Description of the mechanisms underlying

Description of the mechanisms underlying geosminMarc Behr, Tommaso Serchi, Emmanuelle Cocco, Cédric Guignard, Kjell Sergeant, Centre de Recherche Public-Gabriel Lippmann, Département Env

Trang 1

Description of the mechanisms underlying geosmin

Marc Behr, Tommaso Serchi, Emmanuelle Cocco, Cédric Guignard, Kjell Sergeant,

Centre de Recherche Public-Gabriel Lippmann, Département Environnement et Agro-biotechnologies, Belvaux, Luxembourg

Article history:

Received 20 March 2013

Accepted 24 October 2013

A 2D-DIGE proteomics experiment was performed to describe the mechanism underlying the production of geosmin, an earthy-smelling sesquiterpene which spoils wine, produced by Penicillium expansum The strains were identified by sequencing of the ITS and beta-tubulin regions This study was based on a selection of four strains showing different levels of geosmin production, assessed by GC–MS/MS The proteomics study revealed the differential abundance of

107 spots between the different strains; these were picked and submitted to MALDI-TOF–TOF MS analysis for identification They belonged to the functional categories of protein metabolism, redox homeostasis, metabolic processes (glycolysis, ATP production), cell cycle and cell signalling pathways From these data, an implication of oxidative stress in geosmin production may be hypothesized Moreover, the differential abundance of some glycolytic enzymes may explain the different patterns of geosmin biosynthesis This study provides data for the characterisation of the mechanism and the regulation of the production of this off-flavour, which are so far not described in filamentous fungi

Biological significance Green mould on grapes, caused by P expansum may be at the origin of off-flavours in wine These are characterized by earthy–mouldy smells and are due to the presence of the compound geosmin This work aims at describing how geosmin is produced by P expansum This knowledge is of use for the research community on grapes for understanding why these off-flavours occasionally occur in vintages

Keywords:

Geosmin

Penicillium expansum

Proteomics

Oxidative stress

MVA pathway

MEP pathway

Wine is a product for which organoleptic quality is primordial

Wine aroma results from the contribution of volatile

com-pounds originating from the grape microflora and winemaking

practices[1] Although in most cases aroma compounds confer

a special, variety-specific, positive characteristic to a wine,

several grape-derived aroma compounds may alter wine aroma

in a negative way Over the last years, winegrowers have observed organoleptic defects in wine characterized by mush-room, mouldy, camphoric or earthy odours[2] The risk for such defects is high when grapes are infected by rots Indeed, they are produced by Botrytis cinerea (causal agent of grey mould), Penicillium expansum (causal agent of green mould), a

wide-Abbreviations: ITS, internal transcribed spacer (of the rDNA); PTV, programmed temperature vaporization; MEP pathway, methylerythritol phosphate pathway; MVA pathway, mevalonate pathway; PH-like, pleckstrin homology-like

⁎ Corresponding author at: Centre de Recherche Public-Gabriel Lippmann, Département Environnement et Agro-biotechnologies, 41, rue du Brill, 4422 Belvaux Luxembourg Tel.: + 352 47 02 61 441; fax: + 352 47 02 64

E-mail addresses:behr@lippmann.lu(M Behr),serchi@lippmann.lu(T Serchi),cocco@lippmann.lu(E Cocco),guignard@lippmann.lu (C Guignard),sergeant@lippmann.lu(K Sergeant),renaut@lippmann.lu(J Renaut),evers@lippmann.lu(D Evers)

http://dx.doi.org/10.1016/j.jprot.2013.10.034

A v a i l a b l e o n l i n e a t w w w s c i e n c e d i r e c t c o m

ScienceDirect

w w w e l s e v i e r c o m / l o c a t e / j p r o t

Trang 2

spread filamentous fungus responsible of fruit decay including

grapes [3], or a combination of both [4] The compounds

responsible for defects associated with mushrooms are C8

alcohols and ketones such as 1-octen-3-ol and 1-octen-3-one

and they have been reported to be metabolites of various fungi;

in oenology, 1-octen-3-ol has been associated with the presence

of B cinerea on grapes[5] As for the earthy smell, La Guerche et

al.[2]identified the responsible molecule as (−)-geosmin The

olfactory perception threshold for geosmin in wines is 50 ng/L

[6] According to La Guerche et al.[7], geosmin originates from

the metabolism of P expansum on grapes pre-contaminated by

B cinerea; thus, B cinerea would induce the production of

geosmin by P expansum

Little is known about the biosynthesis of geosmin by

P expansum Although several papers report about geosmin

production in Actinobacteria, certain Cyanobacteria,

Myxo-bacteria and higher Fungi, little is known about the genes

implicated in geosmin biosynthesis [8] As suggested by

Bentley and Meganathan [9], geosmin would be derived

from a sesquiterpenoid precursor and would be synthesized

from farnesyl pyrophosphate The use of a geosmin

over-producing strain of Streptomyces citreus over-producing also high

levels of the sesquiterpene alcohol

germacra-1-E,5E-dien-11-ol (germacradienol), indicated that this compound might

be a precursor of geosmin [10] Later studies showed that

germacradienol production was indeed the committed step

in geosmin production [11] According to Gust et al [3]

presenting a study on Streptomyces, sesquiterpene synthase

would be involved in an early step in geosmin biosynthesis

The functional characterization of six sesquiterpene synthases

in a basidiomycete called Coprinus cinereus has been described

[12] As previously said, concerning P expansum, few data are

available on geosmin synthesis in must or culture medium

Dionigi[13]has studied the impact of copper sulphate addition

in Czapek medium on the biosynthesis of geosmin Recently,

a cytochrome P450 monooxygenase gene, gpe1, which may

intervene during the transformation of farnesyl pyrophosphate

to geosmin, has been described[14]

Proteomics techniques are used more and more to unravel

proteins implicated in different pathways[15] Here we report a

comparative proteomic study aimed at identifying differentially

expressed proteins in four P expansum strains producing geosmin

in different amounts in culture medium Some proteins

puta-tively implicated in geosmin production were identified and their

possible implication in geosmin biosynthesis is discussed

2.1 Cultivation of strains

The potential of geosmin production was assessed in triplicate on

fourteen Penicillium strains isolated in vineyards in the

luxembourgish part of the Moselle valley Picking was done on

mature berries between 2007 and 2010 Cultures were conducted

on malt-agar medium in Petri dishes at 25 °C, based on initial

monoconidia production The same conditions were applied for

the assessment of geosmin production and the proteomic studies:

50 mL of malt-peptone media were inoculated with 50μL of the

conidia suspension (106conidia/mL) in a 250 mL Erlenmeyer

flask, plugged with cotton and placed at 120 rpm, at room temperature, during 3 days Geosmin quantification was done in triplicate and protein extraction was done in quadruplicate

2.2 DNA extraction

Pure isolates were sub-cultured in Potato Dextrose Broth (PDB) during one week at 25 °C on a rotary shaker set at 120 rpm The subsequent biomass was lyophilised prior to grinding with metallic beads DNA was extracted with the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany), following instruc-tions of the manufacturer DNA concentration was quantified with the NanoDrop (ND 1000, Thermo Scientific, Waltham, MA) The samples were stored at−20 °C

2.3 Molecular identification of strains

All the strains were identified with four sets of primers:β-tubulin

1 BT1 (a and b),β-tubulin 2 BT2 (a and b)[16], ITS1–ITS4 and ITS U5–ITS R2[17] The PCR reaction mixture contained 10μL of Finnzymes Taq Phusion-buffer Mastermix HF (Thermo

Scientif-ic, Waltham, MA); 1μL of each primer (10 μM); 1 μL of DNA (100 ng/μL) and 7 μL of UltraPure™ DNase/RNase-free distilled water (Invitrogen, Paisley, UK) for a final volume of 20μL Amplification was performed on a Biometra T-professional thermocycler (Biometra, Goettingen, Germany) using the follow-ing programme: an initial denaturation at 98 °C (2 min) followed

by 30 cycles with denaturation at 98 °C (15 s), annealing at 67 °C (β-tubulin) or 63 °C (ITSU5–ITSR2) or 64 °C (ITS1–ITS4) during

20 s and elongation at 72 °C during 20 s (β-tubulin, ITS1–ITS4) or

10 s (ITSU5–ITSR2); the final elongation was performed at 72 °C during 10 min The size, quality and quantity of the amplicons were checked on 3% w/v agarose gel stained with ethidium bromide (1 h; 100 V) under UV transillumination

PCR products were diluted in UltraPure™ DNase/RNase-free distilled water (Invitrogen, Paisley, UK) to reach a concentration of 10–20 ng/μL The sequencing PCR was achieved with these dilutions using Big Dye products (Applied Biosystems, Carlsbad, CA): 5× sequencing buffer (2μL), Big Dye sequencing RR-100 (2μL), primer 10 μM (0.32 μL), 1 μL of the diluted PCR amplicons and 14.68μL of UltraPure™ DNase/ RNase-free distilled water (Invitrogen, Paisley, UK) for a total volume of 20μL Both strands of each amplicon were sequenced The PCR programme consisted in an initial denaturation at 96 °C (1 min) followed by 25 cycles of dena-turation at 96 °C (10 s) and elongation at 60 °C (4 min) The PCR products were cleaned with the BigDye Xterminator Purification kit (Applied Biosystems, Carlsbad, CA) Sequenc-ing was done on the Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems)

2.4 Geosmin measurement

Post culture geosmin abundance of fourteen strains was mea-sured on sterile-filtered media (cellulose acetate membrane, 0.2μm, Sartorius, Goettingen, Germany) When it was required, the cultivation medium was diluted up to 50 fold with fresh sterile malt-peptone broth 2-Ethoxy-3-isopropylpyrazine (IPEP) (TCI, Tokyo, Japan) was used as internal standard Geosmin standard [(±)-Geosmin solution, 100μg/mL in methanol] was

Trang 3

obtained from Sigma-Aldrich (St Louis, MO) The determination

of geosmin was performed by GC–MS/MS using a Trace GC Ultra

coupled to a TSQ Quantum XLS tandem mass spectrometer

(Thermo Scientific, Waltham, MA) Geosmin was

precon-centrated using Headspace Solid-Phase MicroExtraction

(HS-SPME) on a DVB/CAR/PDMS fibre (Supelco,

Sigma-Aldrich, St Louis, MO) The extraction was fully automated

using a PAL Combi-xt autosampler with the following

programme: incubation at 70 °C during 5 min, adsorption at

70 °C during 20 min and desorption in the GC injector during

2 min Injection was done in PTV splitless mode running the

following programme: 56 °C during 0.05 min — ramp of

14.5 °C/s until 270 °C and hold at 270 °C for 17 min The

column was an Rxi-5Sil MS (20 m∗ 0.18 mm ∗ 0.18 μm,

Restek, Bellefonte, PA) and the GC oven was programmed as

follows: 50 °C during 2 min, ramp of 15 °C/min until 100 °C

and 30 °C/min until 250 °C (hold for 4.7 min) Retention times

of geosmin and IPEP were respectively 8.38 and 10.07 min A

first, semi-quantitative approach, based on the calculation of

the geosmin to internal standard ratio, was used to select

four strains within the fourteen, i.e P4, P8, P21 and P23

(Fig 1) P4 and P8 were selected as low geosmin producers

whereas P21 and P23 were considered as high geosmin

producers These four strains were identically resubmitted to

cultivation and geosmin assessment to obtain quantitative data

(using a calibration curve with geosmin standard) The case of P8

was ambiguous: its production during the second round was

much higher than during the first one

2.5 Protein extraction and quantification

Unless stated otherwise, all reagents used for extraction and

subsequent separation were purchased from GE Healthcare

(GE Healthcare, Little Chalfont, UK) Mycelia which were used for protein extraction were the same than those produced for geosmin assessment Mycelia were removed from growing medium and dried on cellulose acetate membrane After-wards mycelia were ground in liquid nitrogen and proteins were precipitated by addition of ice cold TCA/Acetone/DTT (20%/79.9%/0.1% v/v) overnight at −20 °C The mixture was centrifuged at 30,000 g for 45 min at 4 °C The precipitate was washed three times with rinsing buffer (TCA 20%, acetone 80% v/v) and dried in vacuo Pellets were solubilised in 1 mL of lysis buffer (Urea 7 M, Thiourea 2 M, CHAPS 4%, TRIS 30 mM and proteases inhibitors) for about 30 mg of mycelium Quantification of the extracted proteins was achieved by the

(Beckman Coulter, Brea, CA)

2.6 Labelling of proteins and 2D electrophoresis

Prior to labelling, the pH of each sample was checked and, if necessary, adjusted to pH 8.5 30μg of proteins were labelled and separated by 2D-DIGE as reported previously[18] Briefly,

240 pmol of dyes were incubated with the proteins for 30 min

on ice in the dark The reaction was then stopped by addition

of 1μL of 10 mM lysine solution and incubation on ice for 10 additional minutes Samples were labelled either with Cy3

or Cy5; the internal standard, constituted by an equal amount

of each sample, was labelled with Cy2 Dye swap was performed by labelling, in each group, 2 biological replicates with Cy3 and the other 2 biological replicates with Cy5 After labelling, one Cy3 labelled sample was combined with one Cy5 labelled sample and with the Cy2 labelled standard: the mixture was diluted to 450 mL with a solution containing Urea 7 M, Thiourea 2 M, CHAPS 4%, TRIS 30 mM, 9μL of

Fig 1– Preliminary semi-quantitative assessment of geosmin production of 14 strains of Penicillium expansum by GC–MS/MS Values represent the geosmin/internal standard ratio ± SD n = 3 biological replicates

Trang 4

Bio-Lyte pH 3–10 ampholyte buffer (Bio-Rad, Hercules, CA)

and 2.7μL of destreak reagent (GE Healthcare) and traces of

bromophenol blue 24 cm pH 3–10 non-linear strips (Readystrip

IPG, BioRad) were passively rehydrated at room temperature

overnight and focused at 20 °C in an Ettan IPGphor III (GE

Healthcare) system until reaching approximately 100 kVh

Following the first dimension, equilibration of the strips was

carried out firstly during 15 min in the equilibration solution

(provided by Serva, Heidelberg, Germany) containing 1% w/v

DTT and then in equilibration solution supplemented with 2.5%

w/v iodoacetamide solution (w/v) for 15 min The second

dimension was obtained by a run on a 12.5% pre-cast

polyacrylamide gel (Serva, Heidelberg, Germany) Obtained

gels were scanned at a spatial resolution of 100μm with a

9400 Typhoon (GE Healthcare) using the following wavelengths:

excitation at 488 nm, 532 nm, and 633 nm (Cy2, Cy3 and Cy5,

respectively) and emission at 520 nm, 610 nm and 670 nm (Cy2,

Cy3 and Cy5, respectively) Images of the gels were analysed by

DeCyder 2D Differential Analysis v.7.0 software (GE Healthcare)

Maps were calibrated, to obtain experimental molecular weight

and isoelectric point estimations, using the Fusarium reference

map which was produced earlier in our laboratory [19]

Highlighted proteins of interest (fold change ±1.3; t-test≤0.05)

were picked, trypsin digested for 6 h at 37 °C and then spotted

on MALDI disposable targets by the Ettan Spot Handling

workstation (GE Healthcare) Identification of the proteins was

carried out using an AB SCIEX TOF/TOF 5800 System (AB SCIEX,

Framingham, MA) MS spectra were internally calibrated using

trypsin autocleavage signals In MS/MS mode an external

calibration using fragmentation products of Glu-fibrinopeptide

was done For MS, the recorded spectrum was the accumulation

of 1500 shots, for MS/MS this was 3000 Spectra were acquired

using an automated approach defined in the MALDI software

(TOF/TOF Series Explorer™ V4.1.0, AB Sciex); for each spot the 8

highest peaks in the raw MS spectrum were selected for

fragmentation after exclusion of common contaminants, for

instance peaks from trypsin autocleavage products or keratin

Proteins were identified by searching with the MASCOT

algorithm version 2.3 (Matrix Science, www.matrixscience

com, London, UK) against the NCBI database (updated to the

18th of January 2013, with 1,585,852 sequences belonging to

“Other Fungi”), using ProteinPilot™ Software version 4.0 (AB

SCIEX) Searches were carried out allowing a mass window of

100 ppm for the precursor and 0.5 Da for fragment ion masses

The search parameters allowed maximum two missed

cleav-ages; carbamidomethylation of cysteine as fixed modification;

oxidation of methionine and oxidation of tryptophan (single

oxidation, double oxidation and kynurenine) as variable

mod-ifications Proteins with probability-based MOWSE scores

(p≤ 0.05) were considered to be successfully identified

3.1 Identification of the strains

Consensus sequences have been produced and compared to

reference strains through BLAST Each strain has been

successfully amplified and sequenced by each primer set

They were all identified as P expansum with an E value of 0

and 100% of identity The amplicon sizes were 484 bp (BT1),

477 bp (BT2), 716 bp (ITS1–4) and 280 bp (ITS U5-R2) (Behr et al., Journal International des Sciences de la Vigne et du Vin, accepted manuscript)

3.2 Geosmin production of the strains

In order to select the appropriate strains for the proteomic investigations, fourteen isolates were submitted to a prelim-inary geosmin assessment (Fig 1) Based on this screening, four strains, namely P4, P8, P21 and P23, were selected P4 and P8 were supposed to be low geosmin producers, while P21 and P23 exhibited a much higher production during the prelimi-nary screening During the second assessment (Fig 2), a similar geosmin production was measured with the exception

of P8, showing a production comparable to P21 and P23 Malt-peptone broth was found to be a very efficient medium for the induction of geosmin production The determination

of geosmin content is made easier by the liquid form of the medium, which allows a direct extraction by HS-SPME, without the extraction step required by culture on solid media [2] As compared to similar studies realised on other medium (grape juice or malt-agar), the production was much higher Indeed, Morales-Valle et al [4] have described a maximum concentration around 600 ng/L; La Guerche et al [20] have reported, under the same conditions, a maximum concentra-tion of 500 ng/L, while we have reached an average concen-tration superior to 5000 ng/L for P23 Usually, concenconcen-trations reached in wine are lower, generally around 100 ng/L, up to

300 ng/L [21,22] Despite the first objective to select two strains of each phenotype, we decided to keep P8 for the proteomic study, since the examination of such a profile may

be interesting

3.3 Differentially expressed proteins

107 proteins were differentially expressed and were used

to do a Principal Component Analysis (PCA, Fig 3) and a Hierarchical Clustering (HC, Fig 4) A typical gel with differentially expressed proteins is shown (Fig 5) Information concerning the fold-change of the proteins is presented in Table 1 In supplementary data S1, a summary of all relevant information for each identified protein, such as possible

Fig 2– Quantitative assessment of geosmin production (ng/L

of media ± SD) for the strains used in the proteomic study

n = 3 biological replicates

Trang 5

involvements of the proteins in metabolism as suggested by

KEGG databasehttp://www.genome.jp/kegg/pathway.html, is

reported In supplementary data S2, detailed information

about the identification of the differentially expressed

pro-teins, including peptide mass fingerprinting (PMF) and MS/MS

fragmentation data can be found

3.3.1 Clustering of the strains

Only the proteins which have exhibited a fold change of at

least ± 1.3 and a p value below 0.05 and resulted in a single

identification are presented inTable 1 Most of the proteins

were presenting multiple isoforms with sometimes different

behaviours in their relative fold-change It is clear that the two

high geosmin producing strains P21 and P23 can be

distin-guished neither in the PCA nor in the HC, while the low

geosmin producer P4 is well separated from the other strains

The strain P8 was not consistent in its production of geosmin,

so that we could not clearly put it in one of the two categories,

and this can be seen in the proteomic profile, since in the PCA

and in the HC this strain was separated from high producers

and the low producer Hereafter, the classification of the

differentially regulated proteins into different functional

categories will be discussed

3.3.2 Proteins involved in metabolic processes

Several proteins involved in metabolic processes were found to

be differentially abundant between the four strains Some of the

proteins are involved in carbohydrate catabolism, including

enolase and phosphoglycerate kinase (PGKA) Spots containing

these proteins did not show a uniform trend within the groups

Several of them were more abundant in the strain P4 (3 isoforms

of phosphoglycerate kinases PGKA), while those containing

enolase were less abundant Fourteen isoforms of enolases

were found; nine of them were found in their full length

(spots n° 1638, 1643, 1655, 1656, 1663, 1667,1674, 1680 and

1686; 43 kDa, pI from 4.89 to 5.20) while the others were found

to be degradation/processing products of enolase Spot n°

2340 contains the original N-terminus, in all the spots (spots n° 2767, 2768, 2800, 3174 and 4580) the original C-terminus was identified The peaks were extracted from the spectra and used for classification with Speclust (http://bioinfo.thep lu.se/speclust.html) [23] With the peaks-in-common tool, those peaks that were unique to each spectrum were isolated and studied Because the spectra were generally of low intensity, no differences between the molecular forms at the same molecular weight could be found nor could cleavage sites resulting in the observed forms be discerned The nine isoforms presenting an intact form did not display significant differences Four degraded forms were more abundant in the geosmin producing strains

Importantly, acetyl-CoA C-acetyltransferase, the enzyme which constitutes the beginning of secondary metabolism starting from acetyl-CoA [24] was less abundant in P4 This enzyme catalyses the transfer of an acetyl group into acetyl-CoA, producing acetoacetyl-CoA Acetyl-CoA is mainly produced via pyruvate from the glycolysis, or by theβ-oxidation

of the fatty acids Acetoacetyl-CoA is the starting point for the synthesis of farnesyl diphosphate, the common molecule

of pathways leading to sesquiterpenoid products, including geosmin An overview of the glycolytic, methylerythritol phosphate (MEP) and mevalonate (MVA) pathways is shown (Fig 6) Expressions of the ATP synthase enzymes were also changing from one isoform to the other: two were much more abundant in P4 and six were less abundant Other proteins related to ATP synthesis were also differentially abundant: two cytochrome C oxidases (subunit 5a) were less abundant in P4 Cytochrome C oxidase is involved in the ATP production by the mitochondrial respiratory chain Two enzymes may be consid-ered both as metabolic and linked to phytopathogenicity: serine carboxypeptidase and proteinase A They are involved in

Fig 3– PCA analysis of the strains (n = 4 biological replicates) used in proteomic studies Left panel: score plot of spot maps Right panel: loading plot of proteins

Trang 6

nitrogen metabolism and also in the lysis of molecules

encountered during infection of vegetal host cells

(destruc-turation of cell wall, reaction to PR proteins)[25,26] The three

spots corresponding to proteinase A were less abundant in P4,

as were two spots containing serine carboxypeptidases In

contrast, three other spots containing serine

carboxypepti-dases were significantly more abundant in P4

3.3.3 Proteins involved in protein synthesis and folding

Enzymes involved in protein synthesis and folding were also

a major point of differentiation between the groups One protein involved in DNA transcription (a nucleic acid binding protein) was more abundant in P4 Concerning protein synthesis, ribosomal protein S2 (RPS2) and one translation elongation factor containing a glutathione S-transferase

Fig 4– Hierarchical clustering of four strains of Penicillium expansum Red, yellow, green and blue dots represent P4, P8, P21 and P23 respectively Pearson correlation coefficient was used in order to achieve the clustering

Fig 5– Representative gel of Penicillium expansum proteome Whole protein extracts were labelled with CyDyes and separated

in first dimension by 24 cm 3–10 non-linear strips and in second dimension by 12.5% polyacrylamide precast gels The numbers of picked spots are reported The presented image is the standard (Cy2 channel) of the gel which was used as master gel in the experiment Additional images, one representative of each experimental group, are presented in supplementary material S3

Trang 7

Table 1– Differentially abundant proteins identified by MALDI-MS in the four Penicillium expansum strains Theor.

MW/pI— theoretical molecular weight (expressed Da) and isoelectric point (expressed in pH units), Exp MW/pI — experimental molecular weight (expressed in Da) and isoelectric point (expressed in pH units); P8/P4 column and followings: fold change relative to the reported strains with the corresponding p-value are reported: positive values (reported in green) when the numerator is up-regulated– negative values (reported in red) when the denominator is up-regulated

Spot

No

Theor

MW/pI

Exp

MW/pI

Protein name / function

UniProt access

No

Protein Existance (UniProt)

Glucose / TCA cycle metabolism

4.52

66680/

4.53

Amidase family

–1.32;

0.014

1.04;

0.75

1.12;

0.053

1.38;

0.036

1.48;

0.0027

1.07; 0.43

5.26

43255/

Inferred from homology

1.00;

0.89

–1.19;

0.21

–1.51;

0.028

–1.19;

0.15

–1.52;

0.018

–1.27; 0.21

5.26

43322/

1.00;

0.96

–1.21;

0.11

–1.43;

0.036

–1.22;

0.087

–1.43;

0.029

–1.18; 0.31

5.26

47055/

1.32;

0.10

–1.26;

0.080

–1.29;

0.10

–1.66;

0.0036

–1.70;

0.0073

–1.02; 0.72

5.26

43054/

1.04;

0.67

–1.29;

0.15

–1.74;

0.0018

–1.34;

0.10

–1.82;

0.00098

–1.35; 0.16

5.26

42392/

1.28;

0.047

–1.12;

0.17

–1.08;

0.50

–1.44;

0.0036

–1.39;

0.044

1.04; 0.88

5.26

42656/

1.05;

0.42

–1.12;

0.28

–1.30;

0.017

–1.18;

0.11

–1.37;

0.0034

–1.15; 0.25

5.26

42195/

1.09;

0.31

–1.18;

0.16

–1.43;

0.035

–1.29;

0.057

–1.56;

0.016

–1.21; 0.26

5.26

41869/

1.52;

0.0047

1.09;

0.29

1.06;

0.51

–1.39;

0.0097

–1.43;

0.0 093

–1.03; 0.71

5.26

42064/

1.17;

0.12

–1.22;

0.084

–1.39;

0.024

–1.43;

0.0098

–1.63;

0.0043

–1.14; 0.31

5.26

42260/

1.20;

0.21

–1.20;

0.23

–1.47;

0.054

–1.44;

0.015

–1.77;

0.0072

–1.23; 0.18

5.98

41869/

5.51

Phosphoglycerate

–1.12;

0.41

–1.50;

0.015

–1.47;

0.029

–1.34;

0.076

–1.31;

0.12

1.02; 0.93

5.26

30182/

1.46;

0.0096

1.72;

0.00080

1.62;

0.045

1.18;

0.15

1.11;

0.72

–1.06; 0.61

2361 34281/5.44 29948/4.99

Glyoxysomal and mitochondrial malate dehydrogenase

B6GYI7

1.06;

0.54

1.50;

0.018

1.34;

0.11

1.41;

0.033

1.26;

0.20

–1.12; 0.50

8.44

28366/

5.73

Malate

–1.05;

0.60

1.22;

0.088

1.24;

0.022

1.29;

0.083

1.31;

0.039

1.01; 0.82

8.44

27202/

5.43

Malate

–1.43;

0.058

1.32;

0.10

1.23;

0.17

1.88;

0.027

1.76;

0.056

–1.07; 0.76

5.26

23768/

1.41;

0.024

1.45;

0.045

1.53;

0.0042

1.02;

0.95

1.08;

0.54

1.06; 0.65

5.26

23731/

1.22;

0.16

1.42;

0.0023

1.40;

0.00023

1.16;

0.23

1.15;

0.21

–1.02; 0.86

5.93

23548/

4.65

Acetyl–CoA C–

acetyltransferase B6HV94

1.16;

0.43

1.41;

0.011

1.49;

0.00089

1.22;

0.22

1.29;

0.096

1.06; 0.47

5.98

23621/

7.33

Phosphoglycerate

–1.07;

0.66

–1.96;

0.012

–2.07;

0.0034

–1.83;

0.023

–1.93;

0.0078

–1.06; 0.86

5.98

23548/

7.20

Phosphoglycerate

–1.05;

0.78

–1.72;

0.020

–1.71;

0.019

–1.64;

0.017

–1.63;

0.016

1.01; 0.97

5.26

23402/

–1.64;

0.058

2.41;

0.00071

2.04;

0.029

3.95;

0.0012

3.34;

0.0080

–1.18; 0.41

5.45

23221/

4.75

Triosephosphate

–1.02;

0.74

1.46;

0.024

1.47;

0.0024

1.50;

0.20

1.50;

0.049

1.00; 0.88

Trang 8

Table 1 (continued)

Spot

No

Theor

MW/pI

Exp MW/pI

UniProt access

No

P8/P4 P21/P4 P23/P4 P21/P8 P23/P8 P23/P21

5.98

22165/

7.22

Phosphoglycerate

1.03;

0.73

–1.62;

0.020

–1.48;

0.066

–1.68;

0.0031

–1.53;

0.022

1.10; 0.65

6.08

19823/

6.61

Phosphoglycerate

–1.34;

0.064

–1.56;

0.010

–1.68;

0.0056

–1.17;

0.40

–1.26;

0.23

–1.08; 0.65

5.26

19307/

1.27;

0.25

–1.28;

0.12

–1.24;

0.082

–1.63;

0.041

–1.58;

0.031

1.03; 0.72

8.44

12389/

5.94

Malate

–1.26;

0.061

1.17;

0.040

1.14;

0.46

1.48;

0.0096

1.44;

0.073

–1.02; 0.76

6.23

10642/

4.40

Glyceraldehyde–3–

phosphate dehydrogenase

B6HI59

1.40;

0.020

1.62;

0.00053

1.59;

0.0056

1.16;

0.23

1.13;

0.40

–1.02; 0.82

5.26

9269/4

1.32;

0.0010

1.52;

0.00022

1.79;

1.2e–006

1.15;

0.047

1.36;

0.00016

1.18; 0.010

8.44

28944/

5.68

Malate

–1.05;

0.64

1.92;

0.0042

1.65;

0.0027

2.02;

0.0070

1.74;

0.0077

–1.16; 0.41 ATP synthesis

4.47

83112/

–1.21;

0.25

2.93;

8.9e–005

3.17;

2.9e–005

3.56;

0.00036

3.85;

0.00020

1.08; 0.50

5.25

50357/

4.25 F0F1 ATP synthase

–8.15;

3.7e–006

–7.52;

1.1e–006

–7.94;

7.2e–005

1.08;

0.41

1.03;

0.95

–1.06; 0.63

5.25

47770/

–5.00;

0.00021

–2.75;

0.0023

–3.08;

0.0061

1.82;

0.022

1.62;

0.19

–1.12; 0.54

5.25

46311/

1.33;

0.053

1.37;

0.036

1.36;

0.0072

1.03;

0.85

1.02;

0.78

–1.01; 0.97

5.25

44274/

1.41;

0.00026

1.09;

0.56

–1.03;

0.65

–1.29;

0.056

–1.45;

0.0042

–1.13; 0.45

5.44

31668/

1.60;

0.0014

1.25;

0.013

1.18;

0.25

–1.28;

0.017

–1.35;

0.046

–1.06; 0.54

5.44

31718/

1.42;

0.00044

1.24;

0.14

1.14;

0.26

–1.15;

0.27

–1.25;

0.071

–1.09; 0.61

5.26

21455/

–1.32;

0.013

–1.13;

0.20

–1.09;

0.34

1.17;

0.11

1.22;

0.044

1.04; 0.63

5.25

13162/

1.46;

0.051

1.39;

0.021

1.46;

0.0055

–1.05;

0.72

1.00;

0.93

1.05; 0.50

8.44

13020/

2.99;

6.3e–007

–1.06;

0.15

1.02;

0.98

–3.18;

3.1e–007

–2.94;

0.00011

1.08; 0.64

6.73

13020/

6.10 Ribose 5–phosphate

–2.27;

0.0039

–1.85;

0.013

–2.26;

0.0034

1.23;

0.36

1.01;

0.97

–1.22; 0.36

6.17

11662/

4.35 Cytochrome c oxidase

1.73;

0.00023

1.16;

0.071

1.32;

0.0052

–1.48;

0.0014

–1.30;

0.012

1.14; 0.14

6.17

11411/

4.41 Cytochrome c oxidase

1.43;

0.018

1.36;

0.021

1.27;

0.12

–1.06;

0.64

–1.13;

0.37

–1.07; 0.55

Table 1 (continued)

Trang 9

Spot

No

Theor

MW/pI

Exp MW/pI

UniProt access

No

P8/P4 P21/P4 P23/P4 P21/P8 P23/P8 P23/P21

Protein metabolism

5.08

77749/

4.87

Serine carboxypeptidase B6HNT3 Predicted

–2.09;

0.00011

–1.35;

0.047

–1.38;

0.019

1.55;

0.020

1.51;

0.012

–1.02; 0.91

5.08

77388/

4.83

–2.26;

0.0014

–1.52;

0.015

–1.45;

0.013

1.49;

0.032

1.55;

0.014

1.04; 0.66

5.08

76197/

4.79

–1.90;

0.0057

–1.23;

0.23

–1.23;

0.24

1.54;

0.0065

1.54;

0.012

1.00; 0.97

5.08

60005/

4.06

–1.09;

0.44

1.34;

0.027

1.38;

0.0077

1.46;

0.0083

1.50;

0.0020

1.03; 0.70

4.55

54250/

4.47

Protein Disulfide Isomerase (PDIa) K9GF84

–1.39;

0.050

–1.00;

0.94

–1.13;

0.41

1.39;

0.018

1.23;

0.064

–1.13; 0.24

4.99

45598/

4.56 Carboxypeptidase Y,

1.54;

0.0085

1.57;

0.0086

1.56;

0.0078

1.02;

0.87

1.01;

0.91

–1.01; 0.95

4.53

45316/

4.50

UV excision repair

–1.01;

0.94

1.38;

0.027

1.44;

0.016

1.40;

0.029

1.45;

0.017

1.04; 0.59

5.24

37502/

4.68 hypothetical protein

1.45;

0.0082

1.23;

0.11

1.28;

0.011

–1.18;

0.23

–1.13;

0.28

1.04; 0.65

4.79

37155/

4.60 Ribosomal protein S2

1.44;

0.012

1.66;

0.0049

1.77;

0.00045

1.16;

0.23

1.23;

0.021

1.06; 0.44

5.79

35853/

5.78 Nucleic acid binding

–1.20;

0.26

–1.56;

0.068

–1.87;

0.0035

–1.30;

0.23

–1.56;

0.024

–1.20; 0.55

5.08

34169/

4.48

Proteinase A –

–1.43;

0.13

1.32;

0.18

1.51;

0.052

1.90;

0.0019

2.17;

9.3e–005

1.14; 0.30

4.44

24785/

4.38

Elongation factor, C – terminal, alpha helical domain of the GST family

B6H4G8

2.00;

0.10

3.35;

0.0040

3.51;

0.0025

1.68;

0.098

1.75;

0.066

1.05; 0.76

5.08

22617/

4.53

Proteinase A –

1.77;

0.00049

1.58;

0.00077

1.48;

0.0031

–1.12;

0.25

–1.20;

0.11

–1.07; 0.47

5.08

18429/

4.45

Proteinase A –

1.80;

0.022

1.62;

0.0057

1.86;

0.00071

–1.11;

0.69

1.03;

0.72

1.15; 0.23

5.58

16975/

1.10;

0.45

–1.27;

0.11

–1.50;

0.0098

–1.39;

0.016

–1.65;

0.00094

–1.19; 0.065 Protein folding

5.03

62378/

5.02

Heat shock 70kDa

1.03;

0.69

1.41;

0.024

1.36;

0.036

1.36;

0.024

1.32;

0.038

–1.03; 0.81

5.61

51144/

1.40;

0.021

1.33;

0.071

1.54;

0.0095

–1.05;

0.61

1.10;

0.29

1.15; 0.22

5.22

29948/

4.66 Heat shock protein

1.58;

0.0072

1.96;

0.00035

2.27;

0.042

1.24;

0.087

1.44;

0.38

1.16; 0.88

5.32

24978/

4.46

Heat shock 70kDa

1.65;

0.011

1.75;

0.0012

1.76;

0.0043

1.06;

0.56

1.06;

0.66

1.00; 0.95

Table 1 (continued)

(continued on next page)

Trang 10

Spot

No

Theor

MW/pI

Exp MW/pI

UniProt access

No

P8/P4 P21/P4 P23/P4 P21/P8 P23/P8 P23/P21

4.81

22407/

4.66

Heat shock 70kDa

1.42;

0.0096

–1.09;

0.16

–1.06;

0.34

–1.54;

0.014

–1.51;

0.0097

1.02; 0.79

4.50

22303/

4.35 Hsps_p23–like protein B6HKR2 Predicted

1.55;

0.023

1.22;

0.11

1.52;

0.044

–1.27;

0.080

–1.02;

0.86

1.25; 0.17

5.32

18005/

5.10

Heat shock 70kDa

–1.04;

0.89

2.17;

0.0070

1.95;

0.032

2.27;

0.0028

2.04;

0.018

–1.11; 0.56

5.89

17482/

5.21

Peptidyl–prolyl cis–

trans isomerase NIMA–interacting 1 Rotamase

0.052

–1.36;

0.066

–1.58;

0.036

1.09;

0.55

–1.06;

0.75

–1.16; 0.35

5.04

16870/

5.05 Heat shock 70 kDa

–1.06;

0.46

1.43;

0.0070

1.52;

0.00024

1.52;

0.0050

1.61;

0.00037

1.06; 0.35

6.91

14763/

6.85 Peptidyl–prolyl cis–

trans isomerase B6HAJ7

–1.32;

0.034

–1.36;

0.018

–1.68;

0.0026

–1.03;

0.76

–1.28;

0.059

–1.23; 0.074

6.43

9084/4

.87 Chaperonin, putative K9GM71

1.65;

0.029

1.62;

0.00096

2.08;

0.00061

–1.02;

0.94

1.26;

0.24

1.28; 0.12 Redox homeostasis

5.37

66165/

1.02;

0.89

1.57;

0.0055

1.61;

0.00099

1.54;

0.012

1.58;

0.0035

1.03; 0.74

5.37

66992/

–1.02;

0.89

1.45;

0.018

1.32;

0.095

1.48;

0.0087

1.35;

0.063

–1.10; 0.49

5.37

66784/

1.04;

0.64

1.43;

0.020

1.35;

0.044

1.37;

0.011

1.29;

0.032

–1.06; 0.57

8.64

29124/

5.13

Cytochrome c peroxidase, mitochondrial

Q4WPF8

–1.13;

0.34

1.23;

0.027

1.11;

0.22

1.40;

0.035

1.25;

0.25

–1.12; 0.27

5.64

20767/

6.37

Superoxide

–1.57;

0.092

–2.48;

0.0032

–1.88;

0.012

–1.57;

0.18

–1.19;

0.65

1.32; 0.22

8.67

14924/

5.34

Peroxiredoxin 5, atypical 2–Cys peroxiredoxin

0.00058

–2.07;

0.00037

–2.25;

6.9e–005

1.07;

0.61

–1.02;

0.98

–1.09; 0.50

5.31

10126/

–2.64;

0.022

–3.50;

0.0024

–4.36;

0.00085

–1.33;

0.47

–1.65;

0.17

–1.24; 0.26 Cell cycle/cell signaling

4.9

86936/

4.46 Pleckstrin homology–

like domain protein B6HV58 Predicted

–1.52;

0.0051

–1.30;

0.084

–1.35;

0.15

1.17;

0.32

1.13;

0.82

–1.04; 0.72

4.9

86131/

–1.64;

0.0029

–1.60;

0.012

–1.89;

0.0020

1.02;

0.94

–1.15;

0.21

–1.18; 0.30

4.9

85997/

–1.57;

0.0097

–1.69;

0.018

–2.01;

0.018

–1.08;

0.50

–1.28;

0.19

–1.19; 0.40

4.9

86131/

–1.76;

0.0013

–2.56;

0.0063

–3.16;

0.0022

–1.45;

0.095

–1.80;

0.025

–1.24; 0.54

4.9

85864/

–1.66;

0.0012

–1.98;

0.0087

–2.58;

0.0015

–1.19;

0.26

–1.55;

0.029

–1.30; 0.32

Table 1 (continued)

Định dạng
Số trang	16
Dung lượng	2 MB