The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells

$Page 1: The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells$
a 1774 (2007) 413–432www.elsevier.com/locate/bbapap

Biochimica et Biophysica Act

The nuclear proteome and DNA-binding fraction of humanRaji lymphoma cells

Silke Henrich a, Stuart J. Cordwell a, Ben Crossett a, Mark S. Baker b, Richard I. Christopherson a,⁎

a School of Molecular and Microbial Biosciences, University of Sydney, Sydney, NSW 2006, Australiab Australian Proteome Analysis Facility Ltd., Building F7B, Research Park Drive, Macquarie University, North Ryde, NSW 2109, Australia

Received 17 August 2006; received in revised form 26 November 2006; accepted 20 December 2006Available online 12 February 2007

Abstract

Purification of organelles and analysis of their proteins is an important initial step for biological proteomics, simplifying the proteome prior toanalysis by established techniques such as two-dimensional liquid chromatography (2-DLC) or two-dimensional gel electrophoresis (2-DE).Nuclear proteins play a central role in regulating gene expression, but are often under-represented in proteomic studies due to their lowerabundance in comparison to cellular ‘housekeeping’ metabolic enzymes and structural proteins. A reliable procedure for separation and proteomicanalysis of nuclear proteins would be useful for investigations of cell proliferation and differentiation during disease processes (e.g., humancancer). In this study, we have purified nuclei from the human Burkitt's lymphoma B-cell line, Raji, using sucrose density gradient centrifugation.The integrity and purity of the nuclei were assessed by light microscopy and proteins from the nuclear fractions were separated by 2-DE andidentified using matrix assisted laser desorption ionization mass spectrometry (MALDI-MS). A total of 124 unique proteins were identified, ofwhich 91% (n=110) were predicted to be nuclear using PSORT. Proteins from the nuclear fraction were subjected to affinity chromatography onDNA-agarose to isolate DNA-binding proteins. From this purified fraction, 131 unique proteins were identified, of which 69% (n=90) wereknown or predicted DNA-binding proteins. Purification of nuclei and subsequent enrichment of DNA-binding proteins allowed identification of atotal of 209 unique proteins, many involved in transcription and/or correlated with lymphoma, leukemia or cancer in general. The data obtainedshould be valuable for identification of biomarkers and targets for cancer therapy, and for furthering our understanding of the molecularmechanisms underlying lymphoma development and progression.Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved.

Keywords: DNA-binding protein; Burkitt's lymphoma; Nuclear proteome; Two-dimensional gel electrophoresis

1. Introduction

Proteomics is a tool for identifying proteins related to eventsat the phenotypic level. Comparison of protein expressionbetween normal and diseased cells, for example, provides abasis for diagnosis of diseases and elucidation of theirmechanisms. It is essential that a maximum proportion of the

Abbreviations: 2-DE, two-dimensional gel electrophoresis; 2-DLC, two-dimensional liquid chromatography; IPG, immobilized pH gradient; MALDI-TOF, matrix-assisted laser desorption-ionization time-of-flight; MS, massspectrometry; MS-MS, tandem mass spectrometry; pI, iso-electric point; PMF,peptide mass fingerprinting; SDS-PAGE, sodium dodecyl sulfate-polyacryla-mide gel electrophoresis; SPITC, 4-sulfophenyl isothiocyanate⁎ Corresponding author. Tel.: +61 2 9351 6031; fax: +61 2 9351 4726.E-mail address: [email protected] (R.I. Christopherson).

1570-9639/$ - see front matter. Crown Copyright © 2007 Published by Elsevier Bdoi:10.1016/j.bbapap.2006.12.011

proteome from cell extracts is analyzed. A cell extract can befractionated to yield a specific sub-set of the proteome relevantto the biological process under investigation. Proteomicanalysis involving two-dimensional gel electrophoresis (2-DE)is limited by the inability to detect low abundance proteins.Certain proteins, such as hydrophobic integral membraneproteins, those with an extremely acidic or basic isoelectricpoint, or high or low molecular mass, may be poorlyrepresented in traditional 2-DE analysis. Equally, low abun-dance proteins in samples with a high dynamic range of proteinconcentrations may only be identified after fractionation of thesample. Sub-fractionation of complex protein mixtures can bebased on (i) removal of high abundance proteins (e.g., humanserum albumin in plasma [1,2]); (ii) affinity enrichment basedon chemical or functional protein properties [3–6]; (iii)

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http://dx.doi.org/10.1016/j.bbapap.2006.12.011

414 S. Henrich et al. / Biochimica et Biophysica Acta 1774 (2007) 413–432

isoelectric focusing coupled with micro-range or ‘zoom’ IPG 2-DE [7,8]; and (iv) sub-cellular fractionation to enrich forproteins from a particular organelle [3,4,8–21].

Organelle proteomics reduces the complexity of the totalcellular proteome enabling visualization of low abundanceproteins, and allows study of a specific group of proteins centralto the biological problem under investigation. Information can beobtained on protein function and dynamic changes in the pro-teome such as sub-cellular distribution events in signal transduc-tion or apoptosis. For this approach, sub-cellular fractions areprepared that are relatively free of contaminating abundantproteins from the cytosol and other organelles. Commonmethodsfor preparation of sub-cellular fractions include differentialsolubility or ‘sequential extraction’ for separation of hydrophobicmembrane-associated proteins [20], sucrose density gradients[16,17,22], and differential solubility and phase-partitioning [21].Sub-cellular organelles that have been purified for proteomicanalysis include the nucleus [11,12,16–18], mitochondria[3,4,8,19], and membranes [9,20,21].

The nuclear proteome, including transcription factors, playsa central role in cellular activities controlled by gene expression.Cell differentiation and proliferation are regulated by tissue-specific transcription factors and other proteins that controleukaryotic gene expression. These proteins, in combination,control the specific protein expression patterns of particulartissues. Development of procedures for isolating proteinsextracted from pure nuclei is critical to further our under-standing of cellular differentiation, proliferation and malignant

Fig. 1. Light microscopy of Raji cells and isolated nuclei. Cells and nuclei were stainnuclei pink. (A) Raji cells×400, (B) Raji cells×1000, (C, D) Raji nuclei×1000.

growth. Nuclear regulatory proteins, including transcriptionfactors, are the least abundant proteins in eukaryotes, often withonly a few copies per cell, making them difficult to visualizeand identify by 2-DE and MS.

The nuclear proteome has been analysed from human liver[17], fibroblasts [18], neuroblastoma [16], and a variety of otherhuman organs [23]. Most investigations have used a crudenuclear pellet from low-speed centrifugation without furtherpurification or fractionation. Anderson et al. [24,25] minedfurther down into the nuclear proteome of human HeLa cells byenriching for nucleoli. Almost 400 proteinswere identifiedwithinthe human nucleolar proteome. Schirmer et al. [26] and Dreger etal. [10] investigated the nuclear envelope of neuroblastoma cells.Whereas Dreger et al. further purified nuclear extracts forhydrophobic envelope proteins, Schirmer et al. used a “subtrac-tion” method where a microsomal membrane fraction wascollected and ER proteins identified were subtracted from thenuclear envelope proteome. The nuclear localization of proteinsof interest was then confirmed using immuno-staining.

In the present study, we have analysed the nuclear proteomeof the human Burkitt's lymphoma B-cell line, Raji. Burkitt'slymphoma is the most aggressive subtype of non-Hodgkin'slymphoma. Although a rare condition in adults, Burkitt'slymphoma is relatively common in children, making up about30% of all childhood non-Hodgkin's lymphoma. It is one of themost common cancers in Central Africa [27,28] and also foundmore in AIDS patients [28,29]. The nuclear extract isolatedfrom Raji cells has been further purified by affinity chromato-

ed with combined Giemsa–May–Grünwald stain. Cytoplasm is stained blue and

Table 1Reproducibility of nuclear preparations

Replicate Number of spotsdetected

Number of spotsmatched to master gel

Matchrate %

WN1 376 376 100WN2 363 307 85WN3 387 350 90WN4 287 217 76DNA1 233 233 100DNA2 212 197 92DNA3 215 196 91

Gel images derived from four different whole nuclear (WN1–WN4) and threedifferent DNA-affinity preparations (DNA1–DNA3) were subjected to automaticspot detection and matching using the software PDQuest Version 7.3.0 (Bio-Rad).Using the same spot detection parameters, similar numbers of spots were detectedin all preparations, and matching rates of more than 75% for the whole nuclearfraction and more than 90% for the DNA-binding fraction were obtained.

415S. Henrich et al. / Biochimica et Biophysica Acta 1774 (2007) 413–432

graphy on DNA-agarose enabling deeper analysis of this subsetof nuclear proteins.

2. Materials and methods

2.1. Cell culture

Raji cells were grown in RPMI 1640 mediumwith 50 mMHEPES (Sigma, St.Louis, MO, USA), 10% (v/v) fetal calf serum (Gibco, Auckland, NZ) and 50 μg/mL gentamycin (Gibco, Auckland, NZ) in 75 cm2 culture flasks. Cells weregrown at 37 °C and passaged three times a week to maintain exponential growth.

Fig. 2. Protein map for whole nuclei from Raji cells. Proteins were focused on a 17-cSypro–Ruby staining. Protein spots were excised, identified and marked with the nu

2.2. Whole cell extract

All procedures were performed at 0–4 °C. Cells were harvested from cultureat a density of 106 cells/mL by centrifugation (300×g, 8 min, 4 °C), and washedthree times in PBS before being collected by centrifugation. The resulting cellpellet was resuspended in a 2-DE compatible lysis buffer.

2.3. Purification of nuclei

All procedures were performed at 0–4 °C. Reagents were supplied bySigma (St. Louis, MO, USA) unless stated otherwise. Cells were harvestedfrom culture at a density of 106 cells/mL by centrifugation (300×g, 8 min,4 °C), and washed in PBS, followed by a second wash using hypotonic BufferA (10 mM K.HEPES pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT and0.5 mM PMSF). The cells were collected by centrifugation and the resultingpellet resuspended in Buffer A containing 0.5% (v/v) Surfact-Amps® NP-40(Pierce, Rockford, IL, USA) and placed on ice for 5 min. The swollen cellswere then transferred to a 40 mL Dounce glass homogenizer, lysed with 10gentle strokes, centrifuged (800×g, 8 min, 4 °C) and the resulting crudenuclear pellet was resuspended in Buffer B (0.25 M sucrose, 10 mM MgCl2,20 mM Tris–HCl pH 7.4 and 1 mM DTT). To further purify the nuclei, thesuspension was layered onto a two-step sucrose gradient (1.3 M sucrose,6.25 mM MgCl2, 20 mM Tris–HCl pH 7.4, 0.5 mM DTT above 2.3 M sucrosein 2.5 mM MgCl2 and 20 mM Tris–HCl, pH 7.4), then centrifuged (5000×g,45 min, 4 °C, Beckman, SW28 swinging bucket rotor). The purified nuclearpellet was carefully resuspended in Buffer A and centrifuged again (1000×g,5 min, 4 °C).

To examine the purity and integrity of cells and isolated nuclei,preparations were washed twice in PBS, carefully smeared onto glass slidesand allowed to dry. The slides were fixed in 100% (v/v) methanol for 5 min,air dried at room temperature, stained for 5 min with May–Grünwald stain,washed for 1 min in PBS, ‘double-stained’ in 1:20 diluted GIEMSA stain for

m pH 3–10 IPG strip, separated on an 8–18% SDS-PAGE gel, and visualised bymbers listed in Table 2.

Table2

Proteinsidentifiedfrom

thewho

lenu

clearproteomeof

Rajicells

(see

Fig.2)

Spot

number

Protein

name

SWISS-PROT

accession

number

Theoretical

kDa

Theoretical

pICalculated

kDa

Calculated

pINum

berof

peptides

identified

Sequence

coverage

[%]

Mascot

Score

Molecular

functio

n

1Tpr

protein

Q99

968

267.2

5.0

267.5

5.4

2915

151

Unknown(nuclear

pore

protein)

2Splicingfactor

3Bsubunit2

Q13

435

97.6

5.5

162.4

5.0

1318

57Splicing

3Kelch-likeprotein14

[fragm

ent]

Q9P

2G3

73.2

6.2

190.0

4.9

915

72Unknown

4Zincfinger

SWIM

domaincontaining

protein4

Q9H

7M6

116.6

7.8

142.1

4.9

88

70Unk

nown

5Hyp

othetical

proteinDKFZp762

N1910

Q8N

3B3

72.4

5.3

132.4

4.7

1221

66Unk

nown

6–8

Cho

ndroitinsulfateproteoglycan

6Q86

VX4

141.4

6.8

145.1–

145.3

6.4–

6.7

119

60Unk

nown

9,10

Matrin3

P4324

394

.65.9

122.4,

126.4

5.0,

5.2

1418

125

Nuclear

matrixprotein,

mRNA

processing

11Splicingfactor

3subunit1

Q15

459

88.8

5.2

128.1

6.7

1018

65Splicing

12,1

3Elongationfactor

2P13639

95.1

6.4

96.4,96.0

6.4,

6.5

914

66Translatio

nassociated

14DDX1protein

Q6P

JR1

77.8

8.3

89.6

6.4

1120

75mRNA

processing

15,1

6Nucleolin

P1933

876

.24.6

95.6,76

.24.8,

4.6

1622

125

pre-rRNA

transcriptionand

ribosomeassembly

17Structure-specificrecognition

protein1

Q08

945

81.0

6.5

94.4

4.9

1016

68Transcriptio

n18

ATP-dependent

DNA

helicaseII,80

kDasubunit

P13010

82.5

5.6

86.9

5.0

1419

72DNA

repair

19Mito

ticspindleassemblycheckp

oint

proteinMAD1

Q9Y

6D9

83.0

5.6

86.9

5.1

1423

75Cellcycleprotein

20SW

I/SN

F-relatedmatrix-associated

actin-dependent

regulator

ofchromatin

subfam

ilyCmem

ber1

Q92

922

122.8

5.9

172.5

5.0

1412

86Transcriptio

nfactor

21Pre

mRNA

splicingfactor

PRP17

Q5S

RN0

65.5

6.6

92.7

5.8

718

61Splicing

22Splicingfactor,proline-

andglutam

ine-rich

P2324

676

.19.5

105.2

9.8

1323

74Splicing

23ProbableRNA-dependent

helicasep68

P17844

69.1

9.1

71.7

9.9

1117

72RNA-dependent

ATPase

activity

24Nov

elprotein

Q5S

S78

170.7

7.1

88.1

4.8

2215

74Unk

nown

2578

kDaglucose-relatedprotein[Precursor]

P1102

172

.15.0

79.1

4.8

1120

96ERlumen

protein

26Lam

inB1

P2070

066

.25.1

71.3

4.9

1532

135

Structuralcomponent

ofnuclear

lamina

27Heatshockcognate71

kDaprotein

P1114

270

.95.4

73.8

4.9

1433

137

Heatshockprotein

28Mortalin

-2Q8N

1C8

73.8

6.0

74.9

5.0

818

67Heatshockprotein

29DEAD

boxpolypeptide17

isoform

p82variant

Q59F66

81.0

8.2

68.1

6.2

1013

70RNA

helicase

30,3

1Paraspeckle

protein1beta

isoform

Q8W

XE8

58.7

6.3

72.4,65

.36.2,

6.2

1325

90mRNA

processing

orsplicing

32Cleavagestim

ulationfactor,64

kDasubunit,tauvariant

Q9H

0L4

64.4

6.8

726.4

921

65mRNA

processing

33–37

Heterogeneous

nuclearribonucleoproteinL

P14866

60.1

6.7

64.4–64

.757

.6,56

.46.3–

6.8

10.0,10

.012

2570

hnRNP

38,3

9,10

7–13

2Heterogeneous

nuclearribonucleoproteinA2/B1

P2262

637

.49.0

32.1–37

.767

.7,67

.67.5–

10.0

2153

178

hnRNP

40,4

1Lam

inB2

Q03

252

67.6

5.3

4.9,

5.0

1729

113

Structuralcomponent

ofnuclear

lamina

42–45

58Heterogeneous

nuclearribonucleoproteinK

P61978

50.9

5.2

64–64.7

58.2

4.9

–5.0

4.9

1030

125

hnRNP

46–49

Splicingfactor

3Asubunit3

Q12

874

58.8

5.3

59.3–61

.74.9

1436

160

Splicing

50Non

-POU

domain-containing

octamer-binding

protein

Q9B

QC5

54.2

9.0

57.9

5.0

1028

84Multitasking,m

RNAprocessing,

splicing,

transcriptionassociated

51U4/U6sm

allnu

clearribo

nucleoproteinPrp4

O43

172

58.4

7.1

58.1

7.3

915

68Spl

icing

52PRP4pre-mRNA

processing

factor

4ho

molog

Q5T

1M7

58.3

7.1

58.5

7.0

1122

76Splicing

53Nucleoporin

p54

Q7Z

3B4

33.1

6.0

57.5

6.3

832

67Trafficking


54,56

Glutamatedehy

drog

enase

P00

367

61.4

7.7

53.2,53

.26.7,

6.4

1630

183

Metabolism

associated,

mito

chondrial

55Smu-1suppressor

ofmec-8

andun

c-52

homolog

Q9B

U59

57.5

6.7

54.0

6.5

921

71Unk

nown

57PRP19/PSO4ho

molog

Q9U

MS4

55.1

6.1

55.1

6.1

921

87DNA

repair

59Tubulin

alpha-ubiquitous

chain

P68363

49.9

4.9

56.7

4.9

1034

95Structuralcomponent

ofcytoskeleton

60Tubulin

alpha-6chain

Q9B

QE3

49.9

5.0

54.3

4.9

722


ofcytoskeleton

61BAF53

Aprotein

Q6F

I97

47.4

5.5

49.0

4.9

3313

63Transcriptio

nfactor

62SWI/SNF-related

matrix-associated

actin

-dependent

regulatorof

chromatin

subfam

ilyEmem

ber1

Q96

9G3

46.6

4.9

55.3

4.8

1027

51Transcriptio

n

63Histone-binding

proteinRBBP4

Q09

028

47.5

4.7

53.6

4.7

11MSMS

(peptid

e1188

.6,

296–

303;

peptide13

43.6,

103–

113)

2910

2Transcriptio

nregulatoractivity

64Splicingfactor,arginine/serine-rich

6and

Histone-binding

proteinRBBP7

Q13

247

Q16

576

39.5

47.8

11.4

4.6

51.9

4.8

9MALDI-TOF

MSMS

(peptid

e1141

.6,

102–

113;

peptide97

3.6,

296–

303)

2393

Splicing

65ATPsynthase

beta

chain,

mito

chondrial[Precursor]

P06576

56.5

5.3

51.2

4.9

1948

234

Transcriptio

nregulatoractivity

Metabolism

associated,

mito

chondrial

66Tubulin,beta

polypeptide

Q5JP53

47.7

4.7

52.7

4.8

1445


ofcytoskeleton

67,68

HNRPH

IQ6IBM4

49.1

5.8

932

71hn

RNP

69,81

,82

Heterog

eneous

nuclearribonu

cleoproteinD0

Q14

103

35.9

9.0

50.8,

50.5

49.0,45

.4,

44.9

6.1,

6.2

6.3,

6.8,

7.6

621

74hn

RNP

70–78

Heterog

eneous

nuclearribonu

cleoproteinH

P31

943

49.2

5.9

48.0–51

.15.0–

5.5

1434

90hn

RNP

79Ruv

B-like1

Q9Y

265

50.2

6.0

51.1

6.2

823

84Transcriptio

nassociated

80Cleavagestim

ulationfactor,64

kDasubunit

Q05048

48.4

6.1

52.1

6.2

620

54mRNA

processing

83–86

ProbableATP-dependent

helicaseDDX48

P38919

46.8

6.1

45.6–45

.95.8–

6.4

1741

181

Unk

nown

87Elong

ationfactor

Tu,

mito

chon

drial[Precursor]

P49

411

49.5

7.7

43.3

6.3

1545

134

Translatio

nassociated

88RNA

bind

ingmotifprotein4

Q9B

WF3

40.3

6.6

38.9

6.4

1452

142

mRNA

processing

89Poly(rC)-bindingprotein1

Q15365

37.5

6.7

38.0

6.5

836

79posttranscriptionalregulator

90SETprotein-spliceform

beta

Q0110

532

.14.1

40.0

4.1

516

62Multitasking

protein,

involved

inapoptosis,transcription,

nucleosomeassemblyand

histonebinding

91–94

Heterog

eneous

nuclearribonu

cleoproteinF

P52

597

45.7

5.4

42.0–46

.54.9

936

88hn

RNP

95,96

163

ACTBprotein

Q96

E67

40.2

5.6

42.5,

41.9

30.2

4.9,

4.9

4.8

1143

122

Structural,component

ofcytoskeleton

97Interleukinenhancer-binding

factor

2Q12905

44.7

8.3

43.4

4.9

1843

127

Transcriptio

nfactor

98SW1/SN

Frelatedmatrixassociated

actin

-dependent

regulatorof

chromatin,subfam

ilyBmem

ber1

Q12

824

43.1

5.9

42.1

5.0

826

78Transcriptio

nassociated

99Ank

yrin

repeat

andSOCSbo

xprotein15

Q8W

XK1

48.3

5.5

40.6

5.4

712

95Cellcommunication

100–

103

Heterog

eneous

nuclearribonu

cleoproteinA/B

Q99

729

30.6

7.7

39.8–39

.95.9–

6.3

725

82hn

RNP (c

ontin

uedon

next

page)


Table2(contin

ued)

Spot

number

Protein

name

SWISS-PROT

accession

number

Theoretical

kDa

Theoretical

pICalculated

kDa

Calculated

pINum

berof

peptides

identified

Sequence

coverage

[%]

Mascot

Score

Molecular

functio

n

104

Activator

140

kDasubu

nit

P3525

039

.16.0

38.0

5.6

934

67DNA

replication

105

Mito

ticcheckpoint

proteinBUB3

O43684

37.2

6.4

38.5

6.3

1134

102

Cellcommunication

106

Serine/threonineproteinphosphatasePP1

-alpha

catalytic

subu

nit

P6213

635

.16.4

35.6

5.6

835

76Cellcycle

133

40Sribosomal

proteinSA

P0886

531

.84.8

40.9

4.8

829

86Cellcommun

ication

134–

137

156–

158

206–

209

Heterogeneous

nuclearribonucleoproteinC1/C2

P07910

33.3

5.1

39.8–39

.930

.8–31

.137

.0–37

.5

4.8–

4.9

6.5–

7.4

4.8–

4.9

939

73hn

RNP

138,160161

Nucleophosm

inP06748

32.6

4.5

35.7,27

.3,

26.2

4.8,

4.4,

4.4

1342

79Multitasking,m

RNAprocessing

139,

140

Proliferatingcellnu

clearantig

enP6125

828

.84.6

34.1,32

.64.7,

4.7

1044

86DNA

replication

141

Com

plem

entcompo

nent

1,Q

subcom

ponent

bind

ing

proteinmito

chondrial

Q07

021

31.3

4.7

31.7

4.4

741

112

Receptoractiv

ity

142

DNA-directedRNA

polymeraseII33

kDapolypeptide

P19387

31.4

4.7

34.0

4.8

732

60Transcriptio

nassociated

143,

144

Splicingfactor,arginine/serine-rich

7Q16

629

27.4

11.8

35.0,34

.94.8,

4.9

728

89Splicing

145

DNA

directed

RNA

polymeraseI40

kDa

O15

160

38.4

5.4

39.6

4.9

732

75Transcriptio

nassociated

146

Eukaryotic

translationinitiationfactor

3subunit2

Q13347

12.3

5.6

35.5

5.0

740

69Translatio

nassociated

147

60Sacidic

ribo

somalproteinP0

P0538

834

.35.7

35.3

5.0

936

67Ribosom

alsubunit

148

Heterogeneous

nuclearribonucleoproteinD-like

O14979

46.4

9.6

36.5

6.1

58

48hnRNP

149–

153

Heterogeneous

nuclearribonucleoproteinH3

P31942

36.9

6.4

33.3–35

.46.1–

6.2

1663

116

hnRNP

154

A+U-richelem

entRNA

bind

ingfactor

Q7K

Z74

30.2

8.8

36.3

6.3

1023

71mRNA

processing

155

Ribose-ph

osphatepy

roph

osph

okinaseI

P6089

134

.76.6

31.9

6.5

926

89Metabolism

159

Guanine

nucleotid

e-bindingproteinbeta

subunit2-lik

e1

P63246

35.1

7.6

30.5

7.6

925

85Cellcommunication

162

Eukaryotic


6Q96TD5

36.5

4.6

26.6

4.5

529

68Translatio

nassociated

164,

165


1Q07

955

27.6

10.4

31.3,30

.94.8,

4.9

1028

72Splicing

166–

168


2Q01130

25.4

11.9

31.8–31

.94.9

726

66Splicing

169

DnaJhomolog

subfam

ilyCmem

ber9

Q8W

XX5

29.9

5.6

30.5

5.2

931

91Chaperone

170

PQBP-1d

Q9G

ZP2

18.8

4.9

32.6

6.1

441

42Transcriptio

nassociated

171

Voltage-dependent

anion-selectivechannelprotein1

P21796

30.6

8.6

29.7

9.6

522

65Transport

172

Peroxiredox

in1

Q06

830

19.0

6.4

23.8

8.9

949

121

Metabolism

andsign

allin

g17

3Histone

H1.4

P1041

221

.711.0

21.7

105

1959

Histone

174

Histone

H2B

Q99

877

13.8

10.3

18.2

105

4041

Histone

175

Prohibitin

Q6P

UJ7

29.8

5.6

27.4

5.0

1229

72Signalling

176

Spliceosomeassociated

proteinSPF27

O75934

26.1

5.5

26.4

4.9

728

79Splicing

177


9Q13242

25.5

8.7

26.2

4.9

1344

82Splicing


178

Chrom

obox

proteinho

molog

5P4597

322

.25.7

24.1

4.9

1048

90Genesilencing

179

Exosomecomplex

exonucleaseRRP41

Q9N

PD3

26.4

6.1

27.3

5.7

932

109

Exonuclease

180

Nuclear

proteinHcc-1

P8297

923

.56.1

29.1

6.1

727

94Transcriptionregulatoractivity

181

CLE

Q5R

LJ0

28.1

6.0

25.9

6.1

734

69Transcriptio

nassociated

182–

184

SmallheterogeneousnuclearribonucleoproteinF

P6230

69.7

4.7

9.6–

9.7

4.3–

4.7

MALDI-TOF

MS-MS

(peptid

e99

4,66–73

)

hnRNP

185

Nucleoplasm

in-3

O75607

19.2

4.6

23.0

4.5

217

40Chaperone

186,

187

FUSinteractingserine-argininerich

protein1

O75

494

21.0

10.5

23.0,22

.96.2,

6.4

845

97Splicing

188

RNA-binding

protein8A

Q9Y

5S9

18.2

6.5

23.4

4.9

417

68Splicing

189,

190

Chrom

obox

proteinho

molog

3Q13

185

20.8

5.2

23.1,23

.14.87,4.9

5MALDI-TOF

MS-MS

(peptid

e14

89.8,

142–

154)

2468

Genesilencing

191

Program

med

celldeath6

O75340

21.9

5.2

22.4

4.8

422

81Cellcommunication

192

DNA

directed

RNA

polymeraseII16

kDapo

lypeptide

Q9D

7M8

16.3

4.8

21.8

4.7

328

65DNA

replication

193

Smallubiquitin

-related

modifier2[Precursor]

P61956

10.9

5.3

19.5

4.9

363

48Ubiquitin-specific

protease

194,

195

Magonashiproteinho

molog

P6132

615

.75.2

17.4,17

.05.0,

5.5

753

103

Splicing

196

Enh

ancerof

rudimentary

homolog

P8409

012

.35.6

15.0

6.3

941

76Transcriptio

nassociated

197

Calmodulin

P6215

814

.24.1

18.9

4.1

971

88Cellsign

allin

g19

860

Sacidic

ribo

somalproteinP2B

P0538

711.7

4.4

16.9

4.2

1034

95Ribosom

alsubunit

199

U6snRNA-associatedSm-likeproteinLSm8

O95777

10.3

4.3

13.9

4.2

472

91RNA

processing

200


P62310

11.7

4.6

15.4

4.5

422

65RNA

processing

201

ReplicationproteinA

14kD

asubunit

P3524

413

.65.0

12.5

4.8

772

67DNA

replicationandrepair

202

NHP2no

n-histonechromosom

eprotein2-lik

e1

Q6F

HM6

14.2

8.7

14.8

9.4

324

51Unk

nown,

possibly

ribosome

biog

enesis

203

GTP-binding

nuclearproteinRan

P62826

24.4

7.0

24.

7.0

939

86Trafficking

204

Splicingfactor

3Bsubunit5

Q9B

WJ5

10.1

5.9

9.9

5.3

332

50Splicing

205

SETprotein-spliceform

alpha

Q6F

HZ5

33.5

4.2

41.7

4.3

623

66Multitasking

protein,

involved

inapoptosis,transcription,

nucleosome

assemblyandhistonebinding

210

PutativeRNA-binding

protein3

P9817

917

.28.9

20.7

8.2

547

86RNA

processing

,apoptotic

mod

ulatorycapabilities



15 min and washed briefly with deionized water. A cover slip was attachedusing DPX mountant (ProSciTech, Thuringowa, Australia) and microscopywas performed using a Nikon Eclipse 3800 transmission light microscope withdifferential interference and Camware 1.21 software (Cooke Corp., AuburnHills, MI, USA).

2.4. Fractionation of nuclear proteins on DNA-agarose

All procedures were performed at 0–4 °C. A cocktail of protease inhibitors(Sigma, St. Louis, MO, USA, cat. no. P8340) was added to all buffers. DNA-binding proteins were isolated from the nuclear pellet obtained after the first lowspeed centrifugation (800×g, 8 min, 4 °C) of the cell lysate. The crude nuclearpellet was washed once in Buffer A, centrifuged at low speed, resuspended in20mMHEPES (pH 7.6), 350mMNaCl, 5 mMMgCl2, 5 mMNaF, 1mMEDTA,1 mM DTT, 1 mM PMSF and stirred gently for 30 min on ice. Aftercentrifugation (47,800×g, 1 h, 4 °C) the supernatant was diluted 1:1 with 20 mMHEPES (pH 7.6), 20% (v/v) glycerol, 5 mM MgCl2, 5 mM NaF, 1 mM EDTA,1 mM DTT, 1 mM PMSF and subsequently loaded onto a DNA-agarose column(GE Healthcare, Piscataway, NJ, USA). The nuclear protein extract was passedthrough the column twice to ensure maximum binding, followed by extensivewashing with 50 mMHEPES (pH 7.6), 100 mMNaCl, 10% (v/v) glycerol, 0.5%(v/v) Triton-X, 5 mM NaF, 1 mM EDTA, 1 mM DTT and 1 mM PMSF. DNA-binding proteins were eluted with column washing buffer containing 1 M NaCl,then precipitated with 10% (v/v) TCA overnight on ice, and the protein pellet waswashed twice with ice-cold acetone.

2.5. Two-dimensional gel electrophoresis

Whole cells, nuclei or protein pellets of the DNA-binding fractions weresolubilized in a 2-DE compatible buffer containing 5 M urea, 2 M thiourea,2% (w/v) CHAPS, 40 mM Tris–HCl, 65 mM DTT, 2% (w/v) sulfobetaine 3–10, 1% (v/v) Bio-Lyte 3–10 carrier ampholytes and 0.002% (w/v)bromophenol blue. To remove DNA from extracts of whole nuclei, Benzonase(200 U/mL) was added, the extract incubated at room temperature for 25 minand then centrifuged (20,000×g, 10 min, 25 °C) to remove insoluble material.The pellet was resuspended, spun and the supernatants were pooled. Proteinconcentrations of the whole nuclear extract and redissolved DNA-bindingfractions were determined using the 2-D Quant protein assay (GE Healthcare,Piscataway, NJ, USA). Samples (450 μg of protein, 400 μL) were loaded ontoa 17 cm pH 3–10 linear pH gradient IPG gel strip (Bio-Rad, Hercules, CA,USA). After overnight in-gel rehydration, isoelectric focusing was carried outusing a Multiphor II apparatus (GE Healthcare) using a five-step program (3 hat 100 V, 2 h at 300 V, 2 h at 1 kV, 1 h at 2.5 kV and 17 h at 5 kV) for a totalof 90.4 kVh. After focusing, the IPG strips were equilibrated for 20 min in6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol, 5 mM TBP, 2.5% (v/v)acrylamide and 350 mM Tris–HCl (pH 8.8), then transferred onto a seconddimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel (1 mm thick 8–18% linear gradient) and sealed in place with 0.5%(w/v) agarose in cathode buffer with a trace of bromophenol blue. Second-dimension electrophoresis was carried out using a Protean IIxi system (Bio-Rad) at 8 °C using a two-step program, 2 mA per gel for 3 h, followed by10 mA per gel overnight until the bromophenol blue reached the bottom of thegel. The cathode and anode buffer consisted of 192 mM glycine, 25 mM Tris–HCl (pH 8.3) and 0.1% (w/v) SDS. The anode buffer was furthersupplemented with 0.005% (w/v) sodium azide. The gels were fixed in 10%(v/v) methanol and 7% (v/v) acetic acid for a minimum of 1 h, stained withSypro Ruby (Bio-Rad, Hercules, CA, USA) overnight and washed in 10% (v/v) methanol and 7% (v/v) acetic acid for 2 h before being visualized on aMolecular Imager® FX Pro Plus (Bio-Rad). Data were analyzed usingPDQuest Version 7.3.0 (Bio-Rad).

2.6. Protein identification

2.6.1. In-gel digestionTo facilitate spot excision, the gels were stained overnight with colloidal

Coomassie Blue G-250 and washed in 1% (v/v) acetic acid [30]. Spots weremanually excised using a scalpel, destained in 60% (v/v) 50 mM ammonium

bicarbonate buffer (pH 7.8), 40% (v/v) acetonitrile for a minimum of 1 h at roomtemperature and dehydrated in 100% acetonitrile for 1 min before being dried ina vacuum centrifuge. Gel pieces were rehydrated in 10 μL trypsin solution(12 ng/μl porcine modified sequencing grade trypsin in 50 mM ammoniumbicarbonate, pH 7.8) at 4 °C for 1 h. Excess trypsin solution was removed andthe gel pieces were resuspended in 15 μL of 50 mM ammonium bicarbonate (pH7.8) and incubated overnight at 37 °C.

2.6.2. Mass spectrometryFor MALDI-TOF MS analysis, 1 μL of extracted peptides was spotted onto

a target plate with an equal volume of matrix solution (10 mg/mL α-cyano-4-hydroxycinnamic acid in 70% (v/v) acetonitrile, 1% (v/v) TFA), and air-dried atroom temperature. Mass spectra were acquired in the mass:charge range of 800–3500 u on a QSTAR XL mass spectrometer equipped with a MALDI source(Applied Biosystems Inc., Foster City, CA, USA). Mass calibration wasperformed using [Glu1] Fibrinopeptide B as an external calibrant. The generatedmonoisotopic peak masses were subjected to database searching against theMSDB comprehensive non-redundant database (releases 20040106 and20050701) using MASCOT vr 2.0 (Matrix Science, London, UK). The peptidemass fingerprinting (PMF) data were also searched using the MS-Fit program(Protein-Prospector, UCSF Mass Spectrometry Facility). Each PMF spectrumwas manually inspected, and peptides were obtained by manual annotation ofthe spectra. Parameters for protein identification included searching with a masserror tolerance of 65 ppm per peptide, 1 missed tryptic cleavage, and allowingoxidation of methionine as an optional modification. No pI or mass restrictionswere included. Confident matches were defined by the MASCOT score andstatistical significance (p<0.05), the number of matching peptides and thepercentage of total amino acid sequence covered by those matching peptides. Ingeneral, matches required sequence coverage of at least 30% for proteins of 15–60 kDa. This percentage may vary for low and high molecular mass proteins,and those that generate few or many tryptic peptides.

Peptide mixtures that provided poor initial mass spectra were concentratedand desalted using C18 PerfectPure reverse-phase micro-columns (Eppendorf,Düsseldorf, Germany) according to the manufacturer's instructions and elutedin matrix solution directly onto the target plate. MALDI-TOF MS was thenperformed as described above. Proteins that could not be confidently identifiedby PMF were subsequently analyzed by MALDI-Q TOF MS-MS (QSTARXL) to obtain sequences that confirmed the initial PMF data. To facilitateionization and fragmentation of peptides for MS-MS sequencing, peptideswere subjected to N-terminal sulfonation using 4-sulfophenyl isothiocyanate(SPITC) [31,32]. After tryptic digestion, the peptide mixture was mixed 1:1with 10 mg/mL SPITC in 20 mM ammonium bicarbonate and incubated at56 °C for 45 min. The reaction was stopped with 5% (v/v) TFA and peptideswere concentrated prior to MALDI-TOF MS-MS using C18 micro-columns asdescribed above. De novo peptide sequences were obtained by manualannotation of the spectra.

2.7. Prediction of protein sub-cellular location

Identified proteins not previously known to be localized to the nucleus asindicated by a database such as SWISS-PROT, were subjected to PSORTprediction (http://psort.nibb.ac.jp) that predicts sub-cellular location on the basisof protein amino acid composition using a set of candidate localization sites [33].

2.8. SDS-PAGE and Western blotting

Proteins were separated with a Bio-Rad Mini-Protean II gel electrophoresisapparatus utilizing a 5% polyacrylamide stacking gel and a 10% polyacrylamideresolving gel employing the buffer system of Laemmli [34]. Proteins weretransferred onto Immobilon-P PVDF membranes (Millipore Corp., Bedford,MA) with a Criterion™ Blotter (Bio-Rad). The membranes were blockedovernight at 4 °C using a blocking solution (4% gelatin from cold water fish skin(Sigma, St. Louis, MO, USA) in TBS-Tween 20: 20 mM Tris–HCl pH 7.5,150 mM NaCl and 0.1% (v/v) Tween-20), washed twice for 5 min with TBS-Tween 20 and incubated for 1 h at 25 °C with a 1/500 dilution of rabbit anti-IgGpolyclonal antibody (PU.1) or a 1/1000 dilution of mouse anti-IgG1 monoclonalantibody (actin, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Afterwashing with TBS-Tween 20 (3×10 min), membranes were incubated for 1 h at

http://psort.nibb.ac.jp


25 °C with a 1/8000 dilution of bovine anti-rabbit or a 1/10000 dilution of anti-mouse IgG-alkaline phosphatase conjugate (Santa Cruz Biotechnology) andwashed (3×) for 10 min with TBS-Tween 20. Development was carried outusing AttoPhos® (Promega, Madison, WI, USA) and bands were detected on aMolecular Imager® FX Pro Plus (Bio-Rad).

Membranes were stripped using two 30-min incubations at 50 °C in astripping buffer (65 mM Tris–HCl pH 6.8, 2% (w/v) SDS, 0.1 M β-mercaptoethanol) before being washed twice for 5 min in double-distilled waterand twice for 10 min in TBS-Tween 20.

3. Results

3.1. Integrity and purity of nuclear fractions

A major problem with proteomic analyses of purified sub-cellular organelles is contamination with cytosolic, cytoskele-tal, or other proteins. To determine the purity and integrity ofthe nuclear fraction, light microscopy was employed usingGiemsa–May–Grünwald staining. Following sucrose densitygradient centrifugation, preparations of nuclei appeared intactand pure with no visible contamination with cytoplasm (Fig. 1,panels C and D). Each nuclear preparation was verified usingthis technique. Separation of nuclear proteins by 2-DE and

Fig. 3. Protein maps from whole nuclear (A) and DNA-binding fractions (B). To cobiological replicates (gels 1, 2 and 3) were compared and plotted against each other

identification by PMF confirmed the integrity and purity of thenuclei. Proteins from nuclei purified on a sucrose densitygradient were solubilized in 2-DE-compatible sample buffer(see Materials and methods) and separated by 2-DE. Gels (11)from different nuclear preparations were highly reproducible(Table 1, Fig. 3), a representative gel image is shown in Fig. 2.The protein maps for the nuclear fraction showed asubstantially different spot pattern from the unfractionatedsample (whole cell lysate, Fig. 4), indicating that fractionationof nuclei results in sub-organellar enrichment enablingidentification of more low abundance proteins. The mostabundant protein in the whole cell lysate, actin, was reducedby 90%. Nucleophosmin, which is the most abundant nuclearprotein, was enriched 9.1-fold in the nuclear fraction.Although primarily either cytoplasmic or nuclear, bothproteins shuttle between nucleus and cytoplasm and arepresent in both fractions. Image analysis using the programPD-Quest detected approximately 400 spots in the nuclearfraction. Proteins that could be visualized with CoomassieBlue G-250 were excised, digested with trypsin and analysedby MALDI-TOF-MS, followed by database searching usingMASCOT (Table 2). A total of 210 protein spots generated

mpare the quantitative reproducibility of both fractions, the spot intensities of. Linear regression was performed, the resulting R-values are presented.

Table3

Proteinsidentifiedfrom

thenuclearDNA-binding

proteomeof

Rajicells

(see

Fig.3

)

Spo

tnu

mber

Protein

Nam

eSWISS-

PROT

accession

number

Theoretical

kDa

Theoretical

pICalculated

kDa

Calculated

pINum

berof

peptides

identified

Sequence

coverage

[%]

Mascot

Score

Molecular

functio

n

1Antigen

Ki-67

P46013

319.3

9.5

267.2

5.0

209

96Signaltransductio

n,cellcycle

control

2SWI/SN

F-related

matrix-associated

actin

-dependent

regulatorof

chromatin

subfam

ilyCmem

ber2

Q8T

AQ2

132.7

5.4

160.7

5.4

1514

78Activator

and/or

repressor

oftranscription

3SWI/SN

F-related

matrix-associated

actin

-dependent

regulatorof

chromatin

c1Q58

EY4

122.8

5.5

123.0

5.4

1411

86Transcriptio

nfactor

activ

ity

4Transcriptio

nelongatio

nregulator1variant[Fragm

ent]

Q59EA1

122.1

8.7

136.5

6.8

1413

67Transcriptio

nfactor

5Transcriptio

nelongatio

nregulator1

O14776

123.9

8.8

123.0

6.1

87

80Transcriptio

nfactor

6Structuralmaintenance

ofchromosom

e3

Q9U

QE7

141.4

6.8

110.6

6.16

1415

76Unk

nown

7DDX42

protein

Q68

G51

100.9

6.4

88.0

5.8

1019

96RNA

processing

8Staph

ylococcalnu

clease

domaincontaining

protein1

Q7K

ZF4

101.9

6.7

77.0

6.2

2128

154

Transcriptio

nregu

latory

activ

ity9

Poly[A

DP-ribose]

polymerase1

P0987

4112.9

9.0

82.8

6.8

1621

98DNA

repair

10–12

Splicingfactor,proline-

andglutam

ine-rich

P23246

76.1

9.5

74.4–77

.07.6–

9.5

1024

72Splicing

13,14

Nucleolin

P1933

876

.34.6

71.0,70

.04.5,

4.7

915

79RNA

processing

15Celldivision

cycle5-lik

eprotein

Q99459

92.2

8.2

73.6

5.7

819

82Signaltransductio

n16

Interleukinenhancer-binding

factor

3Q12906

74.6

8.4

68.2

6.2

1836

154

Transcriptio

nfactor

activ

ity17

78kD

aglucose-regu

latedprotein[Precursor]

P1102

172

.15.0

61.6

4.9

918

105

Heatshock

18Euk

aryo

tictranslationinitiationfactor

4BP2358

869

.25.5

62.4

5.3

913

79Translatio

n19

,20

Ezrin

Q6N

UR7

69.2

5.9

63.5,63

.15.7,

5.7

1425

132

Cytoskeletalanchoringactiv

ity,

grow

thandmainanence

21–23

KHSRPprotein

Q5U

4P6

72.9

8.0

65.2–65

.56.1–

6.5

1835

174

Transcriptio

nregulatoractiv

ity24

Lam

ina-associated

polypeptide2isoform

alpha

P4216

675

.37.8

63.1

7.0

922

100

Cellcycleregulatio

n,sign

altransductio

n25

DEAD

boxpo

lypeptide17

isoform

p82

variant[Fragm

ent]

Q59

F66

81.0

8.2

64.6

7.7

812

69RNA

helicase

26ProbableATP-dependentRNA

helicaseDDX5

P1784

469

.19.1

57.6

8.6

1018

72RNA

processing

27RNA-binding

proteinFUS

P3563

753

.49.4

59.6

9.0

921

64RNA

processing

28–31

Far

upstream

elem

ent-bindingprotein1

Q96AE4

67.4

7.2

57.8–58

.66.1–

7.3

1127

67Activator

and/or

repressorof

transcript.(m

yc)

3275

kDaglucoseregulatedprotein(M

ortalin

)P38646

73.7

6.0

56.9

5.4

1532

107

Cellproliferationandcellu

lar

aging;

mito

chondrial

33Heatshockcogn

ate71

kDaprotein

P1114

270

.95.4

56.9

5.2

1024

74Heatshock

34Moesin

P26038

67.6

6.1

60.3

5.8

1833

120

Structuralcomponent

ofcytoskeleton

35Cleavagestim

ulationfactor,64

kDasubunit,tauvariant

Q9H

0L4

64.4

6.8

56.9

6.25

614

67mRNA

processing

36ATP-dependent

DNA

helicaseII,70

kDasubunit

P12956

69.7

6.2

57.1

5.8

1229

85DNA

helicase

37Heterog

eneous

nuclearribonu

cleoproteinQ

O60

506

46.7

5.9

55.5

6.6

1947

170

RNA

processing

38–40

Heterog

eneous

nuclearribonu

cleoproteinL

P1486

660

.16.7

53.2

6.4–

6.8

1531

109

pre-mRNA

processing

41SWAP-70

Q9U

H65

69.0

5.7

55.4

5.5

816

70pre-mRNA

processing

42,43

Far

upstream

elem

entbind

ingprotein2

Q92

945

72.7

8.0

62.8,63

.06.1,

6.3

1026

97Transcriptio

nregu

latoractiv

ity44

Cleavagestim

ulationfactor,64

kDasubunit

P33240

60.9

6.4

54.9

5.7

820

93mRNA

processing

45HypotheticalproteinFL

J34411

Q8N

B11

50.5

5.2

54.2

5.4

1025

80Unk

nown

46,47

Paraspeckle

protein1alphaisoform

Q8W

XF1

58.7

6.3

53.1,52

.45.8,

6.0

1238

98mRNA

processing

orsplicing

48Ty

rosyl-tRNA

synthetase,cytoplasm

icP54577

59.0

6.6

48.9

5.6

1232

101

Metabolism,cytoplasmic


49Putative55

kDaprotein

Q9P

0J3

55.0

7.0

48.8

6.8

920

77Unk

nown

50U4/U6sm

allnu

clearribonu

cleoproteinPrp4

O43

172

58.5

6.7

49.7

7.2

1025

121

Splicing

51,52

RCC2protein

Q9P

258

56.0

9.0

48.0,48

.27.2,

7.5

1024

76Mito

sisandcytokinesis

53Serinehydroxym

ethyltransferase2(M

itochondrial)

variant[Fragm

ent]

Q53

ET4

55.9

8.6

46.1

7.6

613

65Metabolism

54–56

Non-POU

domain-containing

octamer-binding

protein

Q15233

54.2

9.0

48.9–49

.68.0–

8.3

1535

66Several

nuclearprocesses;

Splicing,

Transcriptio

nal

regulatio

n57

Polypyrim

idinetract-bindingprotein1

P26599

59.0

9.2

49.2

8.7

1027

92pre-mRNA

splicing

58Translatio

nelongatio

nfactor

1alpha1-lik

e14

Q96RE1

43.0

8.9

42.8

9.0

617

53Translatio

nalelongatio

n59

Eukaryotic


2subunit3

P41091

50.9

8.7

44.0

8.2

716

81Translatio

n60

Splicingfactor

3Bsubunit4

Q15

427

44.4

8.5

42.1

7.9

619

65Splicing

61–63

Regulator

ofchromosom

econdensatio

nP1875

442

.86.2

40.7–40

.96.3–

6.9

621

66Signaltransm

ission

protein

64,65

RuvB-like1

Q9Y

265

50.2

5.9

45.3,45.2

5.7,

5.8

1135

109

Transcriptio

nalactiv

ational

activ

ity66

Cleavagestim

ulationfactor,50

kDasubunit

Q05048

48.3

6.1

44.4

5.8

721

72pre-mRNA

processing

67SDCCAG10

Q6U

X04

53.8

5.6

50.1

5.5

825

67Unknown(possiblymetabolism)

68–71

Heterog

eneous

nuclearribo

nucleoproteinK

Q5T

6W5

47.5

5.5

48.8–51

.95.1–

5.3

1030

73hn

RNP

72,73

PRP19/PSO4ho

molog

Q9U

MS4

55.1

6.1

47.6,47

.55.7,

5.7

1120

118

DNA

repair

74Heterog

eneous

nuclearribo

nucleoproteinK

isoform

avariant

Q59

F98

48.8

5.5

52.1

4.8

616

57hn

RNP

75Dop

aminereceptor

interactingprotein5

Q4W

4Y2

44.2

4.6

49.6

4.5

517

75Unk

nown

76–78

Nucleosom

eassemblyprotein1-lik

e1

P55209

45.3

4.4

43.4–48

.64.4–

4.5

935

73Cellproliferation

79Chrom

atin

assemblyfactor

1subunitC

Q09028

46.1

4.9

45.7

4.6

1034

90Transcriptio

nregulatoractiv

ity80

Tub

ulin,beta

polypeptide

Q5JP53

47.7

4.7

44.8

4.8

1842

121

Cytoskeletalprotein

81Tubulin

alpha6variant[Fragm

ent]

Q53GA7

49.8

5.0

48.2

5.0

621

70Structuralconstituent

ofcytoskeleton

82Splicingfactor

3Asubu

nit3

Q12

874

58.8

5.3

49.4

5.1

716

65Splicing

83–85

Heterog

eneous

nuclearribo

nucleoproteinH

P3194

349

.15.9

43.8–44

.15.5–

5.6

1634

81pre-mRNA

processing

86Elong

ationfactor

1-gamma

P2664

150

.06.3

42.3

5.8

1232

112

Translatio

n87

Proliferation-associated

protein2G

4Q9U

Q80

43.8

6.1

40.6

5.7

1438

97Signaltransductio

n,cellcycle

control

88–90

Heterog

eneous

nuclearribo

nucleoproteinD0

Q14

103

36.2

8.1

37.4–37

.66.8–

7.6

623

61hnRNP;transcriptionfactor

91Heterog

eneous

nuclearribo

nucleoproteinG

P3815

947

.49.6

38.6

9.2

1633

160

hnRNP

92Heterog

eneous

nuclearribo

nucleoproteinA3

P5199

139

.69.1

34.8

9.1

1131

75hn

RNP

93–95

Heterog

eneous

nuclearribo

nucleoproteins

A2/B1

P2262

636

.08.7

31.3–32

.68.2–

8.9

1338

100

hnRNP

96DNA-(apurinic

orapyrim

idinic

site)lyase

P2769

535

.58.3

32.7

8.3

624

71DNA

repair

97Heterog

eneous

nuclearribo

nucleoproteinA0

Q13

151

30.8

9.3

31.7

9.2

519

87hn

RNP

98WD

repeat

domain58

Q86

W42

37.5

7.1

32.0

7.0

933

93Unk

nown

99Poly(rC)-bindingprotein1

Q15365

37.5

6.7

34.6

6.4

941

91RNA

processing

100,101

Mito

ticcheckpoint

proteinBUB3

O43684

37.1

6.4

35.7,35.7

6.1,

6.3

1031

84Signaltransductio

n10

2HnR

NPJK

TBPprotein

Q7K

Z75

33.6

6.9

33.0

6.2

729

64hn

RNP

103

Heterog

eneous

nuclearribo

nucleoproteinH

P3194

349

.15.9

42.3

5.6

1131

70hn

RNP

104

RuvB-like2

Q9Y

230

51.0

5.5

43.3

5.4

825

73Transcriptio

nalactiv

ation

105

BAF5

3Aprotein

Q6F

I97

47.4

5.5

42.9

5.2

1338

98Transcriptio

nregulatoractiv

ity10

6Heterog

eneous

nuclearribo

nucleoproteinF

P5259

745

.55.4

41.4

5.2

623

76hn

RNP

107

RNA

bind

ingmotifprotein17

Q5W

009

44.9

5.8

42.0

5.4

817

69Splicing

108

Actin,b

eta

Q96E67

40.2

5.6

38.4

5.1

1450

156

Actin

(cytoskeletaland

innucleoli)

109

Nuclear

transcriptionfactor

Ysubunitgamma

Q13

952

37.2

4.9

35.2

4.5

417

65Transcriptio

nfactor

(con

tinuedon

next

page)


Table3(contin

ued)

Spot

number

Protein

Nam

eSWISS-

PROT

accession

number

Theoretical

kDa

Theoretical

pICalculated

kDa

Calculated

pINum

berof

peptides

identified

Sequence

coverage

[%]

Mascot

Score

Molecular

functio

n

110

Hyp

othetical

proteinDKFZp564

N1778

Q9N

TK0

38.4

5.0

35.2

4.9

621

83Unk

nown

111

POLR1C

protein[Fragm

ent]

Q96HT3

38.4

5.4

35.9

5.2

930

91DNA-directedRNA

polymerase

112

THO

complex

subunit3

Q96J01

38.7

5.7

34.3

5.5

823

68Transcriptio

nregulatoractiv

ity113

Hyp

othetical

proteinFLJ139

63Q9H

836

45.0

5.4

36.8

5.5

724

66Probably

transcriptionassociated

114

Nucleophosm

inP06748

32.4

4.6

32.9

5.6

423

40Multitasking,m

RNAprocessing

115,116

Heterogeneous

nuclearribonucleoproteinH3

P31942

31.5

6.8

31.97,32.4

5.9,

6.0

617

69hnRNP

117

Eukaryotic


3subunit2

Q13347

36.5

5.4

32.5

5.3

629

87Translatio

n118

Polyglutamine-bindingprotein1

O60828

30.5

5.9

30.7

5.8

836

65Transcriptio

nregulatory

activ

ity119

Nuclear

proteinHcc-1

P82979

23.5

6.1

28.7

5.8

933

96Transcriptio

nregulatory

activ

ity120

DnaJhomolog

subfam

ilyCmem

ber8

O75937

29.8

9.0

30.0

8.4

937

73Chaperone

activ

ity121

Heterogeneous

nuclearribonucleoproteinA1

P09651

38.7

9.3

30.9

9.1

1033

87hnRNP

122

U1sm

allnu

clearribonu

cleoproteinA

P09

012

31.3

9.8

29.1

9.2

628

51hn

RNP

123

U2sm

allnu

clearribonu

cleoproteinA'

P09

661

28.4

8.7

27.9

8.4

834

110

hnRNP

124

DEK

Oncogene

P35

659

42.3

8.7

27.8

9.1

620

78Suspected

rolesin

human

carcinog

enesis,autoim

mune

disease,andviralinfection

125

Siah-interactingprotein

Q5R

371

26.2

8.3

27.6

7.8

1060

91Ubiquitin-specific

protease

activ

ity126,127

Eukaryotic


4HQ15056

25.2

7.8

27.8,25.3

5.8,

6.7

932

63Translatio

n12

8Exo

somecomplex

exon

ucleaseRRP41

Q9N

PD3

26.2

6.1

27.0

5.7

734

88Ribon

uclease

129


1Q07955

27.6

10.4

29.4

5.8

729

59Splicing

130

Microtubule-associatedproteinRP/EBfamily

mem

ber1

Q15691

29.8

5.0

30.0

5.0

526

60Signaltransductio

n131

Nucleophosm

inP06748

32.4

4.6

33.0

4.6

1136

90Multitasking,m

RNAprocessing

132

Proliferatingcellnu

clearantig

enP12

004

28.8

4.6

30.7

4.5

834

60Aux

iliaryproteinof

DNA

polymerasedelta

133

Nascent

polypeptide-associated

complex

alphasubunit

Q13

765

23.4

4.5

30.8

4.4

528

76Chaperone

activ

ity134,159

Com

plem

entcompo

nent

C1q

bind

ingprotein

Q07

021

31.3

4.7

29.9,29

.14.4,

4.4

626

45Chaperone

activ

ity135

Elongationfactor

1-beta

P24534

24.7

4.5

29.0

4.5

522

65Translatio

nelongatio

n13

614

-3-3

proteinzeta/delta

P63

104

29.9

4.7

27.2

4.6

629

73Cellcommun

ication


137,138

Breastcarcinom

aam

plifiedsequence

2variant

[Fragm

ent]

Q53

HE3

26.1

5.5

28.1,26

.95.0,

5.2

536

79Cellcommun

ation,

cellcycle

control

139

DNA-directedRNA

polymeraseII23

kDapolypeptide

P19388

24.6

5.7

25.6

5.5

950

144

RNA

polymerase

140

HMG-1

Q14321

25.0

5.8

24.8

5.8

1042

66Transcriptio

nregulatoractiv

ity14

1Perox

iredox

in1

Q06

830

19.0

6.4

24.4

8.2

944

124

Metabolism

142

Highmobility

groupprotein2

P26583

21.4

6.0

24.9

8.8

936

66Transcriptio

nregulatoractiv

ity14

3U2sm

allnu

clearribonu

cleoproteinB"

P08

579

44.6

9.7

26.8

9.3

528

50hn

RNP

144

Hom

eoboxproteinDUX3

Q9U

ND2

19.4

11.2

26.3

9.3

639

65Transcriptio

nfactor

activ

ity145

Peptid

yl-prolylcis-transisom

eraseB[Precursor]

P23284

22.6

9.2

22.0

9.3

1138

80Chaperone

activ

ity14

6PGBD5protein[Fragm

ent]

Q6P

JN2

14.8

9.5

18.1

8.5

438

47Unk

nown

147

Arginine-rich

protein

P55

145

20.2

8.7

17.8

9.2

522

78Unk

nown

148

Transcriptio

nfactor

BTF3

homolog

3Q13

892

22.2

9.2

22.2

6.9

758

106

Transcriptio

nfactor

activ

ity14

9Nucleic

acid

bind

ing

protein[Fragm

ent]

Q15

410

18.8

11.6

24.1

6.3

423

67Unk

nown

150

Hyp

othetical

protein

NIF3L

1BP1

Q6P

1L3

23.7

5.5

24.3

5.3

637

64Unk

nown

151

Eukaryotic


5AP63241

16.7

5.1

23.8

4.6

431

69Translatio

n152

Eukaryotic


1A,

Y-chrom

osom

alO14602

16.3

5.1

22.5

5.0

555

68Translatio

n

153

RNaseH1sm

allsubunit

Q8T

DP1

17.7

5.0

20.9

4.7

425

59RNA

processing

154

Eukaryotic

initiationfactor

5Aisoform

IvariantD

Q7L

7L3

16.8

5.1

18.4

4.9

538

66Translatio

n15

5Hun

tingtin

interactingproteinHYPK

[Fragm

ent]

O75

408

13.5

4.8

15.0

4.5

554

61Unk

nown

156

DNA-directedRNA

polymerases

I,II,andIII17.1

kDa

polypeptide

P52

434

17.1

4.5

15.3

4.4

649

72RNA

polymerase

157,158


P62310

11.7

4.6

12.1,10.6

4.5,

5.6

548

60m-RNA

processing

160

60SacidicribosomalproteinP2

P05387

11.7

4.4

13.2

4.4

349

58Translatio

n161


O95777

10.3

4.3

10.3

4.3

680

109

m-RNA

processing

162

Smallnu

clearribonu

cleoproteinF

P62

306

9.7

4.7

6.8

4.5

874

59hn

RNP

163

Enhancerof

rudimentary

homolog

P84

090

12.3

5.6

7.9

5.4

425

80Transcriptionregulatory

activity

164

Splicingfactor

3Bsubunit5

Q9B

WJ5

10.1

5.9

6.8

5.6

752

98Splicing

165

LSM2protein

Q6F

GG1

10.8

6.0

6.4

5.8

655

72m-RNA

processing

166

NHP2-lik

eprotein1

P55

769

14.2

8.7

11.4

9.1

631

54RNA

processing

167

Smallnu

clearribonu

cleoproteinSm

D2

P62

316

13.5

9.9

13.2

9.3

547

43Splicing

168,169

Smallnu

clearribonu

cleoproteinG

Q49

AN9

7.1

6.5

5.9,

6.0

7.6,

9.1

339

54hn

RNP


Fig. 4. Protein map for Raji whole cell lysate. Proteins were separated as forFig. 2.


good MS spectra and were positively identified. The spotswere derived from 124 unique primary protein species, withisoforms giving additional spots. The program PSORTpredicted 91% (n=113) of the identified proteins to benuclear. Only around 5% (n=6) of the total identified proteinswere predicted to be of non-nuclear origin (cytoplasmic ormitochondrial), and a further 4% (n=5) were predicted to becytoskeletal.

3.2. DNA-binding fraction of nuclear proteins

While 2-DE analysis of the nuclear fraction enabledidentification of many proteins, there was a lack of lowerabundance proteins, such as transcription factors. To “minedeeper” into the nuclear proteome, nuclear proteins werefractionated by chromatography on DNA-agarose. A nuclearprotein extract prepared as described above was loaded onto aDNA-agarose column and eluted with 1 M NaCl buffer (seeMaterials and methods). Proteins were quantified usingBradford reagent. Approximately 67% of the protein in thenuclear extract bound to the column and 33% passed through. Ina control experiment, 7% of the cytosolic fraction bound to thecolumn, 93% went straight through. No proteins were retainedfrom a whole membrane-enriched fraction obtained bysequential centrifugation. The DNA-binding protein fractionwas then separated by 2-DE (three biological replicates, Table 1,Fig. 3), a representative gel image is shown in Fig. 5. Imageanalysis detected approximately 200 spots, many in the basicpH region. Spots that could be visualized with Coomassie BlueG-250 (173) were excised, digested with trypsin, analyzed byMALDI-TOF MS and the PMF data were analyzed byMASCOT (Table 3). A total of 131 unique proteins wereidentified, 90 were known to be DNA- or RNA-associated, witha further 24 having tentative prediction of nucleic acidinteraction. Only 3.1% of the identified proteins were predictedto be of non-nuclear origin (PSORT), and a further 3.1% werecytoskeleton-associated (Fig. 4).

3.3. Comparison of the whole nuclear and DNA-bindingproteomes

Comparison of protein maps from 2-DE gels for the nuclearand DNA-binding fractions showed little similarity in posi-tions of the spots (Fig. 5). Of the most abundant 400 proteinspots that PDQuest detected in the whole nuclear fraction,32% were matched to protein spots in the DNA-bindingfraction. Due to the very different spot patterns of the twofractions, software matching did not generate useful identifica-tion of protein differences. On closer inspection, manyPDQuest matched spots were mismatched, or matches weremissed. Protein spots matched correctly using PDQuest aresummarized in Table 4. Since the gel images could not bereadily overlapped to determine differences, we identified allthe proteins visualized with Coomassie Blue G-250 in theDNA-binding fraction and compared the protein identificationsfor the two fractions. The two nuclear fractions hadsubstantially different protein compositions, with only 46

proteins common to both (Fig. 6). The whole nuclear proteomecontained 78 proteins not found in the DNA-bindingproteome, while the DNA-binding fraction contained 85proteins not detectable in the whole nuclear proteome. Intotal, 209 unique proteins were identified from the twofractions. Of the proteins identified in both fractions, manyDNA-associated proteins were substantially enriched in theDNA-agarose fraction (e. g. Exosome complex exonucleaseRRP41 enriched 8-fold, DEAD box protein 5 9-fold, Hcc-118-fold, PSP1 25-fold, SFPQ 36-fold and DEAD boxpolypeptide 17 104-fold). Actin was further reduced 10-foldcompared to the whole nuclear fraction (almost 100-foldreduction compared to the whole cell fraction), nucleophosminlevels were unchanged. To determine false positives in PMF-based protein identification, all generated peptide lists weresearched against a randomized version of the MSDB databasewith the same parameters for protein identification (asdescribed in Materials and methods), and an error rate of3.6% false positives was observed. The proteins identifiedwere grouped according to their functions (Fig. 7). In thewhole nuclear fraction, the most abundant groups were RNAprocessing proteins (15.5%), splicing proteins (14.7%),transcription-associated proteins (12.9%) and heterogeneousnuclear ribonucleoproteins (hnRNPs, 10.3%), a group mostlyinvolved in mRNA processing.

Regulatory proteins such as transcription factors may beundetectable using 2DE since they only present as a few copiesper cell, and are masked by more abundant proteins. To provethat such proteins are present in the nuclear fractions and areenriched, we used Western Blotting for the hematopoietictranscription factor PU.1 (Fig. 8). The results show that therewas a substantial enrichment in PU.1, but for many transcription

Fig. 5. Protein map for the DNA-binding fraction from Raji nuclei. Proteins were separated and identified as for Fig. 2 with the spot numbers listed in Table 3.


factors the concentrations were too low for detection. Whereasactin was significantly decreased in both the whole nuclearfraction and the DNA-binding fraction compared to the wholecell lysate, PU.1 was enriched around 6-fold in the nuclearfraction (Fig. 8, panels A and C) and 45-fold in the DNA-binding fraction (panel A).

The identification of a number of transcription-associatedproteins in the total nuclear fraction (Table 2) was encouraging,since some of these proteins are found at low abundance ineukaryotic cells. These proteins included the transcriptionfactors chromobox protein homologues 3 and 5, interleukinenhancer-binding factor 2, nuclear protein Hcc-1, CLE andmembers of the SWI/SNF-related matrix-associated actin-dependent regulator of chromatin sub-family. Furthermore,6.9% of the proteins from the whole nuclear fraction were ofunknown function (spots 1, 3, 4, 5, 6–8, 24, 55, 83–86 and 202).

For the proteins further purified by DNA-affinity chroma-tography, the most prevalent functional group was thetranscription-associated proteins (18.6%), consisting mainlyof lower abundance regulatory proteins. RNA processingproteins (14%) and hnRNPs (13.2%) were also common inthis fraction. Surprisingly, there was little difference in therelative proportions of the functional groupings seen from thetwo fractions, despite the high number of unique proteins fromeach fraction (Fig. 7). A major difference between the two

fractions was the identification of proteins involved intranslation in the DNA-binding fraction (9.3% compared to2.6% for the whole nuclear fraction). Previous studies withHeLa cells suggested that ribosome-associated and transla-tional proteins are abundant in the nucleus, mainly in nucleoli[24].

4. Discussion

A major challenge in proteomics is to separate and identifythe total complement of proteins in a complex mixture. Lowabundance proteins must be detected with the abundantproteins, requiring a wide ‘dynamic range’. The proteinconcentration of the sample must be increased to visualizelow abundance proteins by 2-DE, causing over-loading andpoor resolution of abundant proteins.

The eukaryotic nucleus is highly organized and dynamic, butnot well characterized. The nucleus is the site of DNAreplication and RNA transcription and processing; therefore itsinvestigation promises valuable insights into gene regulation.Study of the nucleus at the proteomic level is hampered byseveral factors. Firstly, regulatory proteins such as transcriptionfactors are usually present at only a few copies per cell.Secondly, while sub-cellular fractionation has been performed toenrich for proteins from several organelles and sub-cellular

Table 4Protein matching between the whole nuclear and the DNA-binding fractions following PDQuest analysis

Spot number(Fig. 2)

Protein name SWISS-PROTaccessionnumber

TheoreticalkDa

TheoreticalpI

CalculatedkDa (Fig. 2)

CalculatedpI (Fig. 2)

Spotnumber(Fig. 3)

CalculatedkDa(Fig. 3)

CalculatedpI(Fig. 3)

22 Splicing factor, proline- and glutamine-rich P23246 76.1 9.5 105.2 9.8 11 74.6 8.325 78 kDa glucose-regulated protein [Precursor] P11021 72.1 5.0 79.1 4.8 17 61.6 4.927 Heat shock cognate 71 kDa protein P11142 70.9 5.4 73.8 4.9 33 56.9 5.235–37 Heterogeneous nuclear ribonucleoprotein L P14866 60.1 6.7 64.4–64.7 6.4–6.8 38–40 53.2 6.4–6.842–44 Heterogeneous nuclear ribonucleoprotein K Q5T6W5 47.5 5.5 64.6–65.1 4.9 69–71 51.5–51.9 5.1–5.361 BAF53A protein Q6FI97 47.4 5.5 49.0 4.9 105 42.9 5.263 Histone-binding protein RBBP4 Q09028 47.5 4.7 53.6 4.7 79 45.7 4.666 Tubulin, beta polypeptide Q5JP53 47.7 4.7 52.7 4.8 80 44.8 4.881 Heterogeneous nuclear ribonucleoprotein D0 Q14103 35.9 9.0 45.4 6.8 89 37.4 7.2113, 114 Heterogeneous nuclear ribonucleoprotein

A2/B1P22626 37.4 9.0 35.7, 35.4 8.7, 9.2 93, 95 31.4, 31.3 8.2, 8.4

138 Nucleophosmin P06748 32.6 4.5 35.7 4.8 131 33.0 4.6140 Proliferating cell nuclear antigen P61258 28.8 4.6 32.6 4.7 132 30.7 4.5141 Complement component C1q binding protein Q07021 31.3 4.7 31.7 4.4 134 29.9 4.4165 Splicing factor, arginine/serine-rich 1 Q07955 27.6 10.4 30.9 4.9 129 29.4 5.8179 Exosome complex exonuclease RRP41 Q9NPD3 26.4 6.1 27.3 5.7 128 27.0 5.7180 Nuclear protein Hcc-1 P82979 23.5 6.1 29.1 6.1 119 28.7 5.8184 Small heterogeneous nuclear

ribonucleoprotein FP62306 9.7 4.7 9.7 4.7 162 6.8 4.5

198 60S acidic ribosomal protein P2B P05387 11.7 4.4 16.9 4.2 160 13.2 4.4199 U6 snRNA-associated Sm-like protein LSm8 O95777 10.3 4.3 13.9 4.2 161 10.3 4.3


structures, the purity of the resulting fractions has rarely beenverified. In this study, we used light microscopy with Giemsa–May–Grünwald staining to verify the purity and integrity of theisolated nuclei (Fig. 1) prior to protein solubilization, 2-DE (Fig.2) and protein identification byMS (Tables 2 and 3). In total, 209

Fig. 6. Venn diagram of the total nuclear and DNA-binding proteomes from Raji B-celproteome of which 46 were also found in the DNA binding fraction. An additional 8their respective SWISS-PROT primary accession numbers.

unique nuclear proteins were identified. Complementaryseparation procedures, including 2-DLC/tandem mass spectro-metry, would probably substantially increase the number ofproteins identified from the whole nuclear and DNA-bindingfractions.

l lymphoma. A total of 124 different proteins were identified in the whole nuclear5 proteins were only found in the DNA-binding fraction. Proteins are listed with

Fig. 7. Categories of proteins identified in Raji cell nuclei. (A) Total nuclear proteome and (B) the DNA-binding fraction. The 124 proteins in A and the 131 proteins inB are grouped according to molecular function.


The purity of these two nuclear fractions was also assessedusing PSORT to predict the sub-cellular location of theidentified proteins. Most of the identified proteins werepredicted to be nuclear (91.9%, n=192), indicating muchbetter enrichment than previously published nuclear proteomes[11,12,16–18,36]. The remaining 8.1% (n=17 proteins) wereabundant cytosolic proteins (e.g. heat shock proteins and78 kDa glucose-regulated protein [Grp78]) or cytoskeletalproteins (e.g. actin and tubulin). However, it is also possiblethat such proteins may shuttle to the nucleus under certaingrowth conditions, or their presence may be due to physicallinks between the nuclear lamina and the cytoskeleton [35].Previous studies have shown that actin is found in the nucleusand might be involved in important nuclear functions,including transcription initiation [36–38]. Also, Grp78 hasbeen reported in the nucleus [16,17]. Therefore, the purity of

our nuclear and DNA-binding fractions could be higher thanpredicted (Fig. 7).

For the DNA-binding protein fraction, only 6.2% (n=8) ofthe identified proteins were not allocated to the nucleus, 69.8%(n=91) were classical DNA/RNA-interacting proteins, afurther 18.6% had possible nucleic acid association. Lowabundance regulatory proteins, such as transcription factors,determine the genetic programs of cells. The analysis ofnuclear proteins was, in part, aimed at these low copy numberproteins. In the whole nuclear protein fraction, 18 differenttranscription-associated proteins were identified, includinginterleukin enhancer-binding factor 2, BAF53 protein, chromo-box protein homologues 3 and 5, enhancer of rudimentaryhomolog, RuvB-like 1, CLE, PQBP-1d, Hcc-1, structure-specific recognition protein 1, chromatin assembly factor 1subunit C and members of the SWI/SNF-related matrix-

Fig. 9. Protein maps for whole nuclei from leukemia and lymphoma cell lines. (A)lymphocytic leukemia) (C) HL-60 (acute myeloid leukemia) (D) Raji (Burkitt's lym

Fig. 8. Demonstration of enrichment for nuclear/DNA-binding proteins usingthe example of the hematopoietic transcription factor PU.1. Protein extracts wereassayed three times using the 2-D Quant protein assay (GE Healthcare,Piscataway, NJ, USA), and 5 μg of total protein was loaded for each fraction(panels A and B; WC=whole cell fraction, WN=whole nuclear fraction,DNA=DNA-binding fraction) onto a 10% SDS-PAGE gel before beingtransferred onto a PVDF membrane. After Western blotting using an antibodyagainst PU.1 (A), the membrane was stripped and blotted against actin (B).Panel C shows a comparison between the whole cell fraction and the wholenuclear fraction with increased loading of 10 μg per lane. PU.1 substantiallyincreased both from the whole cell fraction to the whole nuclear fraction (seepanels A and C), and was enriched around 40- to 50-fold in the DNA-bindingfraction (see panel A).


associated actin-dependent regulator of chromatin sub-family(Table 2). In the DNA-binding fraction, additional proteinspredicted to function in transcriptional events were identified,indicating that purification of DNA-binding proteins from thenuclear fraction enabled detection of lower abundanceregulatory nuclear proteins. Transcription related proteinsonly detected in the DNA-binding fraction included, RuvB-like 2, transcription elongation regulator 1, nuclear transcrip-tion factor Y subunit gamma, homeobox protein DUX3,interleukin enhancer-binding factor 3, transcription factorBTF3 homolog 3, Staphylococcal nuclease domain containingprotein 1, polyglutamine-binding protein 1, KHSRP protein,high mobility group protein 2, THO complex subunit 3 and farupstream element binding proteins 1 and 2. The DNA-bindingfraction contained more proteins of unknown function (8.5% ofall proteins identified).

The nucleus contains many potential disease markers [16],such as the oncoproteins identified in our nuclear 2-DE proteinmaps. Among those of interest as markers are SET, a nuclearprotein first discovered in acute undifferentiated leukemia [39]involved in transcription, apoptosis and hematopoietic differ-entiation [40]. The DEK oncogene, a multi-tasking DNA-binding protein suspected to play a role in carcinogenesis, isover-expressed in a variety of cancers such as retinoblastoma,

CCRF-CEM (T-cell acute lymphocytic leukemia) (B) MEC-1 (B-cell chronicphoma). Proteins (450 μg) were separated as for Fig. 2.


[41], malignant brain tumors [42], melanoma [43] and leukemia[44]. The proteins, RuvB-like 1 and 2, and BAF53, interact withthe transcription factor c-myc as co-factors for oncogenictransformation [45]. Antigen Ki-67 is a marker of humantumors used to determine proliferation rates in cancers,including leukemias [46,47]. Procedures described here foranalysis of the nuclear proteome will enable analysis of proteinsfollowing drug treatments of leukemias and lymphomas. Theseprocedures have also been applied to other leukemia cell lines ofvarious lineages to give similar protein maps (Fig. 9). Most ofthe differences between the leukemia cell lines were in proteinabundance (data not shown), the actual spot patterns weresimilar. Many of the 209 identified nuclear proteins wereinvolved in transcription and/or correlate with lypmphoma,leukemia or other cancers. The data generated may providebiomarkers and targets for cancer therapy, and further ourunderstanding of the molecular mechanisms underlying lym-phoma development and progression.

Acknowledgments

This work was facilitated by access to the AustralianProteome Analysis Facility Ltd. funded under the AustralianCommonwealth Government Major National Research Facil-ities (MNRF) Program. S.H. acknowledges support from theAustralian Department of Education, Science and Training,University of Sydney, and the DAAD Germany. Thanks to Dr.Louise Cole from the Electron Microscope Unit, University ofSydney, Australia, for assistance with the microscopy.

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The nuclear proteome and DNA-binding fraction of human Raji lymphoma cells