deciphering the proteomic profile of rice (oryza sativa) bran: a pilot study

Research Article

Deciphering the proteomic profile of rice(Oryza sativa) bran: A pilot study

The exact knowledge of the qualitative and quantitative protein components of rice bran

is an essential aspect to be considered for a better understanding of the functional

properties of this resource. Aim of the present investigation was to extract the largest

number of rice bran proteins and to obtain their qualitative characterization. For this

purpose, three different extraction protocols have been applied either on full-fat or on

defatted rice bran. Likewise, to identify the highest number of proteins, MS data collected

from 1-DE, 2-DE and gel-free procedures have been combined. These approaches allowed

to unambiguously identify 43 proteins that were classified as signalling/regulation

proteins (30%), proteins with enzymatic activity (30%), storage proteins (30%), transfer

(5%) and structural (5%) proteins. The fact that all extraction and identification proce-

dures have been performed in triplicate with an excellent reproducibility provides a

rationale for considering the platform of proteins shown in this study as the potential

proteome profile of rice bran. It also represents a source of information to evaluate better

the qualities of rice bran as food resource.

Keywords:

2-DE / MS / Proteomics / Rice bran DOI 10.1002/elps.200900469

1 Introduction

Rice is a very important agricultural resource whose main

utilization is for food. Feeding almost one half of the world’s

population, it represents one of the most attractive feed

ingredients in the world [1]. Nevertheless, it may be used for

many other different applications, including the preparation

of paper, clothing, beer and glue [1]. A variety of interesting

by-products, including rice hull/husk, bran and small

brokens whose management may be a problem (owing to

their amount), as well as a great opportunity (owing to their

potential), is obtained in converting raw rice into table/white

rice [1]. Although an extensive commercial utilization of

these by-products would be of great interest for a series of

good reasons (they contain energy, are a renewable resource

and their use can reduce waste problems), a relative little

amount of this material is still utilized [1]. In particular, it

appears wasteful the present use of rice bran, a by-product

obtained from rough rice milling process. Although its

nutritional and pharmaceutical potential have been well

recognized, it is largely discarded or used as animal feed.

This is really a shame since, owing to the excellent quality of

its components (proteins, fibers, vitamins and minerals), it

could be economically much more attractive [2]. For

example, the utilization of rice bran proteins as a precious

source of nutritious components does not seem to be

adequately promoted. Provided that the main criteria that

may determine their adoption from this source are their

nutritive value, functional properties as well as cost

implications, rice bran proteins obviously need to be isolated

and concentrated in purified form for their utilization as

food stuff. The procedure typically adopted for protein

extraction from nonheat-stabilized or stabilized rice bran is

an alkaline treatment [3–12];] although, in some cases, this

process was shown to induce unfavourable reactions

including denaturation (which affects protein functional

properties), hydrolysis and possible extraction of nonprotein

components [13]. To avoid these troubles, a number of

alternative chemical and/or enzymatic procedures have been

developed. Chemical approaches were chosen either by

Trisiriroj et al. [14] or by Anderson and Guraya [15]. In the

former case, proteins were precipitated from rice bran

(previously grounded in liquid nitrogen) by adding 10%

TCA in acetone. In the latter, 10% w/v slurries of rice bran

Fabio Ferrari1

Marco Fumagalli1

Antonella Profumo2

Simona Viglio1

Alberto Sala3

Lorenzo Dolcini1

Caterina Temporini4

Stefania Nicolis2

Daniele Merli2

Federica Corana3

Begona Casado1

Paolo Iadarola1

1Department of Biochemistry,University of Pavia, Pavia, Italy

2Department of GeneralChemistry, University of Pavia,Pavia, Italy

3Centro Grandi Strumenti (CGS),University of Pavia, Pavia, Italy

4Department of PharmaceuticalChemistry, University of Pavia,Pavia, Italy

Received May 18, 2009Revised August 4, 2009Accepted August 28, 2009

Abbreviations: DF, defatted; EP, extraction procedure; FA,

formic acid; FF, full fat; HMG, high master gel; MG, mastergel

Correspondence: Professor Paolo Iadarola, Department ofBiochemistry, University of Pavia, Via T. Taramelli 3, 27100Pavia, ItalyE-mail: [email protected]: 139-382-423108

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com

Electrophoresis 2009, 30, 4083–4094 4083

samples were submitted to homogenization for 10 min at a

pressure of 1.7� 104 kPa. By contrast, Ansharullah et al. [16]

applied an enzymatic procedure (by using Viscozyme L 1.5)

to obtain proteins from full-fat (FF) extruded rice bran.

Wang et al. [17] used a combination of phytase and xylanase

to extract proteins from FF and nonheat-stabilized rice bran.

Tang et al. [18] confirmed the effectiveness of amylase,

viscozyme and celluclast in extracting proteins from heat-

stabilized defatted (DF) rice bran.

While different analytical procedures, including

proteomic studies, have allowed: (i) to differentiate aromatic

from nonaromatic rice varieties [16], (ii) to map protein

patterns in rice leaf, embryo, endosperm, root, stem, shoot

and callus and (iii) to evaluate their quality and fundamental

properties [19–35], to construct a rice proteome database

[36–38], identification and characterization of rice bran

protein isolate is still partial and confined to a very limited

number of publications [14, 23, 39, 40].

The objective of this study was the identification of the

largest number of rice bran proteins. For this purpose, we

have planned this pilot study that, through the application of

three different extraction strategies and the use of a variety of

proteomic procedures, including 1-DE, 2-DE and LC-MS/MS,

allowed to identify an unprecedented number of proteins.

2 Materials and methods

2.1 Reagents

Unless otherwise stated, all reagents were purchased from

Sigma (St. Louis, MO, USA) and were of analytical grade.

Ultra-pure water used for all extraction procedures (EPs),

electrophoretic and chromatographic runs was obtained

from a Millipore (Bedford, MA, USA) Milli-Q purification

system.

2.2 Rice bran

Rice bran was kindly provided by a local milling factory

(Curti Riso, Valle Lomellina, Pavia, Italy). FF and DF bran

were used as source of proteins. Rice bran defatting was

achieved as previously described [41], by shaking (1000 rpm)

for 30 min at room temperature an aqueous suspension of

bran with n-hexane (1:5 v/v). Components extracted in the

organic phase were analyzed by GC/MS as indicated below.

Unless otherwise stated, all procedures described in the

following paragraphs were performed in parallel either on

FF and on DF rice bran.

2.3 Extraction with acetone-TCA (EP 1)

Thirty grams of rice bran were ground in liquid nitrogen

and proteins were precipitated by the addition of ice-cold

acetone (120 mL) containing 10% TCA and 10 mM DTT

[14]. The resulting mixture was placed at –201C for 12 h

followed by centrifugation at 14 000 rpm for 30 min.

Proteins were solubilized in an aqueous solution adjusted

at pH 9.0 by the addition of 0.1 N NaOH and the mixture

was re-centrifuged as indicated above. The protein-contain-

ing supernatant was finally collected, lyophilized and stored

at –801C for further analysis.

2.4 Extraction with sodium bisulfite (EP 2)

Aliquots of rice bran (30 g) were incubated at room

temperature under constant stirring for different times of

incubation (8; 16 and 24 h, respectively) with different

volumes (5, 10 and 20% w/v slurries) of 50 mM sodium

bisulfite pH 8.5 [42] in the presence of a cocktail of protease

inhibitors. These slurries were then centrifuged (8000 rpm

for 30 min), and the insoluble fibre product discarded. By

gentle addition of 6 N HCl to the supernatant, the proteins

were allowed to precipitate at their isoelectric point (about

pH 4.0–4.5). To increase the amount of precipitate, the

suspension was left at 41C overnight. This pellet was finally

collected by centrifugation (8000 rpm for 20 min), dried by

lyophilization and stored at –801C prior to use.

2.5 Extraction with SDS-urea solution (EP 3)

Thirty grams of rice bran were ground in liquid nitrogen.

Proteins were solubilized by adding 100 mL of 50 mM Tris-

HCl buffer pH 6.8 containing 8 M urea, 4% SDS, 5%

2-mercaptoethanol, 20% glycerol [43]. The mixture was left

2 h at 601C and then centrifuged (14 000 rpm for 15 min) to

remove debris. The protein-containing filtrate was lyophi-

lized and finally stored at –801C.

2.6 Quantification of proteins

The Lowry [44] and BCA [45] methods were applied to obtain

an exact quantification of extracted proteins. The assay was

performed on each protein solution before proceeding to

their lyophilization. In both cases, BSA was the standard

protein used for the production of calibration curves.

2.7 Characterization of proteins

2.7.1 1-DE

1-D SDS-PAGE was carried out according to the Laemmli

method [46] using 5–20% polyacrylamide gradient gels and

applying a voltage of 120 V. The gels were stained with

Coomassie Brilliant Blue G-250 [47]. Identical amounts

(typically 25–30 mg) of protein mixtures were loaded on each

lane. To assess the identity of proteins, gel lanes were cut

along the dotted lines indicated in Fig. 2 into five large

Electrophoresis 2009, 30, 4083–40944084 F. Ferrari et al.


slices, approximately of the same dimension. Each gel slice

was cut into tiny (1 mm3) cubes and transferred to

eppendorf tubes, where it was washed, destained and

digested with trypsin following the procedure detailed in

Section 2.7.3. After digestion, supernatants were transferred

to fresh tubes and the resulting tryptic peptides were

extracted by incubating gel pieces two times with 30% ACN

in 3% TFA, followed by dehydration with 100% ACN. The

peptide mixtures were separated and analyzed, at least in

triplicate, by LC-MS/MS as indicated above. For the peptide

sequence searching, mass spectra were processed using the

Swiss-Prot 49.1 database and interpreted using the Peaks

Studio, version 4.5 software. To ensure reproducibility, five

gels were produced and processed independently.

2.7.2 2-DE

One milligram of extracted bran proteins was dissolved in

350 mL of rehydration buffer (8 M urea, 2% CHAPS (w/v),

0.2% DTT (w/v), 0.5% IPG buffer (v/v) and 0.002%

bromophenol blue) and loaded onto 18 cm IPG strips, pH

range 3–10 NL (Amersham Biosciences). Strips were rehy-

drated in the presence of sample solution containing 0.5%

ampholyte buffer under constant low voltage (30 V) for 15 h.

The first-dimensional IEF was carried out at 151C using an

IPG-phore (Amersham Biosciences), programmed with

voltages from 30 V for 8 h, 120 V for 1 h, 500 V for 0.5 h,

1000 V for 0.5 h, and 7950 V, for a total of 60 kVh. After the

first-dimensional IEF, the strips were incubated for 20 min in

equilibration buffer (375 mM Tris-HCl, pH 8.8, 6 M urea,

20% v/v glycerol, 2% w/v SDS, 1% DTT) followed by a second

incubation in the same buffer containing 0.13 M iodoacet-

amide and 0.02% bromophenol blue for 20 min. The second-

dimensional electrophoresis was performed by using the

Protean II xi Multi-Cells (Bio-Rad Laboratories, Hercules, CA,

USA) system on 16% polyacrylamide gels with 10 mA/gel

until the buffer front line was 5–10 mm from the bottom of the

gel. Proteins were stained with Coomassie Brilliant Blue G-250

[47]. The gels were scanned by using the model 3000 of the

Versadoc Imaging system (Bio-Rad) and images were

imported and compared with the PD QUEST, Version 7.2.

software (Bio-Rad).

2.7.3 In situ enzymatic digestion

Enzymatic digestion was performed as previously described

[7, 13]. Briefly, bands/spots of interest were carefully excised

from the gel, placed into eppendorf tubes and broken into

small pieces. This material was then washed twice with

aliquots (200 mL) of 50 mM ammonium bicarbonate buffer pH

7.8, 50% ACN and kept under stirring overnight. Gels were

dehydrated by addition of ACN (100 mL). After removing the

organic solvent, reduction was performed by addition of 30 mL

of 10 mM DTT solution (40 min at 371C). DTT was replaced

with 30 mL of 55 mM iodoacetamide for 45 min at 561C. This

solution was removed and the gel pieces were washed twice

with 200 mL of 25 mM ammonium bicarbonate for 10 min,

while vortexing. The wash solution was removed and gel

dehydrated by addition of 200 mL of ACN until the gel pieces

became an opaque-white color. ACN was finally removed and

gel pieces were dried with air. Gels were rehydrated by using

the N-p-Tosyl-L-phenylalanine chloromethyl ketone (TPCK)-

trypsin (571.0 ng/mL) solution and digestion was performed

incubating overnight at 371C. Following enzymatic digestion,

the resultant peptides were stored at –801C until mass

spectrometric analysis.

2.7.4 In-solution enzymatic digestion

One hundred microgram aliquots of lyophilized extracted

bran proteins were placed in different sets of vials (each set

containing FF or DF proteins) and dissolved in 100 mM

ammonium bicarbonate buffer pH 8.5 (100 mL). After

incubating at 371C for 30 min, to each set was added: (i)

trypsin, (ii) a-chymotrypsin or (iii) endoproteinase Glu-C from

Staphylococcus aureus V8 (50:1 protein/enzyme ratio in all

cases). Digestion was performed overnight and the reaction

was stopped by addition of formic acid (FA) (2 mL). Peptide

mixtures were separated as indicated in the following sections.

2.7.5 LC-MS/MS

All analyses were carried out on an LC-MS (Thermo Finnigan,

San Jose, CA, USA) system consisting of a thermostated

column oven Surveyor autosampler controlled at 251C; a

quaternary gradient Surveyor MS pump equipped with a diode

array detector and an Linear Trap Quadrupole (LTQ) mass

spectrometer mass spectrometer with electrospray ionization

ion source controlled by Xcalibur software 1.4. Analytes were

separated by RP-HPLC on a Jupiter (Phenomenex, Torrance,

CA, USA) C18 column (150� 2 mm, 4 mm, 90 A particle size)

using a linear gradient (2–70% solvent B in 90 min) in which

solvent A consisted of 0.1% aqueous FA and solvent B of ACN

containing 0.1% FA. Flow-rate was 0.2 mL/min. Mass spectra

were generated in positive ion mode under constant instru-

mental conditions: source voltage 4.0 kV, capillary voltage 46 V,

sheath gas flow 40 (arbitrary units), auxiliary gas flow 10

(arbitrary units), sweep gas flow 1 (arbitrary units), capillary

temperature 2501C, tube lens voltage –105 V. MS/MS spectra,

obtained by CID studies in the linear ion trap, were performed

with an isolation width of 3 Th m/z, the activation amplitude

was 35% of ejection RF amplitude that corresponds to 1.58 V.

Data acquisition and processing were performed using

Peaks studio 4.5 and Bioworks version 3.1 softwares. The

mass lists were searched against the SwissProt protein

database with taxonomy of Oryza sativa using MASCOT

(Matrix Science; www.matrixscience.com) search engine

under continued mode (MS plus MS/MS) with the follow-

ing parameters: trypsin, chymotrypsin and V8 protease (E)

specificities, five missed cleavages, cysteine carbamido-

methylation as variable modifications, peptide tolerance at

0.2 Da and MS/MS tolerance at 0.25 Da. Peptide charge 1, 2,

31 and experimental mass values: monoisotopic. MASCOT

scores greater than 65 were considered significant (po0.05).

Electrophoresis 2009, 30, 4083–4094 Proteomics and 2-DE 4085


2.7.6 Analysis by GC-MS of organic phase obtained

after defatting the rice bran

Four milliliters of organic phase obtained from rice bran by

extraction with n-hexane, as we have shown previously, were

treated at 701C for 1 h with 250 mL of methanol containing 3 M

HCl. The mixture of esters was then dried under a nitrogen

flux, redissolved in 260 mL of dichloromethane and analyzed

on a TRACE DSQ Finnigan (Thermo Finnigan) mass

spectrometer (ion source 2501C, 70 eV, acquisition mass range

50–650 Da, full-scale mode) which was connected to a TRACE

Finnigan GC equipped with an Rtx-5MS fused silica capillary

column (30 m, 0.25 mm id, 0.25 mm df). Injector temperature

was 2501C in splitless mode. Carrier gas was He at a flow rate

of 1 mL/min. Injection volume was 1 mL. The oven tempera-

ture was held at 1001C for 1 min, then programmed at 201C/

min up to 3001C and held isothermically at 3001C for 15 min.

3 Results and discussion

Aim of the present study was to carry out a systematic and

exhaustive survey of proteins contained in commercial rice

bran. To date, relatively few studies have been specifically

designed to identify rice bran proteome. Attempts performed

by other authors to address this issue by using IEF-2DE have

not been completely successful; only a limited number of

proteins being partially characterized [23, 24]. To identify the

largest possible number of proteins, three different protocols

for protein extraction have been applied in this study. In

parallel with an extraction protocol previously employed by

other authors [14], two additional procedures never reported

before for rice bran have been explored. The schematic

diagram of the proteomic workflow that illustrates the key

steps as outlined in this study is shown in Fig. 1. To

minimize sample handling artifacts, a number of variables

including pH, temperature and time of extraction have been

investigated for each approach considered.

3.1 EPs

In the present work, the three extraction protocols (EP 1, EP 2

and EP 3) described in the following paragraphs were used.

EP 1. The addition of cold acetone containing TCA and

DTT was used on both DF and FF rice bran as previously

described [14]. In our hands, the highest recovery of proteins

(around 17%) was achieved when the procedure was applied

Figure 1. Schematic diagram ofthe proteomic workflow showingthe key steps as outlined in thisreport.



to FF rice bran. In this case 30 g of rice bran produced 5.2 g

of a protein precipitate with the aspect of a pale-yellow

powder. A lower amount of proteins (around 12%) was

obtained from DF rice bran. The recovery was lower than

the average value of 14–16% previously reported in the

literature [14, 15].

EP 2. Sodium bisulfite solutions had never been used

before for rice bran protein extraction. Nevertheless, on the

basis of the good extraction yields previously reported for

soybeans and other vegetable sources [42], this procedure

looked promising and its effectiveness was tested also on

our material. Therefore, a series of attempts were performed

to optimize the pH, the bran to bisulfite solution ratios

(5; 10 and 20% w/v slurries were prepared) and the time of

incubation (8; 16 and 24 h, respectively). The highest yield

(14.40%) of soluble proteins could be achieved incubating

for 24 h at room temperature a 20% bran slurry prepared in

50 mM sodium bisulfite buffer pH 8.5 and precipitating

proteins at their isoelectric point (around pH 4.0) by addi-

tion of HCl. No differences in the precipitation yield were

observed between DF and FF rice bran. Forty gram of rice

bran yielded in both cases 5.9 g of a protein mixture with the

aspect of soft white flakes.

EP 3. SDS-urea was previously employed for rice grains

only [43]. Thus, its use was extended also to rice bran. In our

hands, the extraction performance of this method was higher

with DF rather than with FF rice bran. To extract proteins, rice

bran was ground in liquid nitrogen as indicated in the

experimental section. Proteins were precipitated by addition of

a solution made of 50 mM Tris-HCl buffer pH 6.8 containing

8 M urea, 4% SDS, 5% 2-mercaptoethanol and 20% glycerol.

This procedure resulted for DF rice bran in protein yield

(12%) similar to that previously observed with EP 1, i.e. 30 g of

DF rice bran produced 3.6 g of a precipitate with a pale-brown

colour. The same treatment on FF rice bran produced 3.1 g of

a protein precipitate, with a yield of 10.3%.

3.2 Protein patterns by 1-DE

Not surprisingly, the composition of the soluble protein

mixture obtained from the various procedures varied with

the type of processing applied. Although identical amounts

of protein mixtures were loaded on the gel, the major

differences were apparently quantitative rather than quali-

tative. Indeed, the visual inspection of 1-DE profiles shown

in Fig. 2 evidenced few differences in the distribution

pattern of protein bands. The profiles of proteins obtained

from extraction of FF rice bran with EPs 1, 2 and 3 are

shown in lanes 3, 6 and 9, respectively. Despite the poor

separation of very high and very low MW protein bands,

most of them were consistently present, although with

different intensity, in most of the experiments carried out.

As expected, these profiles were also very similar, if not

practically identical to those of proteins obtained submitting

DF rice bran to the above procedures (lanes 4, 7 and 10,

respectively). The reproducible occurrence of protein

profiles qualitatively similar, in our opinion provided a

rationale for plausibly considering the extractability of

proteins comparable among the adopted procedures.

3.3 Protein identification

To assess the identity of proteins, 1-DE gel lanes were cut

into slices (along the dotted lines indicated by scissors in

Fig. 2), which were processed as indicated in details in the

experimental section. Data from each single slice were then

combined to obtain the composition of the whole lane.

Although achieved on complex protein samples, with the

presence of impurities from the gel-based separation step

prior to MS analysis, taken collectively, experimental

findings were encouraging. Based on the number of

proteins that were consistently identified for each lane

analyzed, the reproducibility of extraction within each

procedure was excellent. The number of identified proteins

extracted from DF rice bran by treatment with acetone-TCA

(EP 1), sodium bisulfite (EP 2) and SDS-urea (EP 3) was 12,

7 and 8, respectively. Likewise, the number of identified

proteins extracted from FF rice bran with the same

procedures was 9, 7 and 19, respectively. In total, 23 unique

proteins were identified and their distribution (shown in

Table 1) confirmed the good similarity of patterns among

different procedures. The use of SDS-urea (EP 3) on FF rice

bran was an exception to this uniform distribution. This

extraction technique was apparently the most ‘‘productive’’

since it allowed to identify the highest number of proteins.

In particular, different isoforms of type A and B glutelins

Figure 2. Mono-dimensional separation of rice bran proteinsextracted by applying acetone-TCA (EP1), Na2SO3 solution (EP2)and SDS-urea (EP3) on FF (lanes 3, 6 and 9) and DF (lanes 4, 7and 10) bran. Scissors and dotted lines indicate the level atwhich gel slices have been cut. Detailed information on allexperimental procedures are described in the text.



and of prolamins were identified. This finding was

interesting, if not surprising. In fact, owing to their

structure, glutelins and prolamins tend to aggregate

forming large, insoluble complexes. Most likely, the use of

dissociating agents promoted their dissociation and allowed

to identify each single component of these complexes.

3.4 2-DE

Since current 2-DE technologies can resolve many more

protein/peptide spots than the number of protein/peptide-

containing bands of current 1-D technology, a series of

attempts (not shown) were performed in which the pH

gradient in the first dimension and the gel porosity in the

second were optimized. A porosity of 16% and a non-linear

3–10 pH gradient were, in our hands, the best answers to

these questions. Thus, 2-DE gel analyses were performed in

triplicate, under the above experimental conditions, on all

protein samples obtained from each EP applied, to produce

18 gels in total (nine relative to FF and nine to DF rice bran

proteins). Consistently, more than 700 protein spots

(typically, 6507140 for EP1: 6007150 for EP2 and

5807160 for EP3) were observed on gels. Two of these

2-DE gels, containing typical protein patterns obtained from

FF and DF rice bran and representative of all other gels, are

shown in Fig. 3, panels A and B, respectively.

All three gels within a group of samples were scanned

and interpreted with the PD Quest 7.2 software. Spot

detection was achieved using the spot detection wizard tool

Table 1. List of rice (Oryza sativa) bran proteins obtained applying the three EPs indicated and extracted from 1-DE gel slices shown in

Fig. 2

Extraction procedurea)

TCA-acet-

one

Na2SO3 SDS-

urea

No. Accession

no.

Protein identified Function/location DF FF DF FF DF FF

1 P07728 Glutelin A1 Seed storage protein in endosperm | | | | | |2 P07730 Glutelin A2 Seed storage protein in endosperm | | | |3 Q09151 Glutelin A3 Seed storage protein in endosperm | | |4 Q02897 Glutelin B2 Seed storage protein in endosperm |5 P14614 Glutelin B4 Seed storage protein in endosperm | | |6 P29835 19 kDa globulin Seed storage protein in endosperm | | | | | |7 Q42465 Prolamin PPROL 14P Seed storage protein in endosperm |8 Q0DJ45 Prolamin PPROL 14E Seed storage protein in endosperm | |9 Q9ZRG9 Putative globulin (cupin family protein) Seed storage protein in endosperm | | | | |10 A3AHG5 Late embryogenesis abundant protein 1 Salt stress response in seedling roots and seedling

leaves

|

11 O64937 Elongation factor 1-a Protein biosynthesis/cytoplasm |12 P27777 16.9 kDa class I heat shock protein 1 Stress response/cytoplasm. |13 P29421 a-Amylase/subtilisin inhibitor Inhibits independently subtilisin and T. castaneum

a-amylase.

| | | |

14 Q84Q77 17.4 kDa class I heat shock protein 3 Stress response/nucleus |15 A2XL05 Oleosin 18 kDa Structural role to stabilize the lipid body during

desiccation/surface of oil bodies

| | | | | |

16 Q42980 Oleosin 16 kDa Structural role to stabilize the lipid body during

desiccation/surface of oil bodies

| | | | | |

17 Q75KH3 Glucose and ribitol dehydrogenase Oxidoreductase involved in embryonic carbohydrate

pathway

| |

18 P48494 Triosephosphate isomerase cytoplasmatic Glycolysis and gluconeogenesis/cytoplasm |19 P0C5C9 1-Cys peroxiredoxin A Antioxidant protein. It seems to inhibit germination

during stress/nucleus

| | | |

20 A2YQT7 Glyceraldeide 3-P-dehydrogenase cytosolic Glycolysis/cytoplasm |21 P17784 Fructose bisphosphate aldolase

cytoplasmatic isozyme

Glycolysis/cytoplasm |

22 P37833 Aspartate aminotransferase cytoplasmatic Amino acids, Krebs-cycle, nitrogen, carbon and

energy metabolism/cytoplasm

|

23 P55142 Glutaredoxin-C6 glutathione-disulfide oxidoreductase/cytoplasm |

Identification was achieved by means of LC-MS/MS.

a) DF, defatted; FF, full-fat; |, indicates the sample in which each protein was identified.



after defining and saving a set of detection parameters. After

spot detection, the original gel scans were filtered and

smoothed to clarify spots, remove vertical and horizontal

streaks and remove speckles. Three-dimensional Gaussian

spots were then created from filtered images. Three images

were created from the process: the original raw 2-D scan, the

Filtered image and the Gaussian image. A match set for

each group of samples was then created for comparison

after the gel images had been aligned and automatically

overlaid. Thus, for each EP a virtual image (master gel

(MG)) was produced, for a total of six MGs (three of which

relative to FF and three to DF rice bran), which included

protein spots only if present at least in two out of the three

gels. The MGs from DF and FF bran showed patterns of

proteins similar for each EP and they could be matched to

each other. This allowed to generate the final virtual image

indicated as high master gel (HMG) comprehensive of all

matched spots derived from MGs. A scheme that

summarizes the series of steps performed and the virtual

image HMG is shown in Fig. 4.

The spots on HMG were matched to reference gels and

selected for protein identification based on a combination of

the following criteria: spot quantity (spot density in blue-

coomassie stained pattern) and spot quality (shape and

compactness of spots, nonoverlap with others). From among

the 63 spots excised and analyzed, at the most seven spots

might be expected to be false positive. The remaining 56

spots gave interpretable spectra with the following annota-

tions: MS signals from 35 of these spots allowed to identify

unique proteins, 23 of which were the same proteins

previously observed with 1-DE and 12 were new proteins.

Signals from other 21 spots were interpreted as natural

isoforms of the same protein (in particular. glutelins and

prolamins) or different products of a single gene due to

PTMs or modifications caused by sample preparation.

Interestingly, all single gel spots analyzed contained a single

protein.

The characteristics and functions of the newly identified

proteins are summarized in Table 2.

3.5 In-solution digestion

In an effort to assess the actual total number of proteins

present in rice bran, some additional insights were

generated from procedures other than gel electrophoresis.

Thus, in-solution enzymatic digestion with different

proteases, separation and identification of peptides by LC-

MS/MS were performed ‘‘in parallel’’ (as indicated in the

experimental section) on 100 mg of each DF and FF protein

mixture dissolved in 100 mM ammonium bicarbonate pH

8.5 (final concentration 1 mg/mL). The lack of impurities and

salts, typically present in in-gel digested samples, that may

suppress the signal of peptide ions, resulted for LC-MS/MS

in the identification of a higher number of proteins either in

DF or in FF samples. In fact, in addition to the 35 proteins

previously reported, an average of 1–8 unique proteins,

whose characteristics and functions are summarized in

Table 3, were newly identified. In comparing protein

identification from in-gel MS and LC-MS/MS it was found

that 35 (81.4%), out of the 43 proteins identified by LC-MS/

MS, matched the proteins identified on gel spots.

Taken together, these results demonstrate that, while

the 2-DE gel-based image analysis for identification of

proteins is generally reliable, it may have some bias for the

detection of less abundant proteins or of proteins with

extreme pI values. They also suggested that the utilization of

only one or two of the three procedures to select for rice bran

proteins was not sufficient to reveal the ‘‘entire’’ proteome.

The results from these three parallel approaches have been

Figure 3. Two-dimensional gels obtained using a gel porosity of16% and a non-linear 3–10 pH gradient and containing typicalprofiles of FF and DF rice bran proteins are shown in panels Aand B, respectively. Given the similarity of profiles, these twogels may be considered representative of all 18 gels (not shown)produced for all FF and DF rice bran proteins extracted.Experimental conditions used for the production of gels aredetailed in the text.



combined in a table (Table S1 of Supporting Information),

which contains details about identification data of all 43

proteins identified, including protein specifications such as

accession number, percent of sequence coverage; number of

peptides identified and MOWSE score.

A second table (Table S2 of Supporting Information)

shows the primary sequences of all peptides found for each

protein identified.

3.6 Proteome profiling

Proteins identified in DF and FF groups for each EP applied

were characterized for their molecular function by assessing

Gene Ontology (http://www.amigo.geneontology.org). As

shown in Fig. 5 (panel A), patterns were reasonably similar

with the following exceptions. Sodium bisulfite solutions had

fewer transfer constituents compared with other two extrac-

tion methods. Enzymes were lower in DF samples from SDS-

urea and in FF samples from acetone-TCA than in each of the

other groups. The highest number of proteins was isolated in

FF samples from SDS-urea EP. Once collated, the informa-

tion from GO analysis revealed that signalling/regulation

proteins, proteins with enzymatic activity and storage

proteins were present in identical percent (30%) followed

by transfer and structural proteins (5%) (Fig. 5, panel B).

Representatives of the category of signalling/regulation

proteins were a series (n 5 4) of proteins belonging to the

late embryogenesis abundant proteins and one protein that

belongs to the small hydrophilic plant seed protein family,

and which acts as a cytoplasm protectant during essication.

Moreover, two proteins that belong to the small heat shock

proteins (HSP20) family were also identified. They are

produced in response to stress and both form oligomeric

structures. Interestingly, a different protein identified (salt

stress-induced protein) is produced in response to salt and

related osmotic stresses and another one promotes the GTP-

dependent binding of aminoacyl-tRNA to the site of

ribosomes during protein biosynthesis. Finally, a good

number (n 5 4) of a-amylase/subtilisin inhibitors (belonging

to the protease inhibitor 13 or 16 families or to the Bowman-

Birk serine protease inhibitor family) were identified and

classified in this category.

Among the proteins with catalytic activity, the enzymes

of glycolysis were predominant. Other have oxidoreductase

activity (glutaredoxin-C6, malate dehydrogenase) or are

antioxidant proteins (1-Cys peroxiredoxin A and peroxir-

edoxin 2E-1) that seem to contribute to the inhibition of

germination during stress or are involved in chloroplast

redox homeostasis. An interesting enzyme (lactoylglu-

tathione lyase) was also found, which belongs to the

glyoxalase I family and catalyzes the conversion of

Figure 4. Schematic representation that summarizes the series of steps followed to interpret 2-DE gels. First line (top to bottom): 2-DEgels obtained in triplicate for all protein samples (FF and DF) from each EP applied (EP1; EP2 and EP3). Second line: after matching thesegel images, a virtual image (MG) was produced for each EP, which included protein spots only if present at least in two out of the threegels. A total of six MGs (three of which relative to FF and three to DF rice bran proteins) were thus generated. Third line: based on thesimilarity of patterns of proteins for each EP, MGs from FF and DF bran could be matched to each other and the final virtual image,indicated as HMG and comprehensive of all matched spots derived from MGs was generated. The numbering of spots inside HMGreflects the numbers assigned to proteins in Tables 1–3.



hemimercaptal, formed from methylglyoxal and gluta-

thione, to S-lactoylglutathione. Particularly interesting

seems to be the pyrophosphate energized vacuolar

membrane proton pump, which belongs to the H1-trans-

locating pyrophosphatase (TC 3.A.10) family and establishes

a proton gradient of similar and often greater magnitude

than the H1-ATPase on the same membrane.

As expected, a large number of storage proteins were

also identified, i.e. type A (A1; A2 and A3) and B (B1; B2; B3

and B4) glutelins, belonging to the 11S seed storage protein

Table 3. List of rice (Oryza sativa) bran proteins obtained applying the three EPs indicated and submitted to gel-free tryptic digestion

# Accession no Protein identified Function/location Extraction procedurea)

TCA-

acetone

Na2SO3 SDS-

urea

DF FF DF FF DF FF

1–35 See content of Tables 1 and 2

36 A2WJU9 Peptidyl-prolyl cis-trans

isomerase

It catalyzes the cis-trans isomerization of proline imidic peptide

bonds in oligopeptides

|

37 Q6H6C7 Phosphoglycerate kinase Glycolisis/Cytoplasm |38 Q01882 Seed allergenic protein RAG2 Belongs to the a-amylase/trypsin inhibitor family | | | |39 A2WK50 Bowman-Birk-type bran trypsin

inhibitor

Protease inhibitor | | | | |

40 Q01MB6 Lectin N-Acetyl-D-glucosamine binding lectin |41 A2XMB2 10 kDa Prolamin Seed storage protein/Vacuole \gt aleurone grain | |42 P17048 13 kDa Prolamin C Seed storage protein/Vacuole \gt aleurone grain | |43 Q7XDC8 Malate dehydrogenase Tricarboxylic acid cycle/Cytoplasm |

Identification of proteins was achieved by means of LC-MS/MS. Only proteins not identified before in the 1-DE and 2-DE gels have been

reported here.

a) DF, defatted; FF, full fat; |, indicates the sample in which each protein was identified.

Table 2. List of rice (Oryza sativa) bran proteins extracted by applying the three EPs indicated and separated on 2-DE gels

No. Accession

no.

Protein identified Function/location Extraction procedurea)

TCA-

acetone

Na2SO3 SDS-

urea

DF FF DF FF DF FF

1–23 See content of Table 1

24 P14323 Glutelin B1 Seed storage protein in endosperm | |25 Q6ERU3 Glutelin B 5 Seed storage protein in endosperm | |26 P0C5A4 Late embryogenesis abundant

protein, group3

Salt stress response in seedling roots and seedling leaves | | |

27 P46520 Embryonic abundant protein 1 May act as a cytoplasm protectant in dry seeds and immature

embryos /cytoplasm

| | | |

28 Q01881 Seed allergenic protein RA5 Belongs to the a-amylase/trypsin inhibitor family | |29 Q0JMY8 Salt stress-induced protein In response to salt and related osmotic stresses | |30 Q01883 Seed allergenic protein RAG1 Belongs to the a-amylase/trypsin inhibitor family | |31 Q42971 Enolase Glycolysis/cytoplasm | |32 Q69TY4 Peroxiredoxin 2E-1 chloroplastic Reduces hydrogen peroxide and alkyl hydroperoxides |33 Q948T6 Lactoylglutathione lyase Secondary metabolite metabolism; methylglyoxal degradation | | | |34 A2ZHF1 Non-specific lipid transfer protein 1 May play a role in wax or cutin deposition in the cell walls and

certain secretory tissues.

| | | |

35 A2XBN5 Non-specific lipid transfer protein 2 May play a role in plant defense or in the biosynthesis of cuticle

layers

|

Identification of proteins was achieved by means of LC-MS/MS. Only proteins not identified before in the 1-DE gels have been reported

here.

a) DF, defatted; FF, full fat; |, indicates the sample in which each protein was identified.



(globulins) family; a 19 kDa globulin and the globulin 1-S

allele, and four proteins belonging to the prolamin family

(prolamins PPROL 14E, 14P, 10 kDa prolamin and 13 kDa

prolamin C). Two oleosins (16 and 18 kDa) that may have a

structural role to stabilize the lipid body during dessication

of the seed by preventing coalescence of the oil were

included in the category of structural proteins. Other two

proteins, belonging to the plant lipid transfer protein family,

which transfer lipids across membranes and may play a role

in plants defense or in the biosynthesis of cuticle layers,

were classified as transfer proteins.

Finally, a fragment of RNA-binding protein was identi-

fied and considered regulatory proteins.

3.7 Comparison of our data with results described in

the literature

Although the number of proteins to date identified in rice

bran is rather limited, remarkable progresses in proteomic

technologies have been made over the past few years,

which allowed researchers to identify and catalogue a large

number of proteins from several rice tissues and organelles

[23, 26]. By using 2-D-PAGE, automated sequencing

and MS techniques, proteomics of rice embryo and

endosperm [19], root [20], green and ethiolated shoot [21],

cultured suspension cells [22], anther [29], leaf sheath [25],

Golgi organelles [30] and mitochondria [31] have been

systematically investigated. This prompted Komatsu et al.[36–38] to construct the rice proteome database (see

the web site: http://gene 64.dna.affrc.go.jp/RPD) that is an

excellent source of information on the progress of rice

proteome research. As expected, many of the proteins

identified in the above tissues and organelles were common

also to rice bran. The variety of proteins shared by bran

and other rice tissues is evidenced in the central column

of Tables T1, T2 and T3 of this report. Not surprisingly, a

number of these proteins have been previously identified

in rice bran by other authors, in particular by Trisiriroj

et al. [14] who, in an interesting paper, described the

Figure 5. Panel A. Ontology of proteinsidentified in the DF and FF groups foreach EP applied based on their mole-cular function. Panel B. Categorizationof the identified rice bran proteinsaccording to their GO terms.



application of 2-DE combined with MALDI-MS and

N-terminal sequencing to investigate protein patterns of

14 rice cultivars. They studied aromatic and nonaromatic

rice bran with the aim of finding marker proteins to classify

aromatic rice. The observation that different varieties of

aromatic and deepwater rice are characterized by different

prolamin polypeptides allowed them to assume that the

regulatory expression of prolamins could be associated with

rice variety characteristics. Interestingly, a number of

prolamin peptide sequences determined with our study

(see Table S2) perfectly overlapped those previously

published by Trisiriroj et al. [14].

Likewise, embryo-specific proteins and globulin/puta-

tive globulin had been previously identified in rice bran by

Yano and Kuroda [40] as putative targets of thioredoxin.

Their findings seemed to suggest that thioredoxin controls

the lifetime of specific proteins effectively by regulating

the redox reactions coordinately. Finally, the 20 kDa

bifunctional subtilisin/a-amylase inhibitor was previously

identified also by Ohtsubo and Richardson [48]. After

performing the complete amino acid sequence of this

protein, they observed a high-sequence similarity with other

bifunctional inhibitors previously isolated from wheat and

barley, which are related to the Kunitz family of proteinase

inhibitors from legume seeds. The identification in rice bran

of proteins never observed before may be useful for

completing the knowledge of proteins expressed in this

model plant.

3.8 GC-MS analysis of organic phase

Although the study of the composition of the oil produced

by treating rice bran with n-hexane was outside the scope of

this article, it seemed worthwhile to introduce these few

comments about our data. The major components of this oil

reported in the literature [40] are oleic (42.5%) and linoleic

(39.1%) acids, respectively. Palmitic, stearic and linolenic

acids are less represented (15, 1.9 and 1.1%, respectively),

whereas arachidic and behenic acids are present only in

traces, ranging between 0.2 and 0.5%. Unexpectedly, the

fatty acid composition of rice bran considered in this study,

while being similar to that above indicated, in terms of type

of components, was found to differ in their relative

proportion. In fact, if the amounts of linoleic acid (37%)

were comparable, the amount of palmitic (44%) was three-

fold higher and that of oleic acid (11%) was four-fold lower

than that reported. All other components were present in

amounts comparable with the values previously indicated.

The finding of higher amounts of palmitic and lower

amounts of oleic acids allowed to speculate that, in terms of

health benefits, the composition of this oil was better

compared with others. Palmitic acid in fact has been shown

to have a favourable effect on blood lipids and cardiovascular

diseases, while a lower amount of oleic acid should decrease

the risk of breast cancer associated with high levels of

monounsaturated fatty acids.

4 Concluding remarks

The use of rice bran in human nutrition is still limited

although this compound has potentially favourable nutri-

tional and technological characteristics, which may permit

to use especially its protein components as ingredients in

the formulation of a series of food products. Obviously, the

exact knowledge of the qualitative and quantitative protein

components of rice bran is an essential aspect to be

considered for a better understanding of the functional

properties of this resource.

This pilot study intended to extract and identify the

largest possible number of rice bran proteins through the

application of three different extraction protocols, either on

FF or on DF rice bran. Our experimental results demonstrate

that a single EP would have not extracted all proteins. In fact,

similar, but not identical, protein patterns are obtained

according to the extraction medium and a comprehensive

protein complement would thus require combining various

extraction methods. The combination of data collected from

1-DE, 2-DE and gel-free procedures and analyzed by LC-MS/

MS allowed to identify a good number of proteins. Obviously,

we cannot definitively rule out the possibility that the number

of proteins might have been underestimated. For example

proteins with extreme pI values could have been lost during

focusing in the first dimension and proteins that are much

less abundant than others could appear in the second

dimension as such faint spots that produce non-interpretable

MS signals. Although the unfiltered spot number in the

original gels was very high, it is a matter of fact that

the number of spots does not necessarily correspond to the

number of proteins. Thus, the filtration procedure adopted

allowed to work on a considerably reduced number of spots,

after ignoring a number of very faint spots, of spots with

undefined shape and of false positive due to a variety of

staining and/or sample manipulation artifacts. On the other

hand, given the results of in-solution digestion, the question

arose whether the limited percent (around 20% of total

proteins) of newly identified proteins might provide a ratio-

nale for speculating that the research of new rice bran

proteins was going to ‘‘completion’’. In this context, it

seemed to be plausible that the sum of proteins observed by

following the three procedures could be regarded as the

potential protein platform that defines rice bran proteome.

The fact that all extraction and identification procedures have

been performed in triplicate with an excellent reproducibility

of data, in terms of number of proteins identified, strength-

ened in us this conviction. In our opinion, these findings

have greatly enhanced the current knowledge on rice bran

protein extracts. Forty-three proteins have been unambigu-

ously identified and classified as signalling/regulation

proteins (30%), proteins with enzymatic activity (30%),

storage proteins (30%), transfer (5%) and structural (5%)

proteins. Although characterized only from a qualitative point

of view, this set of proteins may represent a source of infor-

mation by which the qualities of rice bran as food resource

should be reconsidered and, hopefully, evaluated better.



The authors acknowledge the Curti Riso S.p.A.(ValleLomellina, Pavia, Italy) for the essential contribution given tothe realization of this work.

Drs. Serena Giuliano and Ileana Passadore (Department ofBiochemistry, University of Pavia) are kindly acknowledged fortheir invaluable support with the preparation and interpretationof 2-DE data.

The authors declare no financial or commercial conflict ofinterest.

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deciphering the proteomic profile of rice (oryza sativa) bran: a pilot study

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