS bound), which have been implicated in polysaccharide and/or cell wall synthesis

406

45 .

407

NTR/Trx, including Cu/Zn superoxide dismutase (spot 1468, 3 Cys), glutathione reductase (spot

408

579, 7 Cys), glyoxalase (spot 900, 5 Cys) and the plant stress-response enzyme, which is an

409

aldo-keto reductase (spot 1504, 7 Cys).

410

The urea-extractable protein fraction of feed with NTR/Trx was less crowded compared to the

411

water-extractable fraction (Fig. 5). Evidently, the majority of the identified proteins were those

412

already identified in the water-extractable fraction, which suggests that not all known Trx

413

target proteins were solubilized. It remains to be studied whether the increase in protein

17,19 .

Trxs have been reported to act as a signal early in germination

42–44 .

In addition Trxs have also been reported to

Several stress-related enzymes were identified in the water-extractable protein fraction with


Page 19 of 47


414

solubility is dependent on the extent of reduction (increasing concentration of NTR/Trx

415

system). Enzyme inhibitors dominated the protein identifications, including -amylase/trypsin

416

inhibitor CMd (spots 2082, 2108, 2124 and 2212) and CMb (spots 2202, 2279, 2234 and

417

2245), -amylase inhibitor BMAI-1 (spots 2163 and 2204) and subtilisin-chymotrypsin

418

inhibitor CI-1A (spot 2562). Several storage proteins increased in abundance in the presence of

419

NTR/Trx, including B hordeins (spots 1060 and 1140, 8 Cys), D hordeins (spot 2388, 7 Cys)

420

and embryo globulin (spot 2303, 6 Cys), in accordance with the role of Trx to enhance the

421

solubility of several storage proteins in cereals 14,19,46. Other previously identified Trx targets

422

showing an increase in spot intensity in presence of Trxs included inorganic diphosphatase

423

(spot 1795, 2 Cys), osmotically inducible protein (spot 2113, 2 Cys). In addition, two spots

424

showing an increase in spot intensity in presence of Trxs were identified as the outer

425

membrane protein X (spot 2169 and 2189). These hydrophobic membrane proteins predicted to

426

contain two Cys residues have not previously been reported as Trx targets.

427

In summary, the differential proteomics data provide valuable insight into the protein

428

dynamics and the molecular mechanisms occurring in liquid barley feed over a steeping period

429

of 48 h. Identification of full length storage and stress-/defense-related proteins that increased

430

>1.5-fold in abundance in feed with protease inhibitors, illustrates the major reorganization of

431

seed storage reserves taking place during soaking. Application of NTR/Trx system to liquid

432

feed increased the solubility of both known and potentially new Trx target proteins. Thus

433

application of a functional NTR/Trx system shows potential as feed additive in combination

434

with other additives such as recombinant protease 9. 2-DE is proved to be a powerful technique

435

for resolving individual proteins, proteoforms and protein isoforms, which would otherwise be

436

lacking when running 1D-gels. In addition, the generated proteomic datasets can complement

437

in vivo studies/in vivo digestibility, and provide a deeper insight into various physiological



Page 20 of 47

438

processes at the proteome level. Similar approach can be applied to study other cereals used as

439

feed and the effect of other feed enzymes/additives.

440 441

Associated content

442

Supporting information

443

Supplemental Figure S1. Densitometric analysis of the indicated bands (whole rows) of

444

water-extractable (A) and urea-extractable (B) proteins of liquid feed added water only,

445

protease inhibitors in DMSO or DMSO after 2 and 48 h of incubation.

446

Supplemental Figure S2. Displaying four replicate 2D-gels of water-extractable protein

447

extracts of liquid feed with protease inhibitors in DMSO, DMSO and water, at 2 and 48

448

h of incubation.

449

Supplemental Figure S3. Displaying four replicate 2D-gels of urea-extractable protein

450

extracts of liquid feed with protease inhibitors in DMSO, DMSO and water, at 2 and 48

451

h incubation.

452

Supplemental Figure S4. Displaying four replicate 2D-gels of water-extractable and

453

urea-extractable protein extracts of liquid feed with NTR/Trx h system, at 2 and 48 h

454

incubation.


Page 21 of 47


455

Supplemental Table S1-S2. Detailed list of protein identifications by MALDI-TOF-TOF

456

MS and MS/MS of water-extractable and urea-extractable protein extracts of liquid

457

feed with protease inhibitors in DMSO.

458

Supplemental Table S3-4. Detailed list of protein identifications by MALDI-TOF-TOF

459

MS and MS/MS of water-extractable and urea-extractable protein extracts of liquid

460

feed with NTR/Trx h system.

461 462

Author information

463

Corresponding author

464

*Phone: +45 24893141, E-mail address: [email protected].

465 466

The authors declare no competing financial interest.

467 468

Funding

469

This work was funded by the framework of Directorate for Food, Fisheries and Agri

470

Business (DFFE) through the project “Exploiting barley first wave enzyme activities for

471

better feed” and a 1/3 PhD stipend from the Technical University of Denmark (DTU).

472

MALDI-TOF MS was funded by the Danish Center for Advanced Food Studies (LMC)



Page 22 of 47

473

and Enzyme and Protein Chemistry, DTU Bioengineering. S (Denmark) is thanked for

474

providing the barley grains.

475 476

Acknowledgements

477

A.S. and C.F. conceived the study and designed the experiments. J.B.C. prepared the liquid

478

feed samples. A.S. and B.A. performed the sample preparations and proteomics experiments.

479

A.S. analyzed the data. B.S. and H.D.B gave critical input. A.S. and C.F. critically evaluated

480

the results and wrote the paper.

481 482

Abbreviations used

483

CM, chloroform/methanol; ENO, enolase; FPA, fructose biphosphate aldolase; GH, glycoside

484

hydrolase; HSP, heat shock protein; NTR, NADPH-dependent thioredoxin reductase; PCA,

485

principal component analysis; PTM, post translational modification; TPI, triosephosphate

486

isomerase, Trx, thioredoxin.

487 488

References

489

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Figure captions Fig. 1. The protein concentration measurements of barley determined by the method of Popov et al. (1975) 26. Water-extractable (A) and urea-extractable (B) proteins from liquid feed supplemented with either protease inhibitors in DMSO or DMSO, at 2 and 48 h of incubation. Each histogram represents the mean ± standard deviations obtained from four biological samples. Significant differences (p < 0.05) by ANOVA are indicated with asterisk (*). SDSPAGE loaded with equal volumes (10 μL) of water-extractable (C) and urea-extractable (D) proteins from liquid feed samples after 2 and 48 h. Arrows highlight the observed changes in band intensity across feed samples. H2O represent feed added water.

Fig. 2. 2D-gels of water-extractable (A) and urea-extractable (C) proteins from liquid feed samples with or without protease inhibitors in DMSO and DMSO, at 2 and 48 h of incubation (250 μg, pI 4−7). Numbered boxes indicate selected regions of 2D-gels showing differences across all the samples. Close-up views of the boxes across all the samples for water-extractable (B) and urea-extractable (D) protein fractions. H2O represent feed added water.

Fig. 3. Principal components analysis (PCA) plot over the water-extractable (A) and ureaextractable (B) protein fractions. The enclosed spots represent the gels and the numbers the protein spots on the gels. Pink circles: feed added water at 2 h; Blue: feed with protease inhibitors in DMSO at 2 h; Purple: feed with DMSO at 2 h; Yellow: feed with water only at 48 h; Turquoise: feed with protease inhibitors in DMSO at 48 h; Orange: feed with DMSO at 48 h.


Page 31 of 47


Fig. 4. 2-DE of the water-extractable (A) and urea-extractable (B) proteins of liquid barley feed supplemented with protease inhibitors in DMSO after 48 h of steeping period (250 μg, pI 4−7). Numbered circles represent protein spots that changed in abundance (Table 2). The black circles represent an increase and the red circles a decrease in relative spot intensity compared to feed added water. Molecular weight markers and pI range are as indicated.

Fig. 5. The protein concentration measurements of water-extractable (top histogram) and ureaextractable (bottom histogram) proteins from liquid feed supplemented with thioredoxin system (NTR/Trx) or water only, at 2 and 48 h of incubation. Significant differences (p < 0.05) by ANOVA are indicated with asterisk (*). 2-DE of water-extractable (A−D) and ureaextractable (E−H) proteins from liquid feed supplemented with NTR/Trx, at 2 and 48 h of incubation (250 μg, pI 4−7). H2O represent feed with water. Molecular markers and pI range are as indicated.

Fig. 6. 2-DE of the water-extractable (A) and urea-extractable (B) proteins of liquid barley feed supplemented with NTR/Trx system after 48 h of steeping period (250 μg, pI 4−7). Numbered circles represent protein spots that increase in relative intensity compared to feed added water (Table 4). Molecular markers and pI range are as indicated.



Page 32 of 47

Tables Legends Table 1. Identification of proteins in spots resolved by 2-DE of the water-extractable fraction of liquid barley feed with protease inhibitors in DMSO compared to feed added water (> 1.5fold spot volume ratio change, ANOVA < 0.05) by MALDI-TOF-TOF MS and MS/MS. Probability-based MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification.

Table 2. Identification of proteins in spots resolved by 2-DE of the urea-extractable fraction of liquid barley feed with protease inhibitors in DMSO compared to feed added water (> 1.5-fold spot volume ratio change, ANOVA < 0.05) by MALDI-TOF-TOF MS and MS/MS. Probability-based MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification.

Table 3. Identification of proteins in spots resolved by 2-DE of the water-extractable fraction of liquid barley feed with NTR/Trx at 48 h compared to feed added water (> 1.5 fold spot volume ratio change, ANOVA < 0.05) by MALDI-TOF-TOF MS and MS/MS. Probabilitybased MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification.

Table 4. Identification of proteins in spots resolved by 2-DE of the urea-extractable fraction of liquid barley feed with NTR/Trx at 48 h compared to feed added water (> 1.5 fold spot volume ratio change, ANOVA < 0.05) by MALDI-TOF-TOF MS and MS/MS. Probability-based MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification.


Page 33 of 47


Fig. 1. The protein concentration measurements of barley determined by the method of Popov et al. (1975) 26. Water-extractable (A) and urea-extractable (B) proteins from liquid feed supplemented with or without protease inhibitors in DMSO or DMSO, at 2 and 48 h of incubation. Each histogram represents the mean ± standard deviations obtained from four replicates. Significant differences (p < 0.05) by ANOVA are indicated with asterisk (*). SDSPAGE loaded with equal volumes (10 μL) of water-extractable (C) and urea-extractable (D) proteins from liquid feed samples after 2 and 48 h. Arrows highlight the observed changes in band intensity across feed samples. H2O represent feed added water.



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Fig. 2. 2D-gels of water-extractable (A) and urea-extractable (C) proteins from liquid feed samples with or without protease inhibitors in DMSO and DMSO, at 2 and 48 h of incubation (250 μg, pI 4−7). Numbered boxes indicate selected regions of 2D-gels showing differences across all the samples. Close-up views of the boxes across all the samples for water-extractable (B) and urea-extractable (D) protein fractions. H2O represent feed added water. 34 ACS Paragon Plus Environment

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(A)

(B)

Fig. 3. Principal components analysis (PCA) plot over the water-extractable (A) and urea-extractable (B) protein fractions. The enclosed spots represent the gels and the numbers the protein spots on the gels. Pink circles: feed added water at 2 h; Blue: feed with protease inhibitors in DMSO at 2 h; Purple: feed with DMSO at 2 h; Yellow: feed added water at 48 h; Turquoise: feed with protease inhibitors in DMSO at 48 h; Orange: feed with DMSO at 48 h. 35 ACS Paragon Plus Environment


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Fig. 4. 2-D gels of the water-extractable (A) and urea-extractable (B) proteins of liquid feed supplemented with protease inhibitors in DMSO after 48 h of steeping period (250 μg, pI 4−7). Expression profiles of protein spots (increasing/decreasing in intensity upon protease inhibitor treatment) in the water-extractable protein fraction (C) and urea-extractable protein fraction (D). Each trace correspond to one protein spot and each node correspond to one replicate. Numbered circles represent protein spots that changed in 36 ACS Paragon Plus Environment

Page 37 of 47


abundance (Table 2). The black circles represent an increase and the red circles a decrease in relative spot intensity compared to feed added water. Molecular weight markers and pI range are as indicated.



Page 38 of 47

Fig. 5. The protein concentration measurements of water-extractable (top histogram) and urea-extractable (bottom histogram) proteins from liquid feed supplemented with thioredoxin system (NTR/Trx) or water only, at 2 and 48 h of incubation. Significant differences (p < 0.05) by ANOVA are indicated with asterisk (*). 2-DE of water-extractable (A−D) and urea-extractable (E−H) proteins from liquid feed supplemented with NTR/Trx, at 2 and 48 h of incubation (250 μg, pI 4−7). H2O represent feed with water. Molecular markers and pI range are as indicated.


Page 39 of 47


Fig. 6. 2-DE of the water-extractable (A) and urea-extractable (B) proteins of liquid feed supplemented with NTR/Trx system after 48 h of steeping period (250 μg, pI 4−7). Expression profiles of protein spots in the water-extractable protein fraction (C) and urea39 ACS Paragon Plus Environment


Page 40 of 47

extractable protein fractions (D). Each trace correspond to one protein spot and each node correspond to one replicate. Numbered circles represent protein spots that increase in relative intensity compared to feed added water (Table 4). Molecular markers and pI range are as indicated.


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Table 1. Identification of proteins in spots resolved by 2DE of the water-extractable fraction of liquid feed with protease inhibitors in DMSO at 2 and 48 h of incubation compared to feed added water (> 1.5-fold spot volume ratio change, ANOVA < 0.05) by MALDI-TOF-TOF MS and MS/MS. Probability-based MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification. Spot numbers correspond to gel image shown in Fig. 4A. Comb: combined; Theor: theoretical. Spot no.a

Fold changeb

Accession no.

Protein

Mw theor.

pI theor.

73772 19425 64741 64741 68872 54288 48709 33708 41667 31917 29675 50018 36396 47163

8.31 11.40 5.62 5.62 5.80 8.48 8.54 9.15 8.60 6.37 6.51 6.01 9.23 9.31

PMF score

Comb. score

Sequence coverage (%)

112 74 151 206 110 73 93 85 106 91 94 79 70 127

19 34 25 25 31 18 16 31 25 28 45 18 23 19

2 h Feed with protease inhibitors in DMSO 1745 1746 1862 1866 1942 2442 2488 2502 2947 3199 2704 2705 4318 3352

6.3 7.6 9.9 5.9 8.7 7.5 7.5 14.4 11.2 9.6 28.6 6.3 13.2 15.9

TC200068 TC217857 TC230560 TC230560 TC233233 TC218799 TC204315 TC223074 TC203374 TC216236 TC216967 TC229089 TC216254 TC220483

Cytosolic NADP malic enzyme Chromosome chr10 scaffold_81 Disulfide-isomerase Disulfide-isomerase Beta-amylase Fructose-bisphosphate Serpin Phosphoglycerate kinase Short-chain dehydrogenase/reductase SDR Predicted (Uncharacterized protein LOC100835371) Malate dehydrogenase Protein z-type serpin Nucleoporin-like protein, partial (22%) Globulin-like protein

48 h Feed with protease inhibitors in DMSO 2118

10.1

gi|326490934

Predicted protein (Enolase)

48601

5.39

91

282

26

2099 2997

4.9 6.5

gi|326490934 gi|326498119

Predicted protein - enolase Predicted protein (Ferritin like)

48601 33991

5.39 4.90

52 47

192 326

22 26

3239

6.2

gi|326497219

Predicted protein (Heat shock 70 kDa protein)

72202

5.14

48

246

15

3246

16.2

gi|2507469

Triosephosphate isomerase, cytosolic

26948

5.39

98

429

37

3248

4.5

gi|326497219


72202

5.14

68

432

14

3255

7.0

gi|326497219


72202

5.14

55

242

14

3367

8.4

gi|2507469

26948

5.39

108

334

47

3379 3392

3.3 11.1

TC227462 gi|2507469

Triosephosphate isomerase, cytosolic 27K protein complete Triosephosphate isomerase cytosolic

24946 26948

5.54 5.39

76

124 326

12 30

3460

3.8

gi|476003

HSP70

67146

5.76

60

3



Page 42 of 47

3573 3759 3780 3885 3926

5.7 12.3 12.4 4.1 2.5

gi|229610865 gi|10799810 gi|10799810 TC216046 TC206011

Granule bound starch synthase Cold-regulated protein Cold-regulated protein Globulin-2 precursor Predicted protein (Glyoxalase)

22612 17659 17659 53721 16113

5.06 4.93 4.93 7.14 5.42

4392 4405

6.6 4.0

gi|124129 gi|124127

Subtilisin-chymotrypsin inhibitor CI-1C Subtilisin-chymotrypsin inhibitor CI-1B

8253 8958

6.78 5.33

TC209835 TC203837 TC198009 gi|32651579 gi|297259335 gi|29725933 TC201243

Protein disulfide-isomerase precursor Enolase HSP70 Predicted protein Predicted (Zinc finger protein 512B-like) Predicted (Zinc finger protein 512B-like) Lactoylglutathione lyase

68582 58053 51999 41606 32274 32274 42078

5.37 5.82 9.78 6.49 5.79 5.79 8.46

77 92 104 96

317 327 79 401

47 75 75 33 48

82 161

36 62

48 h Feed added water 1917 2306 2370 2746 2786 2802 2963

7.9 3.9 5.3 4.5 5.1 4.3 11.5

158 96 79 89 83 75 94

31 27 20 5 46 36 40

a) Spot numbers correspond to gel image shown in Fig. 4A. b) Spots changing in relative intensity.


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Table 2. Identification of proteins in spots resolved by 2DE of the urea-extractable fraction of liquid feed with protease inhibitors in DMSO at 48 h of incubation compared to feed added water (> 1.5-fold spot volume ratio change, ANOVA < 0.05) by MALDI-TOFTOF MS and MS/MS. Probability-based MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification. Comb: combined; Theor: theoretical.

1463 1551 1573 1604 1611 1719 1760 1766

Fold changeb 2.6 5.5 3.6 4.6 7.0 4.3 3.0 3.7

Accession no. gi|1310677 gi|1310677 gi|1310677 gi|1310677 gi|1310677 gi|1310677 TC208227 TC228218

Mw theor. 43307 43307 43307 43307 43307 43307 30146 27336

pI theor. 5.61 5.61 5.61 5.61 5.61 5.61 8.16 5.24

Comb. score 166 602 595 404 411 151 80 574

Sequence coverage (%) 21 48 43 26 29 20 15 69

1767

2.7

gi|326496021

Protein Z-type serpin Protein Z-type serpin Protein Z-type serpin Protein Z-type serpin Protein Z-type serpin Protein Z-type serpin B hordein Late embryogenesis abundant protein Predicted protein (glutathione S-transferase, DHAR1)

23436

5.71

1825 1839 2070

4.3 7.9 2.7

BE194281 gi|2507469 gi|326490934

B hordein Triosephosphate isomerase, cytosolic Predicted protein (enolase)

100

23

30146 26948 48601

8.16 5.39 5.39

59 49

63 338 217

13 26 18

2082

4.5

gi|2507469

Triosephosphate isomerase, cytosolic

26948

5.39

91

321

26

2316 2408

1.8 2.2

gi|167004 gi|229464991

Embryo globulin Heat shock 70 kDa protein

72551 70871

6.80 5.22

126

339 100

10 6

2422

3.7

TC163934

26610

7.16

84

2460 2476

3.7 4.4

gi|167004 TC163934

Globulin-2 precursor partial (18%) Embryo globulin Globulin-2 precursor partial (18%)

72551 26610

6.80 7.16

84

2572

6.4

gi|123970

Alpha-amylase inhibitor BDAI-1

17045

2584

6.1

gi|123970

Alpha-amylase inhibitor BDAI-1

17045

Spot no.a

Protein

PMF score 75 139 118 96 108 118

6 113

10 6

5.36

72

15

5.36

70

15

a) Spot numbers correspond to gel image shown in Fig. 4B. b) Spots changing in relative intensity.



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Table 3. Identification of proteins in spots resolved by 2DE of the water-extractable fraction of liquid barley feed with NTR/Trx h at 48 h of incubation compared to feed added water (> 1.5-fold spot volume ratio change and ANOVA < 0.05) by MALDI-TOF-TOF MS and MS/MS. Probability-based MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification. Comb: combined; Theor: theoretical. Spot no.a

Fold changeb

Accession no.

505 541 579 694 705 726 731 741 747 775

3.0 3.1 2.1 2.3 4.8 4.3 2.0 3.8 3.5 2.6

gi|29134857 gi|38349539 gi|157362219 gi|326497219 gi|1310677 TC229089 gi|1310677 gi|1310677 gi|1310677 gi|326511289

763 798 814 826 843 851 900 917 918 1266 1354 1405 1406 1468 1483 1504

3.8 2.7 2.7 2.4 1.9 3.2 2.2 3.5 2.8 2.9 4.9 8.6 2.0 3.3 19.7

TC195313 gi|326511289 gi|326493440 gi|326511741 TC205675 TC205675 gi|326493416 gi|7431022 gi|7431022 gi|585291 gi|326515048 gi|2506771 gi|2506771 gi|408795920 gi|225103 gi|255311878

1554

7.2

gi|123970

Protein Endosperm-specific beta-amylase 1 Beta-amylase 1 Cytosolic glutathione reductase Predicted protein Protein Z-type serpin Protein Z-type serpin Protein Z-type serpin Protein Z-type serpin Protein Z-type serpin Predicted protein Fructose-1,6-bisphosphate aldolase Reversibly glycosylated polypeptide Predicted protein (Fructose-1,6-bisphosphate aldolase) Predicted protein Predicted protein Os05g0273800 protein Os05g0273800 protein Predicted protein (Glyoxalase I) Glucose and ribitol dehydrogenase Glucose/ribitol dehydrogenase Alpha-amylase/trypsin inhibitor CMd Predicted protein Alpha-amylase inhibitor BMAI-1 Alpha-amylase inhibitor BMAI-1 Chloroplast Cu/Zn SOD1 Trypsin/amylase inhibitor pUP38 Chain A, Crystal structure of the plant stress-response enzyme Akr4c8 (Aldo-keto reductases) Alpha-amylase inhibitor BDAI-1

Mw theor.

pI theor.

PMF score

Comb. score

Sequence coverage (%)

59886 57883 53445 72202 43307 44262 43307 43307 43307 39243

5.58 5.65 6.07 5.14 5.61 5.42 5.61 5.61 5.61 6.39

63 89 82 74

118 349 126 173 84 61 217 72 57 291

24 25 29 21 11 11 27 11 10 30

41499 39243 36130 40517 38699 38699 32811 31912 31912 19140 19507 16376 16376 19698 12417 37110

5.82 6.39 8.2 5.5 6.07 6.07 5.34 6.54 6.54 6.07 5.69 5.36 5.36 5.31 4.94 6.97

60 79

209 156 213 186 148 206 282 322 349 118 169 86 248 159 114 87

25 30 15 32 44 41 48 34 39 30 40 23 36 26 29 16

17045

5.36

40

80

28

63 99

55 93 86 111 78 118 68

45


Page 45 of 47


1542

8.4

gi|186972808

1408 1459 1596 1691 1070 1683

6.2 26.8 6.9

gi|2506771 gi|585289 gi|123970 gi|124127 gi|326502266 gi|124127

3.7 3.0

Chain A, Crystal structure of barley Thioredoxin h isoform 2 in the oxidized state. Alpha-amylase inhibitor BMAI-1 Alpha-amylase/trypsin inhibitor CMa Alpha-amylase inhibitor BDAI-1 Subtilisin-chymotrypsin inhibitor CI-1B Predicted protein Subtilisin-chymotrypsin inhibitor CI-1B

13271

5.12

16376 16060 17045 8958 26423 8958

5.36 5.87 5.36 5.33 5.51 5.33

51

36 96

183

52

157 61 74 178 304 235

36 6 28 62 40 62

a) Spot numbers correspond to gel image shown in Fig. 6A. b) Spots changing in relative intensity.



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Table 4. Identification of proteins in spots resolved by 2DE of the urea-extractable fraction of liquid barley feed with NTR/Trx at 48 h of incubation compared to feed added water (> 1.5 fold spot volume ratio change, ANOVA < 0.05) by MALDI-TOF-TOF MS and MS/MS. Probability-based MOWSE scores above the calculated threshold value (p < 0.05) were considered for protein identification. Spot no.a 1060 1140 1795 1865 2039 2082 2108 2113

Fold changeb 1.8 3.7 3.2 7.3 4.2 1.8 2.0 2.4

Accession no. gi|82548223 gi|82548223 gi|304396928 gi|372274053 gi|1536911 gi|585291 gi|585291 gi|308185833

2124 2163 2169 2202 2204 2234 2189 2212 2245 2273 2279 2303 2388 2397

2.1 4.3 12.9 2.0 2.1 2.4 9.8 6.2 2.6 7.8 1.8 2.4 2.2 8.1

gi|585291 gi|2506771 gi|372276145 gi|585290 gi|2506771 gi|585290 gi|327393264 gi|758343 gi|585290 gi|2707920 gi|585290 gi|167004 gi|671537 gi|186972808

2515 2562

4.4 6.4

TC206849 gi|124125

Protein name B Hordein B Hordein Inorganic diphosphatase Type VI secretion system, core protein 17 kDa class I small heat shock protein Alpha-amylase/trypsin inhibitor CMd Alpha-amylase/trypsin inhibitor CMd OsmC gene product Osmotically inducible protein Alpha-amylase/trypsin inhibitor CMd Alpha-amylase inhibitor BMAI-1 Outer membrane protein X Alpha-amylase/trypsin inhibitor CMb Alpha-amylase inhibitor BMAI-1 Alpha-amylase/trypsin inhibitor CMb Outer membrane protein X CMd preprotein Alpha-amylase/trypsin inhibitor CMb BTI-CMe4 protein Alpha-amylase/trypsin inhibitor CMb Embryo globulin D Hordein Chain A, Crystal structure of barley Thioredoxin h isoform 2 in the oxidized state Cold-shock DNA-binding domain protein Subtilisin-chymotrypsin inhibitor CI-1A

Mw Theor. 30593 30593 19897 19703 16832 19140 19140 15061

pI Theor. 8.16 8.16 5.00 4.78 5.83 6.07 6.07 5.28

Comb. score 157 122 321 110 257 138 55 206

Sequence coverage (%) 14 8 27 35 30 28 19 38

19140 16376 18318 17199 16376 17199 18407 17894 17199 16743 17199 72551 51154 13271

6.07 5.36 4.97 5.77 5.36 5.77 5.36 5.24 5.77 8.05 5.77 6.80 7.60 5.12

84 111 174 106 127 121 112 147 53 83 105 86 88 180

39 23 30 32 23 32 36 40 11 24 22 6 7 52

9271 8877

5.02 5.24

58 106

26 62

a) Spot numbers correspond to gel image shown in Fig. 6B. b) Spots changing in relative intensity.


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For Table of Contents Only


1 1 Quantitative proteomics analysis of barley-based liquid feed and

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