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Proteomic comparison between human young and old brains by

Int.J.Devl Neuroscience21(2003)

209–216

two-dimensional gel electrophoresis and identi?cation of proteins

Wen Chen,Jianguo Ji,Xiaoman Xu,Sizhi He,Binggen Ru?

National Key Laboratory of Protein Engineering,College of Life Sciences,Peking University,Beijing100871,PR China

Received22January2003;received in revised form5March2003;accepted6March2003

Abstract

To investigate molecular mechanisms of human brain aging,brain proteins were isolated from postmortem human young and old brains and pro?led by two-dimensional gel electrophoresis(2-DE).With the help of special software,?ve down-regulated protein spots in two-dimensional gel electrophoresis gels of old brains were found compared with young brains,four of which was identi?ed as a protein similar to peroxiredoxin2(accession-numbered as gi|13631440),two of stathmin(phosphoprotein p19)and apolipoprotein A-I precursor (apo-AI)by matrix-assisted laser desorption ionization-time of?ight mass spectrometry(MALDI-TOF MS).Eight common proteins, whose expressions were not altered between young and old brains,were also identi?ed.The possible relevance of changes was analyzed. This study shows that the contribution of proteomics could be valuable in experimental gerontology?eld.

Keywords:Brain;Aging;Proteomics;Two-dimensional gel electrophoresis;Mass spectrometry

1.Introduction

Proteomic study means the analysis of the entire protein complement expressed by a genome(Wilkins et al.,1996). Generally,proteomic analysis,which can not cover the total number of proteins existing in a biological system at a given moment,involves the expression pro?le of many proteins at a time.

Two-dimensional gel electrophoresis(2-DE)(O’Farrel, 1975)with its recent developments has been seen as an ideal tool for proteome analyses.Although it has shortcomings, for example,poor ability to separate hydrophobic proteins and trace-quantity expressed ones,immobilized pH-gradient (IPG)strips used in the?rst-dimensional gel electrophore-sis provide a basis for reproducible separation according to proteins’isoelectric points.Advanced computer graphics and laser ray detection systems have offered a possibility for quantitative detection of protein spots,in addition to edition and storage of the2-DE images.Proteome comparison be-Abbreviations:2-DE,two-dimensional gel electrophoresis;DTT, dithiothreitol;IPG-IEF,immobilized pH-gradient-isoelectric focusing; MALDI-TOF MS,matrix-assisted laser desorption ionization-time of?ight mass spectrometry;PMSF,phenylmethylsulfonyl?uoride;SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis;TFA,tri?uo-roacetic acid

?Corresponding author.Tel.:+86-10-62751842;

fax:+86-10-62751842.

E-mail address:rulab@https://www.doczj.com/doc/41147664.html,(B.Ru).tween tissues in the different situations can cast a light on some protein spots,although not all proteins,which are dif-ferently expressed in quantity under different surroundings. Nowadays,mass spectrometry such as electrospray ion-ization mass spectrometry(von Brocke et al.,2001)and matrix-assisted laser desorption ionization-time of?ight mass spectrometry(MALDI-TOF MS)(Nordhoff et al., 2001),can routinely identify low femtomole quantities of proteins.Peptide mass?ngerprinting identi?es a protein based on the molecular weights of its peptides obtained by MS after digestion by a speci?c protease,classically trypsin (Mann et al.,1993).With the help of speci?c softwares,the list of experimental molecular weights is compared with the theoretical peptide?ngerprinting of protein sequences exist-ing in the current databases.The developed bioinformatics (Tripathi,2000)allows the identi?cation of most proteins. Classically,proteome analysis consists of three steps: 2-DE used for proteins pro?ling,MS for critical con?r-mation of the molecular weights and bioinformatics for protein identi?cation.Although there is much improvement for investigation at the mRNA level,such as low-density DNA arrays(Zammatteo et al.,2002),only approaches at the protein expression level give information about the set of molecular tools used by a cell at a given moment. The characterization of post-translational modi?cations of proteins will facilitate insights into biological events. Protein arrays are becoming widely available to be used as diagnostic tools to easily and quickly monitor the expression

210W.Chen et al./Int.J.Devl Neuroscience21(2003)209–216

of speci?c proteins(Ge,2000;Walter et al.,2000).But the correction of protein arrays is highly dependent on the availability of speci?c antibodies which limits their power. Comparative proteome analysis is a good alternative strategy to discover proteins which undergo changes in expression level and may underlie the differences of phenotype.

The age-dependent deterioration of cell function appears in both replicative and post-mitotic cells in living organ-isms.Post-mitotic cells in the brain,heart,liver and many other organs have tissue-speci?c functions.Although there may be common factors upstream of the cascade in cellular aging,such as the accumulation of DNA and protein dam-age under environmental attacks including oxidative stress, a change downstream of the cascade may be cell-type spe-ci?c,and may proceed in a given intracellular environment that contains all constituents of the proteome(Toda,2000). Damage and cellular morphology changes accumulate progressively with aging.Age-related post-translational modi?cations of proteins could be investigated,like phos-phorylation(Heydari et al.,1989)and glycosylation (Meheus et al.,1987).Oxidation is another post-translational modi?cation.The contribution of proteomics could be extremely valuable to geronto-toxicology and https://www.doczj.com/doc/41147664.html,pared to other large expression analysis approaches,proteomics has currently the monopoly for the investigation of protein post-translational modi?ca-tion.This unique feature opens new gerontological per-spectives(Dierick et al.,2002).Research on proteins up-regulated or down-regulated in aging was initiated by Toda et al.who established the TMIG-2D PAGE database at http://www.tmig.or.jp/2D(Toda et al.,1988).

In this report,using proteomics methodology developed previously in our laboratory(Chen et al.,2002),we chose brains of different ages as experimental materials to in-vestigate molecular mechanisms of brain aging.The brain proteins were pro?led by2-DE.Changes in the relative intensity of protein spots appearing in a proteome pro?ling, may be the results of various molecular alterations that occur during aging.With the help of the software Image-Master2D v3.10,the expression-changed protein spots in the aged brains were found and identi?ed by MALDI-TOF MS.These expression-altered proteins may be responsible for or the results of the functional deterioration in senescent brain cells.Some common protein spots were identi?ed by MALDI-TOF MS too.

2.Experimental procedures

2.1.Materials

Immobilized pH-gradient strips were purchased from Amersham Pharmacia Biotech,Sweden.Dithiothreitol (DTT),acrylamide,N,N -methylenebisacrylamide,CHAPS detergent,TEMED,TPCK-treated trypsin and protease in-hibitors were obtained from Sigma,USA.Tri?uoroacetic acid(TFA)was purchased from Aldrich and acetonitrile was from Fisher(New Jersey,USA).

2.2.Human brain tissues

The proteins of six postmortem human brains including white and gray matter were analyzed in this study.These temporal,frontal and parietal lobes were obtained from301 Hospital in Beijing.The brains were from six individuals:a 25-week-old Chinese male postmortem fetus,a28-week-old Chinese male postmortem fetus,a23-year-old Chinese fe-male who was dead of epileptic attack,a73-year-old Chinese male dead of multiple system organ failure,a74-year-old Chinese male dead of respiratory failure,and a84-year-old Chinese male dead of kinesioneurosis and respiratory fail-ure.The brain tissues were removed during autopsy within the time of24h after postmortem,and stored at?70?C un-til further processed.

2.3.Preparation of protein samples

Whole temporal,frontal or parietal lobes were suspended and homogenized in40ml of0.2M NH4HCO3/protease inhibitors at4?C.Then,these samples were centrifuged at 19,000×g for1h.Three volumes of cooled ethanol were added to the supernatant.After stored at?20?C overnight, the protein pellets were obtained by centrifugation and then lyophilized.

2.4.Two-dimensional gel electrophoresis

Two-dimensional gel electrophoresis was performed es-sentially as reported(Chen et al.,2002).Just prior to the?rst dimension of2-DE,the same quantities of temporal,frontal or parietal lobe proteins(25?g for silver-stained2-DE,5mg for Coomassie Brilliant Blue-stained2-DE)from different experimental individuals were,respectively mixed with a re-hydration solution that contained urea(8M),CHAPS de-tergent(2%(w/v)),IPG buffer(0.5%(v/v)),dithiothreitol (1(w/v)).The samples were often sonicated brie?y to fa-cilitate protein solubilization.After incubation at room tem-perature for1h,the samples were centrifuged for15min at 19,000×g,and the supernatant was directly applied to the 13cm pH4–7IPG strip holder.

Two-dimensional gel electrophoresis was performed with a horizontal IPGphor Isoelectric Focusing(IEF)system (Pharmacia,Uppsala,Sweden),using pre-cast pH4–7im-mobilized linear-gradient strips(130mm×3mm×0.5mm) for the?rst dimension,and sodium dodecyl sulfate(SDS) 12.5%polyacrylamide gels for the second dimension (sodium dodecyl sulfate-polyacrylamide gel electrophore-sis,SDS-PAGE).SDS-PAGE was run in a Hoefer SE600 Series(Pharmacia,Uppsala,Sweden).

IPG-IEF can be simpli?ed by use of an integrated sys-tem,the IPGphor where rehydration with sample solution and IEF are performed in a one-step procedure.Initial

W.Chen et al./Int.J.Devl Neuroscience21(2003)209–216211

rehydration was set for12h.The IEF was then performed

at20?C with the following voltage program:500V(gradi-

ent over1h),1000V(gradient over1h),5000V(gradient

over1h),8000V(?xed for4h),at50?A/strip.Prior to the

second-dimensional gel separation,the IPG strips were equi-

librated for2×15min with gentle shaking in10ml of SDS

equilibration buffer[Tris–Cl,pH8.8(50mM),urea(6M),

glycerol(87%(v/v))30%(v/v),SDS(2%),bromophenol

blue(trace)].DTT(1(w/v))was added to the?rst,and

iodoacetamide(4%(w/v))to the second equilibration step.

The second-dimensional SDS-PAGE with a12.5%run-

ning gel was carried out without a stacking gel.After

placing the IPG gel strip on top of the second-dimensional

polyacrylamide gel,electrophoresis was performed at a

constant power of30mA/gel until the bromophenol blue

reached the bottom of the gel.Protein markers were added

to the marker well.

2.5.Gel scanning and image analysis

Coomassie Brilliant Blue-or silver-stained gels were

scanned with a SHARP JX-330laser https://www.doczj.com/doc/41147664.html,put-

erized2-D gel analysis(spot detection,spot quanti?cation,

pattern matching,database construction)was performed

with the help of the software ImageMaster2D version3.10.

Protein spot expression levels are given as the mean percent ±S.D.Only statistically signi?cant results were considered (P<0.05).P-values were caculated using t-test comparing

means to a hypothetical value of0.00.

2.6.Sample preparation for mass spectrometry

The protein spots of interest were excised from the

Coomassie Brilliant Blue-stained gel,minced with a scalpel,

and placed in a1.5ml siliconized Eppendorf centrifuge

tube.Add100?l of25mM NH4HCO3/50%acetonitrile

and vortex for10min to remove Coomassie Brilliant Blue

stain.Repeat the dye-washing step until Coomassie Brilliant

Blue was completely rinsed.The gel pieces were then dehy-

drated.For trypsin digestion,the gel pieces were re-swollen

in3×V12.5ng/?l trypsin in25mM NH4HCO3,covered

with25mM NH4HCO3and incubated overnight at37?C.

After completion of digestion,the supernatant was trans-

ferred into another tube,and the gel pieces were sonicated

with100?l of50%acetonitrile/5%formic acid for10min

at room temperature.The extract was transferred to the pri-

mary supernatant,and the extraction was repeated one more

time.The extracted digests were evaporated to dryness in a

vacuum centrifuge.

2.7.MALDI-TOF MS

The digests were re-dissolved in15?l0.1%TFA.

The solution was vortexed and centrifuged.One micro-

liter of supernatant was mixed with1?l of10mg/ml ?-cyano-4-hydroxycinnamic acid in50%acetonitrile/0.1%TFA and the mixed solution was spotted on96-spot MALDI target.

Samples were analyzed on a Micromass MALDI-TOF mass spectrometer(Micromass UK Limited)or BRUKER auto?ex MALDI-TOF MS(BRUKER DALTONICS com-pany).All spectra were acquired in a positive-ion re?ector. The resulting peptide mass?ngerprints,together with the p I and MW values(estimated from2-DE gels),are used to search the SWISS-PROT or NCBInr protein database with a special search tool which compares the experimentally deter-mined tryptic peptide masses with theoretical peptide masses calculated for proteins contained in the SWISS-PROT or NCBInr protein database.

3.Results

3.1.2-DE of human temporal,frontal and

parietal lobes’proteins

Two-dimensional gel electrophoresis maps were con-structed for human temporal,frontal and parietal lobes’proteins.We made separate maps for the pH range4–7.The second-dimensional gel electrophoresis was performed with a12.5%SDS-PAGE.2-DE map of human23-year-old brain proteins is shown in Fig.2.For each sample,over1000pro-tein spots were resolved in a2-DE gel(16cm×18cm)with silver-stained by computer-aided image analysis,and over 800protein spots were resolved with Coomassie Brilliant Blue-staining.

3.2.Signi?cant changes in proteins between human young and old brains

Eight independent paired2-DE gels of human23-year-old and73-year-old temporal,frontal or parietal lobe proteins were run and subsequently Coomassie Brilliant Blue-or silver-stained(one paired2-DE gels for silver-stained and seven for Coomassie Brilliant Blue-stained):2-DE runs of human23-year-old and73-year-old brain right tem-poral proteins repeated four times,2-DE runs of human 23-year-old and73-year-old brain left temporal and left frontal proteins once,respectively,2-DE runs of human 23-year-old and73-year-old brain left and right parietal proteins once,respectively.Furthermore,six indepen-dent paired2-DE gels of human25-week-old fetus and 74-year-old brain temporal proteins,human28-week-old fetus frontal and84-year-old brain temporal proteins were run and subsequently silver-stained:2-DE runs of human 25-week-old fetus left temporal and74-year-old brain right temporal proteins repeated three times,2-DE runs of human 28-week-old fetus left frontal and84-year-old brain right temporal proteins repeated three times.

With the help of the software ImageMaster2D v3.10, protein spots in2-DE gels were compared between human young and old brains in these14independent experiments.

212W.Chen et al./Int.J.Devl Neuroscience 21(2003)209–216

Table 1

Signi?cantly quantitative variation of protein spots between 2-DE gels of human young and old brains Spot No.a

Observed Differences b

Identity

p I

MW (kDa)c Down-regulated in old brains (5spots)(Decreased)1 4.917.9(?)78±25%Unidenti?ed

2 5.619.0(?)203±56%Stathmin (Phosphoprotein p19)

3 5.918.8(?)95±20%Stathmin (Phosphoprotein p19)

4 5.422.8(?)110±58%Similar to peroxiredoxin 2

5.4

24.6

(?)274±62%

Apolipoprotein A-I precursor(Apo-AI)

a Numbering as shown in Fig.2.

Presented results,shown as the means ±S .D .,are summaries of 14independent paired experiments performed as described in the Section 3.All P -values <0.05.

c Estimate

d from th

e 2-DE gel.

On summary of 14experiments’results,the distinct varia-tions with a signi?cance value of P <0.05were con?rmed as down-regulated in old brains shown in Table 1depend-ing on the extent of protein expression.Fig.1A–C zoom in all expression-changed protein spots.Each of protein spots which were selected as differential expression in this pa-per,has a consistent decrease in all 14experiments,and the range of changes are all larger than 50%.

A total of ?ve spots have down-regulated expression in old brains.Four of ?ve protein spots that were down-regulated in human old brains,were identi?ed as a protein similar to per-oxiredoxin 2(accession-numbered as gi |13631440),two of stathmin (phosphoprotein p19)and apolipoprotein A-I pre-cursor (Apo-AI).These spots were marked with numbers

https://www.doczj.com/doc/41147664.html,parison between two-dimensional gel electrophoresis maps (pH 4–7)of young and old brains.(A–C)All protein spots in the 2-DE gels,which differed signi?cantly in the expression level between young and old brains,are zoomed in.The left maps represents young brain,the right represents old brain.The numbers refer to Fig.2.

Fig.2;the estimated p I s,estimated molecular masses,per-cent differences and identities of varied spots are summa-rized in Table 1.3.3.MALDI-TOF MS

The data for the protein spot letter c as an example are shown in Fig.3.Fig.3A shows the MALDI-TOF MS pep-tide mass ?ngerprint spectrum of trypsin-digested protein spot letter c.Fig.3B lists the matching peptides.Thirty-two peptides were matched with glial ?brillary acidic protein accession-numbered as P14136in the protein database SWISS-PROT.These 32peptides are indicated in the pro-tein sequence shown in Fig.3C .

W.Chen et al./Int.J.Devl Neuroscience 21(2003)209–216

213

Fig.2.Indication of identi?ed protein spots and variably expressed protein spots between human young and old brains.Numbering as noted in Table 1.Lettering as noted in Table 2.

3.4.Identi?cation of expression-changed proteins and common ones between human young and old brains Four expression-different protein spot and eight common ones between human young and old brains,were analyzed and identi?ed with MALDI-TOF https://www.doczj.com/doc/41147664.html,rmation about these identi?ed protein spots is summarized in Table 2.These identi?ed protein spots are lettered or numbered as shown in Fig.2.

Table 2

Proteins identi?ed from two-dimensional electrophoresis gels (pH 4–7)of human brain proteins by MALDI-TOF MS Spot No.a Identity (theoretical mass in kDa)

Accession number b MALDI-TOF MS coverage (%)c a Enolase 1(47.2)

gi |450357121.90b Heat-shock 20kDa like-protein (22.4)

O4341650.00c Glial ?brillary acidic protein,astrocyte (44.9)P14136

61.81d Hypothetical XM 041154(41.4)gi |1475767720.30e Pyruvate kinase-3(57.9)gi |450583919.00f Gamma enolase (47.1)

P09104

30.72g Glial ?brillary acidic protein (49.9)

gi |450397971.99h Mitochondrial stress-70protein precursor (73.8)P3864641.972d Stathmin (Phosphoprotein p19)(pp19)P1694938.003d Stathmin (Phosphoprotein p19)(pp19)P16949

38.004Similar to peroxiredoxin 2(21.9)

gi |1363144031.005d

Apolipoprotein A-I precursor (Apo-AI)

P02647

22.00

a The numbering and lettering corresponding to the two-dimensional gel electrophoresis image in Fig.2.b

Accession number in NCBInr,SWISS-PROT.

c Percentage of the protein sequence covere

d by th

e matched peptides.d Means that the identity refers to the article (Langen et al.,1999).

4.Discussion

In this paper,we reported comparative proteome stud-ies between postmortem human young and old brains.The pH 4–7range in the ?rst-dimensional gel electrophoresis we used here separates part of the soluble proteins in the brain,and focuses on high-resolution and more unambigu-ous comparison of proteins whose p I s are between pH 4–7.All paired samples,which would be compared each other,

214W.Chen et al./Int.J.Devl Neuroscience21(2003)209–216

Fig.3.Analysis of spot letter c from human brain2-DE map(pH4–7)by MALDI-TOF MS.(A)MALDI-TOF MS peptide mass?ngerprint spectrum obtained from crude peptide mixture after in-gel tryptic digest of spot letter c.(B)The list of matching peptides between the experimental and theoretical values.(C)The sequence of glial?brillary acidic protein identi?ed.The matched peptides are shadowed in the sequence.

W.Chen et al./Int.J.Devl Neuroscience21(2003)209–216215

were run together at the same conditions.This avoids any arti?cial differences between samples due to different run-ning conditions.

The2-DE patterns of the different-aged brains we ana-lyzed were compared each other with the help of the soft-ware ImageMaster2D v3.10,and differently expressional protein spots were found.The threshold,at least0.5times up-or down-regulation,was chosen arbitrarily to exclude proteins that differ in intensity due to small random varia-tions during the performance of experiments.Each of pro-tein spots selected in this paper as differentially expressed has a consistent decrease in all14independent paired exper-iments.Five protein spots were found down-regulated in old brains compared with young ones.The p I s and molecular masses of these key proteins were estimated based on their positions in the2-DE map.These proteins could probably possess some down stream proteins of aging.

Nine protein spots were excised from the gel and digested with trypsin.Molecular weights of the tryptic peptides were determined by MALDI-TOF MS.Obtained protein scores are signi?cant(P<0.05)in the protein database search. 19–72%coverages of protein sequences were obtained in the analyses of MALDI-TOF MS.Four altered protein in the expression level was identi?ed.Also eight common proteins whose expressions did not differ signi?cantly were identi-?ed;these can be used to enrich a2-DE gel database(a reference map)of human brain proteins.Such a database is needed for control/neurodegenerative diseases,such as Alzheimer’s disease,comparison studies.

As antioxidant enzymes in humans,peroxiredoxins ap-pear to be involved in the redox-regulation of cellular sig-naling and differentiation,displaying in part opposite effects (Hofmann et al.,2002).In this paper a protein similar to peroxiredoxin2is found down-regulated in old brains com-pared with young brains.This result gives us the idea that oxidants may play an important role in brain aging.

Two different protein spots,number2and number3,were both identi?ed as stathmin(phosphoprotein p19)(pp19).In-terestingly,the quantity of stathmin representing number2 was under-expressed by(?)203%in old brains comparing with young brains,while the quantity of stathmin repre-senting number3was under-expressed by(?)95%in old brains.From the2-DE gels’image,protein spots of number 2and number3have similar molecular weight,while the observed p I s of number2and number3are5.6and5.9,re-spectively.These results probably show that protein spot of number2is the phosphorylated form of stathmin and protein spot of number3is the unphosphorylated one.Stathmin is a cytosolic protein that binds tubulin and destabilizes cellu-lar microtubules,an activity regulated by phosphorylation. Liedtke et al.(2002)reported that aging stathmin(?/?) mice developed an axonopathy of the central and peripheral nervous systems.As the lesions progressed,degeneration of axons,dysmyelination,and an unusual glial reaction were observed.At the functional level,electrophysiology record-ings demonstrated a signi?cant reduction of motor nerve conduction velocity in stathmin(?/?)mice.Furthermore, expression of stathmin is reduced by aging,and is also changed in age-related neurodegenerative conditions such as Alzheimer’s disease in humans(Mori and Morii,2002). Our?ndings that stathmin is down-regulated in human old brains reveal and con?rm that stathmin is related with bain aging.

Apolipoprotein(apo)A-I alone or as a component of high density lipoprotein particles has antiatherogenic properties (Nakamura et al.,1999).Apolipoprotein E(apoE),a protein with three common isoforms,has a large impact on longevity and successful aging(Smith,2002).Impairments in cogni-tive performance have been observed in aged apolipoprotein E(apoE)-de?cient mice(Law et al.,2003).In this study, apolipoprotein(apo)A-I is found to be down-regulated in human old brains.The age-dependent decline in abundance of this protein may contribute to brain aging and underlie the higher risk for developing diseases in older individuals. Human brain proteins identi?ed in this study include some enzymes,such as enolase1,pyruvate kinase-3and gamma enolase,which play important roles in several metabolic pathways and some regulating proteins,for ex-ample,heat-shock20kDa like-protein,glial?brillary acidic protein and mitochondrial stress-70protein.

Proteome pro?lings of postmortem human brains in 2-DE gels(pH4–7)and protein spots identi?ed in this report will also serve as the studies on the molecular mech-anisms of many diseases associated with the brain,such as Alzheimer’s disease,through further functional analyses of the altered proteins between the proteome of nondemented and dysfunctional brain tissues.

Acknowledgements

We thank Professor Luning Wang in301Hospital in Beijing for kindly providing postmortem human brain tis-sues.This work was supported by a grant from the Ninth Five Years Plan of National Key Science and Technique Foundation,No.96-C02-01-09.

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