Proteomics Analysis of Ovarian Cancer Cell Lines and Tissues Reveals Drug Resistance-associated
Proteins
Isa N. Cruz1*,Helen M. Coley2*, Holger B.Kramer3,ThumuluruKavitah Madhuri2,4,Nur A.
M.Safuwan2,Ana Rita Angelino1 andMin Yang1
1
Department of Pharmaceutical & Biological Chemistry, UCL School of Pharmacy, London, WC1N1AX,
UK;
2
Faculty of Health and Medical Sciences, University of Surrey, Guildford, Surrey, GU2 7XH, UK;
3
Department of Physiology, Anatomy and Genetic, University of Oxford, South Parks Road, Oxford,
OX1 3PT, UK;
4
Section of Gynaecological Oncology, Royal Surrey County Hospital, Guildford, Surrey.
Correspondence to: Min Yang, Department of Pharmaceutical & Biological Chemistry, UCL School of
Pharmacy, London, WC1N1AX, UK. Tel.: +44 2077535876, e-mail: [email protected].
*These Authors contributed equally to this work
Notes
The Authors declare no competing financial interest.
Table S1 List of the primary antibodies used for target validation in the ovarian cancer study. The suppliers and
respective product codes, dilutions and blocking agents used for each antibody are also shown.
Antibody
Dilutio
n
Blocking
Agent
Supplier
(UK)
Product Code
Anti-14-3-3 antibody
1:1000
Milk
Abcam
ab9063
Anti-MMS2 antibody
1:100
Milk
Abcam
ab13906
Anti-PGAM1
antibody
1:500
Milk
Abcam
ab96622
Anti-TRX antibody
1:1000
Milk
Abcam
ab86255
1:1000
Milk
SigmaAldrich
SAB210262350UG
1:100
BSA
Abcam
ab1801
Anti-UBE2K
antibody
Anti-Actin antibody
(loading control)
Table S2 CarboRatio for Pathway analysis spectral counts
Protein
14-3-3 protein theta
14-3-3 protein zeta/delta
26S protease regulatory subunit 4
60S acidic ribosomal protein P0
Abhydrolase domain-containing protein 10, mitochondrial
Actin, cytoplasmic 1
Adenylosuccinate synthetase isozyme 2
ADP-sugar pyrophosphatase
A-kinase anchor protein 12
Annexin A5
Aspartate aminotransferase, mitochondrial
Calumenin
Cathepsin D
Chloride intracellular channel protein 4
Complement component 1 Q subcomponent-binding protein,
mitochondrial
EF-hand domain-containing protein D2
Elongation factor 1-gamma
Eukaryotic translation initiation factor 5A-1
Fatty acid-binding protein, epidermal
Galectin-1
Galectin-7
GIPC PDZ domain containing family, member 1
Glucosidase 2 subunit beta
Glutathione S-transferase omega-1
Glutathione S-transferase P
GTP-binding nuclear protein Ran
Guanine nucleotide-binding protein subunit beta-2-like 1
Hemoglobin subunit beta
High mobility group nucleosome-binding domain-containing
protein 5
Hypoxanthine-guanine phosphoribosyltransferase
Lactoylglutathione lyase
Macrophage-capping protein
Microtubule-associated protein 4
Microtubule-associated protein RP/EB family member 1
Myosin light polypeptide 6
Myristoylated alanine-rich C-kinase substrate
Nascent polypeptide-associated complex subunit alpha
Peroxiredoxin-6
Platelet-activating factor acetylhydrolase IB subunit gamma
Profilin-1
Accession
Number
1433T_HUMAN
1433Z_HUMAN
PRS4_HUMAN
RLA0_HUMAN
ABHDA_HUMAN
ACTB_HUMAN
(+1)
PURA2_HUMAN
NUDT5_HUMAN
AKA12_HUMAN
ANXA5_HUMAN
AATM_HUMAN
CALU_HUMAN
CATD_HUMAN
CLIC4_HUMAN
fold
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
C1QBP_HUMAN
EFHD2_HUMAN
EF1G_HUMAN
IF5A1_HUMAN
FABP5_HUMAN
LEG1_HUMAN
LEG7_HUMAN
GIPC1_HUMAN
GLU2B_HUMAN
GSTO1_HUMAN
GSTP1_HUMAN
RAN_HUMAN
GBLP_HUMAN
HBB_HUMAN
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
HMGN5_HUMAN
HPRT_HUMAN
LGUL_HUMAN
CAPG_HUMAN
MAP4_HUMAN
MARE1_HUMAN
MYL6_HUMAN
MARCS_HUMAN
NACA_HUMAN
PRDX6_HUMAN
PA1B3_HUMAN
PROF1_HUMAN
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
Prohibitin
Prohibitin-2
Proteasome subunit alpha type-3
Proteasome subunit alpha type-6
Proteasome subunit beta type-3
Proteasome subunit beta type-7
Protein DJ-1
Protein S100-A7
Protein SET
Ran-specific GTPase-activating protein
Rho GDP-dissociation inhibitor 1
Serine protease HTRA2, mitochondrial
Serine/arginine-rich splicing factor 1
Serpin B4
Single-stranded DNA-binding protein, mitochondrial
Superoxide dismutase [Mn], mitochondrial
Thioredoxin
Ubiquitin-conjugating enzyme E2 K
UMP-CMP kinase
Uroporphyrinogen decarboxylase
UV excision repair protein RAD23 homolog B
Voltage-dependent anion-selective channel protein 2
L-lactate dehydrogenase B chain
Proliferation-associated protein 2G4
Annexin A1
Heterogeneous nuclear ribonucleoprotein H3
Protein S100-A9
Serpin B3
14-3-3 protein beta/alpha
Alcohol dehydrogenase 1B
Alpha-1-antichymotrypsin
Annexin A3
Coiled-coil-helix-coiled-coil-helix domain-containing protein 3,
mitochondrial
Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial
DnaJ homolog subfamily C member 9
Enoyl-CoA hydratase, mitochondrial
F-actin-capping protein subunit beta
G-rich sequence factor 1
Heat shock protein beta-1
Histidine-tRNA ligase, cytoplasmic
Leukocyte elastase inhibitor
Malate dehydrogenase, cytoplasmic
Nitrilase homolog 1
Nucleophosmin
Nucleoside diphosphate kinase A
PHB_HUMAN
PHB2_HUMAN
PSA3_HUMAN
PSA6_HUMAN
PSB3_HUMAN
PSB7_HUMAN
PARK7_HUMAN
S10A7_HUMAN
SET_HUMAN
RANG_HUMAN
GDIR1_HUMAN
HTRA2_HUMAN
SRSF1_HUMAN
SPB4_HUMAN
SSBP_HUMAN
SODM_HUMAN
THIO_HUMAN
UBE2K_HUMAN
KCY_HUMAN
DCUP_HUMAN
RD23B_HUMAN
VDAC2_HUMAN
LDHB_HUMAN
PA2G4_HUMAN
ANXA1_HUMAN
HNRH3_HUMAN
S10A9_HUMAN
SPB3_HUMAN
1433B_HUMAN
ADH1B_HUMAN
AACT_HUMAN
ANXA3_HUMAN
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-5.1
-3.6
-2.9
-2.7
-2.1
-1.1
1.0
1.0
1.0
1.0
CHCH3_HUMAN
ECH1_HUMAN
DNJC9_HUMAN
ECHM_HUMAN
CAPZB_HUMAN
GRSF1_HUMAN
HSPB1_HUMAN
SYHC_HUMAN
ILEU_HUMAN
MDHC_HUMAN
NIT1_HUMAN
NPM_HUMAN
NDKA_HUMAN
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Peptidyl-prolyl cis-trans isomerase FKBP4
Peroxiredoxin-5, mitochondrial
Poly(rC)-binding protein 2
Proteasome subunit alpha type-1
Proteasome subunit alpha type-7
Proteasome subunit beta type-6
Purine nucleoside phosphorylase
Translationally-controlled tumor protein
Tropomyosin alpha-3 chain
FKBP4_HUMAN
PRDX5_HUMAN
PCBP2_HUMAN
PSA1_HUMAN
PSA7_HUMAN
PSB6_HUMAN
PNPH_HUMAN
TCTP_HUMAN
TPM3_HUMAN
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Tubulin beta-6 chain
Voltage-dependent anion-selective channel protein 1
Adenosylhomocysteinase
TBB6_HUMAN
VDAC1_HUMAN
SAHH_HUMAN
EF1A1_HUMAN
(+1)
EF1D_HUMAN
CALR_HUMAN
NP1L1_HUMAN
HNRPC_HUMAN
ENOA_HUMAN
DESP_HUMAN
KTHY_HUMAN
TPIS_HUMAN
IF4A1_HUMAN
PRDX1_HUMAN
S10A8_HUMAN
HNRPD_HUMAN
VIME_HUMAN
PRDX2_HUMAN
PGK1_HUMAN
ALBU_HUMAN
TAGL2_HUMAN
HNRH1_HUMAN
PDIA3_HUMAN
TBA1A_HUMAN
ALDOA_HUMAN
ROA2_HUMAN
MDHM_HUMAN
G3P_HUMAN
TBB5_HUMAN
PEPD_HUMAN
COF1_HUMAN
H2B1M_HUMAN
(+7)
PPIA_HUMAN
UB2V2_HUMAN
1.0
1.0
1.1
Elongation factor 1-alpha 1
Elongation factor 1-delta
Calreticulin
Nucleosome assembly protein 1-like 1
Heterogeneous nuclear ribonucleoproteins C1/C2
Alpha-enolase
Desmoplakin
Thymidylate kinase
Triosephosphate isomerase
Eukaryotic initiation factor 4A-I
Peroxiredoxin-1
Protein S100-A8
Heterogeneous nuclear ribonucleoprotein D0
Vimentin
Peroxiredoxin-2
Phosphoglycerate kinase 1
Serum albumin
Transgelin-2
Heterogeneous nuclear ribonucleoprotein H
Protein disulfide-isomerase A3
Tubulin alpha-1A chain
Fructose-bisphosphate aldolase A
Heterogeneous nuclear ribonucleoproteins A2/B1
Malate dehydrogenase, mitochondrial
Glyceraldehyde-3-phosphate dehydrogenase
Tubulin beta chain
Xaa-Pro dipeptidase
Cofilin-1
Histone H2B type 1-M
Peptidyl-prolyl cis-trans isomerase A
Ubiquitin-conjugating enzyme E2 variant 2
2.4
4.2
4.6
4.6
8.6
8.6
8.9
10.5
12.3
13.1
14.5
20.8
21.1
23.9
27.1
29.2
44.3
57.9
73.8
76.3
76.3
86.9
109.5
109.5
111.2
153.9
178.5
417.2
679.7
679.7
679.7
60 kDa heat shock protein, mitochondrial
78 kDa glucose-regulated protein
Adenylyl cyclase-associated protein 1
Aldehyde dehydrogenase family 1 member A3
Aldehyde dehydrogenase X, mitochondrial
Alpha-actinin-4
Annexin A11
Annexin A2
Apolipoprotein A-I
ATP synthase subunit alpha, mitochondrial
Bifunctional purine biosynthesis protein PURH
C-1-tetrahydrofolate synthase, cytoplasmic
Caprin-1
Cytosol aminopeptidase
D-3-phosphoglycerate dehydrogenase
Dihydrolipoyllysine-residue succinyltransferase component of 2oxoglutarate dehydrogenase complex, mitochondrial
Elongation factor Tu, mitochondrial
Endoplasmin
Far upstream element-binding protein 1
Fumarate hydratase, mitochondrial
Heat shock 70 kDa protein 1A/1B
Heat shock cognate 71 kDa protein
Heterogeneous nuclear ribonucleoprotein F
Heterogeneous nuclear ribonucleoprotein K
Heterogeneous nuclear ribonucleoprotein L
Heterogeneous nuclear ribonucleoprotein Q
Ig gamma-1 chain C region
Inosine-5'-monophosphate dehydrogenase 2
Isoform 2 of Shootin-1
Lamin-B1
LIM and SH3 domain protein 1
L-lactate dehydrogenase A chain
Neutral alpha-glucosidase AB
Nuclear autoantigenic sperm protein
Nucleolin
Nucleoporin p54
Paraspeckle component 1
PDZ and LIM domain protein 1
Phosphoglycerate mutase 1
Poly(rC)-binding protein 1
Polymerase I and transcript release factor
Prelamin-A/C
Proliferating cell nuclear antigen
Protein disulfide-isomerase
CH60_HUMAN
GRP78_HUMAN
CAP1_HUMAN
AL1A3_HUMAN
AL1B1_HUMAN
ACTN4_HUMAN
ANX11_HUMAN
ANXA2_HUMAN
APOA1_HUMAN
ATPA_HUMAN
PUR9_HUMAN
C1TC_HUMAN
CAPR1_HUMAN
AMPL_HUMAN
SERA_HUMAN
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
ODO2_HUMAN
EFTU_HUMAN
ENPL_HUMAN
FUBP1_HUMAN
FUMH_HUMAN
HSP71_HUMAN
HSP7C_HUMAN
HNRPF_HUMAN
HNRPK_HUMAN
HNRPL_HUMAN
HNRPQ_HUMAN
IGHG1_HUMAN
IMDH2_HUMAN
sp|A0MZ662|SHOT1_HUMA
N (+5)
LMNB1_HUMAN
LASP1_HUMAN
LDHA_HUMAN
GANAB_HUMAN
NASP_HUMAN
NUCL_HUMAN
NUP54_HUMAN
PSPC1_HUMAN
PDLI1_HUMAN
PGAM1_HUMAN
PCBP1_HUMAN
PTRF_HUMAN
LMNA_HUMAN
PCNA_HUMAN
PDIA1_HUMAN
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
Pyruvate kinase isozymes M1/M2
Serine hydroxymethyltransferase, mitochondrial
Serum deprivation-response protein
Stress-70 protein, mitochondrial
Stress-induced-phosphoprotein 1
T-complex protein 1 subunit beta
T-complex protein 1 subunit eta
T-complex protein 1 subunit theta
Transitional endoplasmic reticulum ATPase
tRNA-splicing ligase RtcB homolog
Tropomyosin alpha-1 chain
Tryptophan-tRNA ligase, cytoplasmic
Tubulin alpha-1B chain
Tubulin alpha-4A chain
UDP-glucose 6-dehydrogenase
KPYM_HUMAN
GLYM_HUMAN
SDPR_HUMAN
GRP75_HUMAN
STIP1_HUMAN
TCPB_HUMAN
TCPH_HUMAN
TCPQ_HUMAN
TERA_HUMAN
RTCB_HUMAN
TPM1_HUMAN
SYWC_HUMAN
TBA1B_HUMAN
TBA4A_HUMAN
UGDH_HUMAN
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
Table S3 TaxolRatio for Pathway analysis spectral counts
Protein
Abhydrolase domain-containing protein 10, mitochondrial
Adenosylhomocysteinase
Adenylosuccinate synthetase isozyme 2
ADP-sugar pyrophosphatase
A-kinase anchor protein 12
Aspartate aminotransferase, mitochondrial
Desmoplakin
EF-hand domain-containing protein D2
Elongation factor 1-alpha 1
Elongation factor 1-delta
Elongation factor 1-gamma
Eukaryotic initiation factor 4A-I
Eukaryotic translation initiation factor 5A-1
Fructose-bisphosphate aldolase A
GIPC PDZ domain containing family, member 1
Glucosidase 2 subunit beta
Hemoglobin subunit beta
Heterogeneous nuclear ribonucleoprotein D0
Heterogeneous nuclear ribonucleoproteins A2/B1
Heterogeneous nuclear ribonucleoproteins C1/C2
High mobility group nucleosome-binding domain-containing protein 5
Malate dehydrogenase, mitochondrial
Microtubule-associated protein 4
Microtubule-associated protein RP/EB family member 1
Myristoylated alanine-rich C-kinase substrate
Phosphoglycerate kinase 1
Accession
Number
ABHDA_HUMAN
SAHH_HUMAN
PURA2_HUMAN
NUDT5_HUMAN
AKA12_HUMAN
AATM_HUMAN
DESP_HUMAN
EFHD2_HUMAN
EF1A1_HUMAN
(+1)
EF1D_HUMAN
EF1G_HUMAN
IF4A1_HUMAN
IF5A1_HUMAN
ALDOA_HUMAN
GIPC1_HUMAN
GLU2B_HUMAN
HBB_HUMAN
HNRPD_HUMAN
ROA2_HUMAN
HNRPC_HUMAN
HMGN5_HUMAN
MDHM_HUMAN
MAP4_HUMAN
MARE1_HUMAN
MARCS_HUMAN
PGK1_HUMAN
Fold
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-1000
Prohibitin-2
Proliferation-associated protein 2G4
Serine protease HTRA2, mitochondrial
Single-stranded DNA-binding protein, mitochondrial
Superoxide dismutase [Mn], mitochondrial
Transgelin-2
Uroporphyrinogen decarboxylase
Annexin A5
Protein S100-A7
Fatty acid-binding protein, epidermal
Serpin B3
Complement component 1 Q subcomponent-binding protein,
mitochondrial
78 kDa glucose-regulated protein
Alcohol dehydrogenase 1B
Aldehyde dehydrogenase family 1 member A3
Aldehyde dehydrogenase X, mitochondrial
Alpha-1-antichymotrypsin
Alpha-actinin-4
Annexin A11
Apolipoprotein A-I
ATP synthase subunit alpha, mitochondrial
Bifunctional purine biosynthesis protein PURH
C-1-tetrahydrofolate synthase, cytoplasmic
Caprin-1
Cytosol aminopeptidase
D-3-phosphoglycerate dehydrogenase
Dihydrolipoyllysine-residue succinyltransferase component of 2oxoglutarate dehydrogenase complex, mitochondrial
Endoplasmin
Far upstream element-binding protein 1
Fumarate hydratase, mitochondrial
Heat shock 70 kDa protein 1A/1B
Heat shock cognate 71 kDa protein
Heterogeneous nuclear ribonucleoprotein F
Heterogeneous nuclear ribonucleoprotein L
Heterogeneous nuclear ribonucleoprotein Q
Histidine-tRNA ligase, cytoplasmic
Inosine-5'-monophosphate dehydrogenase 2
Isoform 2 of Shootin-1
Lamin-B1
LIM and SH3 domain protein 1
Neutral alpha-glucosidase AB
Nuclear autoantigenic sperm protein
Nucleoporin p54
PHB2_HUMAN
PA2G4_HUMAN
HTRA2_HUMAN
SSBP_HUMAN
SODM_HUMAN
TAGL2_HUMAN
DCUP_HUMAN
ANXA5_HUMAN
S10A7_HUMAN
FABP5_HUMAN
SPB3_HUMAN
-1000
-1000
-1000
-1000
-1000
-1000
-1000
-38.6
-4.9
-4.2
-4.2
C1QBP_HUMAN
GRP78_HUMAN
ADH1B_HUMAN
AL1A3_HUMAN
AL1B1_HUMAN
AACT_HUMAN
ACTN4_HUMAN
ANX11_HUMAN
APOA1_HUMAN
ATPA_HUMAN
PUR9_HUMAN
C1TC_HUMAN
CAPR1_HUMAN
AMPL_HUMAN
SERA_HUMAN
-1.4
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
ODO2_HUMAN
ENPL_HUMAN
FUBP1_HUMAN
FUMH_HUMAN
HSP71_HUMAN
HSP7C_HUMAN
HNRPF_HUMAN
HNRPL_HUMAN
HNRPQ_HUMAN
SYHC_HUMAN
IMDH2_HUMAN
sp|A0MZ662|SHOT1_HUMA
N (+5)
LMNB1_HUMAN
LASP1_HUMAN
GANAB_HUMAN
NASP_HUMAN
NUP54_HUMAN
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Paraspeckle component 1
PDZ and LIM domain protein 1
Prelamin-A/C
Pyruvate kinase isozymes M1/M2
Serine hydroxymethyltransferase, mitochondrial
Serum deprivation-response protein
Stress-70 protein, mitochondrial
Stress-induced-phosphoprotein 1
T-complex protein 1 subunit beta
T-complex protein 1 subunit eta
Transitional endoplasmic reticulum ATPase
UDP-glucose 6-dehydrogenase
Voltage-dependent anion-selective channel protein 1
Nascent polypeptide-associated complex subunit alpha
Alpha-enolase
Proteasome subunit beta type-3
Protein DJ-1
Platelet-activating factor acetylhydrolase IB subunit gamma
Glutathione S-transferase omega-1
Proteasome subunit beta type-7
Calumenin
Protein S100-A9
Galectin-7
Protein SET
PSPC1_HUMAN
PDLI1_HUMAN
LMNA_HUMAN
KPYM_HUMAN
GLYM_HUMAN
SDPR_HUMAN
GRP75_HUMAN
STIP1_HUMAN
TCPB_HUMAN
TCPH_HUMAN
TERA_HUMAN
UGDH_HUMAN
VDAC1_HUMAN
NACA_HUMAN
ENOA_HUMAN
PSB3_HUMAN
PARK7_HUMAN
PA1B3_HUMAN
GSTO1_HUMAN
PSB7_HUMAN
CALU_HUMAN
S10A9_HUMAN
LEG7_HUMAN
SET_HUMAN
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.9
2.0
2.2
2.2
2.3
2.6
2.6
2.7
3.0
3.4
4.1
Serum albumin
Hypoxanthine-guanine phosphoribosyltransferase
Peroxiredoxin-6
Proteasome subunit alpha type-6
L-lactate dehydrogenase B chain
60S acidic ribosomal protein P0
Serpin B4
Annexin A1
Glyceraldehyde-3-phosphate dehydrogenase
Ran-specific GTPase-activating protein
Rho GDP-dissociation inhibitor 1
Calreticulin
Nucleosome assembly protein 1-like 1
UV excision repair protein RAD23 homolog B
14-3-3 protein zeta/delta
14-3-3 protein theta
Protein S100-A8
Serine/arginine-rich splicing factor 1
Proteasome subunit alpha type-3
GTP-binding nuclear protein Ran
Prohibitin
ALBU_HUMAN
HPRT_HUMAN
PRDX6_HUMAN
PSA6_HUMAN
LDHB_HUMAN
RLA0_HUMAN
SPB4_HUMAN
ANXA1_HUMAN
G3P_HUMAN
RANG_HUMAN
GDIR1_HUMAN
CALR_HUMAN
NP1L1_HUMAN
RD23B_HUMAN
1433Z_HUMAN
1433T_HUMAN
S10A8_HUMAN
SRSF1_HUMAN
PSA3_HUMAN
RAN_HUMAN
PHB_HUMAN
4.7
4.9
4.9
4.9
5.3
5.5
6.2
6.7
6.8
6.9
6.9
7.0
7.0
7.0
7.8
8.3
8.4
8.4
9.4
9.6
9.8
Thymidylate kinase
Chloride intracellular channel protein 4
Heterogeneous nuclear ribonucleoprotein H3
Macrophage-capping protein
Triosephosphate isomerase
Actin, cytoplasmic 1
Cathepsin D
Peroxiredoxin-1
Vimentin
26S protease regulatory subunit 4
Peroxiredoxin-2
Protein disulfide-isomerase A3
Tubulin alpha-1A chain
Lactoylglutathione lyase
Glutathione S-transferase P
Galectin-1
Thioredoxin
Profilin-1
Heterogeneous nuclear ribonucleoprotein H
Myosin light polypeptide 6
UMP-CMP kinase
Ubiquitin-conjugating enzyme E2 K
Tubulin beta chain
Voltage-dependent anion-selective channel protein 2
Xaa-Pro dipeptidase
Histone H2B type 1-M
Peptidyl-prolyl cis-trans isomerase A
Ubiquitin-conjugating enzyme E2 variant 2
Guanine nucleotide-binding protein subunit beta-2-like 1
Cofilin-1
14-3-3 protein beta/alpha
60 kDa heat shock protein, mitochondrial
Adenylyl cyclase-associated protein 1
Annexin A2
Annexin A3
Coiled-coil-helix-coiled-coil-helix domain-containing protein 3,
mitochondrial
Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase, mitochondrial
DnaJ homolog subfamily C member 9
Elongation factor Tu, mitochondrial
Enoyl-CoA hydratase, mitochondrial
F-actin-capping protein subunit beta
G-rich sequence factor 1
Heat shock protein beta-1
Heterogeneous nuclear ribonucleoprotein K
KTHY_HUMAN
CLIC4_HUMAN
HNRH3_HUMAN
CAPG_HUMAN
TPIS_HUMAN
ACTB_HUMAN
(+1)
CATD_HUMAN
PRDX1_HUMAN
VIME_HUMAN
PRS4_HUMAN
PRDX2_HUMAN
PDIA3_HUMAN
TBA1A_HUMAN
LGUL_HUMAN
GSTP1_HUMAN
LEG1_HUMAN
THIO_HUMAN
PROF1_HUMAN
HNRH1_HUMAN
MYL6_HUMAN
KCY_HUMAN
UBE2K_HUMAN
TBB5_HUMAN
VDAC2_HUMAN
PEPD_HUMAN
H2B1M_HUMAN
(+7)
PPIA_HUMAN
UB2V2_HUMAN
GBLP_HUMAN
COF1_HUMAN
1433B_HUMAN
CH60_HUMAN
CAP1_HUMAN
ANXA2_HUMAN
ANXA3_HUMAN
CHCH3_HUMAN
ECH1_HUMAN
DNJC9_HUMAN
EFTU_HUMAN
ECHM_HUMAN
CAPZB_HUMAN
GRSF1_HUMAN
HSPB1_HUMAN
HNRPK_HUMAN
10.4
11.7
12.6
12.8
16.2
16.9
16.9
19.4
19.5
24.4
27.8
37.3
37.3
37.9
42.0
50.1
50.1
53.9
57.9
64.6
70.2
78.1
80.4
148.4
162.4
167.4
167.4
167.4
245.7
268.5
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
Ig gamma-1 chain C region
Leukocyte elastase inhibitor
L-lactate dehydrogenase A chain
Malate dehydrogenase, cytoplasmic
Nitrilase homolog 1
Nucleolin
Nucleophosmin
Nucleoside diphosphate kinase A
Peptidyl-prolyl cis-trans isomerase FKBP4
Peroxiredoxin-5, mitochondrial
Phosphoglycerate mutase 1
Poly(rC)-binding protein 1
Poly(rC)-binding protein 2
Polymerase I and transcript release factor
Proliferating cell nuclear antigen
Proteasome subunit alpha type-1
Proteasome subunit alpha type-7
Proteasome subunit beta type-6
Protein disulfide-isomerase
Purine nucleoside phosphorylase
T-complex protein 1 subunit theta
Translationally-controlled tumor protein
tRNA-splicing ligase RtcB homolog
Tropomyosin alpha-1 chain
Tropomyosin alpha-3 chain
Tryptophan-tRNA ligase, cytoplasmic
Tubulin alpha-1B chain
Tubulin alpha-4A chain
Tubulin beta-6 chain
IGHG1_HUMAN
ILEU_HUMAN
LDHA_HUMAN
MDHC_HUMAN
NIT1_HUMAN
NUCL_HUMAN
NPM_HUMAN
NDKA_HUMAN
FKBP4_HUMAN
PRDX5_HUMAN
PGAM1_HUMAN
PCBP1_HUMAN
PCBP2_HUMAN
PTRF_HUMAN
PCNA_HUMAN
PSA1_HUMAN
PSA7_HUMAN
PSB6_HUMAN
PDIA1_HUMAN
PNPH_HUMAN
TCPQ_HUMAN
TCTP_HUMAN
RTCB_HUMAN
TPM1_HUMAN
TPM3_HUMAN
SYWC_HUMAN
TBA1B_HUMAN
TBA4A_HUMAN
TBB6_HUMAN
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
Table S4 Protein fold regulation using two methods of spot intensity analysis. Spot intensities considering and
ignoring the spot position on the gel were used to determine the number of up-regulated, down-regulated and
unchanged/inexistent proteins in the resistant samples, when compared to the sensitive samples.
Considering Spot Position
Fold Regulation
Not found in
resistant
Down-regulated in
resistant
No change/Not
found
Up-regulated in
resistant
Not found in
sensitive
Total
Ignoring Spot Position
TaxR/S
ens
CarbR/
Sens
SOV-2/
SOV-3
TaxR/S
ens
CarbR/S
ens
SOV-2/
SOV-3
-1000
33
63
10
31
59
10
<0
5
6
2
6
7
4
1
42
24
21
42
24
21
>1
65
34
3
69
40
1
+1000
40
58
2
37
55
2
185
185
38
185
185
38
Table S5 Top
5 canonical pathways most related to the genes that codify for the proteins
in the analysed datasets. Protein fold change was calculated using spot intensity data of
the proteins identified in the sensitive and resistant ovarian cancer cell lines. Pathway
analysis was performed using IPA software. Statistical significance was determined by using
Fisher’s exact test (P<0.001). Arrows next to each gene name indicate up- or downregulation, = indicates no change or not found.
Proteins
Ingenuity Canonical
Pathway
Pvalue
Ratio
Glycolysis I
<
0.001
7/22
(0.318)
Gluconeogenesis I
<
0.001
7/22
(0.318)
Protein Ubiquitination
Pathway
<
0.001
16/263
(0.061)
Remodelling of
Epithelial Adherens
Junctions
<
0.001
9/66
(0.136)
14-3-3-mediated
Signalling
<
0.001
10/118
(0.085)
TaxR
CarbR
ALDOA, TPIS, G3P, PGK1,
PGAM1, ENOA, KPYM=
MDHC, MDHM, ENOA,
PGAM1, PGK1, G3P,
ALDOA
UB2V2, DNJC9, ENPL=,
CH60, GRP75=, GRP78=,
HSPB1, HSP7C=, PSA1,
PSA3, PSA6, PSA7, PSB3,
PSB6, PSB7, PRS4
TBA1A, TBA1B, TBA4A,
TBB5, TBB6, ACTB,
ACTN4=, NDKA, MARE1
PDIA3, VIME, 1433B,
1433T, 1433Z, TBA1A,
TBA1B, TBA4A, TBB5,
TBB6
ALDOA, TPIS, G3P, PGK1,
PGAM1, ENOA, KPYM
MDHC=, MDHM, ENOA,
PGAM1, PGK1, G3P,
ALDOA
UB2V2, DNJC9=, ENPL,
CH60, GRP75, GRP78,
HSPB1=, HSP7C, PSA1=,
PSA3, PSA6, PSA7=, PSB3,
PSB6=, PSB7, PRS4
TBA1A, TBA1B, TBA4A,
TBB5, TBB6=, ACTB,
ACTN4, NDKA=, MARE1
PDIA3, VIME, 1433B=,
1433T, 1433Z, TBA1A,
TBA1B, TBA4A, TBB5,
TBB6=
Selection of protein hits
Mass spectrometry data were used for the protein identification searches in two ways. Firstly,
database searches were performed manually using two online search engines, MASCOT (Matrix
Science) (1) and X!Tandem (The GPM) (2). In order to minimise the chances of false-positive
identification of contaminant proteins, protein hits were accepted if:
MASCOT protein score > 100 and at least one of the theoretical MW and pI values matched the
experimental value. Experimental MW and pI were estimated based on relative positions to MW
markers and corresponding vertical alignment to pH values on 2D gels.
OR
20 < MASCOT protein score < 100 and the theoretical MW and pI values were both similar to the
experimental values.
OR
20 < MASCOT protein score < 100 and at least one of the theoretical MW and pI values was similar to
the experimental values.
AND
First hits in the MASCOT search result corresponded to the same first hits in the X!Tandem search
results or the protein identified belonged to the same family (isoforms).
The criteria used for protein hit selection was: Min Peptide – 95 %, Min # Peptides – 2, Min Protein –
99 % and 1 % false discovery rate (FDR) (3, 4). This search method resulted in a list of 189 proteins
reliably identified.
Use of ovarian cancer cells lines
PEO1
PEO1 TaxR
PEO1 CarbR
Figure S1 Representative 2D-PAGE silver stained gels of the ovarian cancer cell lines PEO1, PEO1
TaxR, PEO1 CarbR. Proteins (approximately 50 µg) were separated on pH 3-10 non-linear 11 cm IPG
strips in the first dimension (IEF) and by precast gels (Criterion TGX Precast Gels, any kD, IPG + 1 well
comb, 11 cm IPG strip) in the second dimension (SDS-PAGE). Resulting gels were silver stained. Differentially
expressed proteins were determined by 2D image analysis using PDQuest Advanced 8.0.1.
Gel image analysis of the PEO1 cell lines
PEO1 TaxR
PEO1
PEO1 CarbR
Figure S2 Spots in the ovarian cancer cell line gels that were selected for excision and further analysis by LC-MS/MS.
Only those spots that were exclusively presence, 2 fold changes above (by PDQuest Advanced 8.0.1) were selected for
excision and further analysis by LC-MS/MS.
Figure S3. Protein Venn diagram. The diagram shows the number of proteins that have been
identified in each category (sensitive, partial and resistant) and in the overlap between
categories. Sensitive category includes PEO1 cancer cell line. Resistant category includes
PEO1 TaxR and PEO1 CarbR cancer cell lines.
pH 10
pH 3
1 - Prohibitin
1
2
2 – Glutathione S-transferase P
3
3 – Triosephosphate isomerase
PEO1
pH 3
pH 10
4
4 – Alpha-enolase
PEO1 CarbR
Figure S4. Examples of proteins that were identified in more than one spot on the same gel and the respective spots
where they were identified.
To avoid errors in the interpretation of spot intensity data, which would influence the analysis of
differences in protein expression between distinct samples, spot intensity was studied using two
different approaches. Firstly, considering the spot position on the gel, where only spots located in
the same position on different gels, in which a certain protein was identified, were used for
quantitative purposes. Theoretically, spots located in the same place on different gels correspond to
the same protein and are given the same SSP number. In this case, only spots with the same SSP
number were compared between samples.
Secondly, ignoring the spot position on the gel, where all the spots on one gel, in which a certain
protein was identified, were considered for quantitation purposes. In this case, the total presence of
a protein in a gel or sample was considered as a sum of the intensities of all the spots in which this
protein was identified. The total intensity values were then compared between samples.
With the aim of identifying differentially expressed proteins that could possibly be related to
resistance, for either of the semi-quantitative approaches described, fold regulation was calculated
for each protein using the corresponding spot intensity data as follows:
Quantity value of the spot/protein in the resistant sample
Quantity value of the spot/protein in the sensitive (control) sample
Five results were possible:
Fold regulation = -1000 (or 0) – not found in the resistant sample considered
Fold regulation < 0 (or >0, <1) – down-regulated in the resistant sample
Fold regulation = 1 – no change in expression or not found in any of the samples considered
Fold regulation > 1 – up-regulated in the resistant sample
Fold regulation = +1000 – not found in the sensitive sample
Fold regulation was validated by confirmation with the gel images. As an example, for a protein
identified in the three cell line samples, if that protein was up-regulated in the resistant cell lines
(TaxR and CarbR), then the corresponding spots on the resistant gels should be more intense than
the one on the sensitive gel. Nevertheless, for proteins that were identified in many spots on the
same gel/sample, this confirmation was not possible.
shows the number of up-regulated, down-regulated and unchanged/inexistent proteins in the
resistant samples, when compared to the sensitive samples, for the cell line and tissue proteins. This
determination was done using spot intensity data considering and ignoring the spot position on the
gel.
TaxR vs Sensitive
Pathways
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
CarbR vs Sensitive
Pathways
P1
P2
P3
P4
P5
P6
P7
P8
P9
P10
P11
P12
P13
P14
P15
Figure S5. Stacked bar charts illustrating the pathways most associated with the
genes that codify for the proteins in the analysed datasets. Canonical pathways are
displayed along the x-axis. P1, Glycolysis I; P2, Gluconeagenesis I; P3, Protein
Ubiquitination Pathway; P4, Remodelling of Epithelial Adherens Junctions; P5, 14-3-3mediated Signaling; P6, NRF2-mediated Oxidative Stress Response; P7, Aspartate
Degradation II; P8, Aldosterone Signalling in Epithelial Cells; P9, TCA Cycle II (Eukaryotic);
P10, Guanine and Guanosine Salvage I; P11, Purine Nucleotides De Novo Biosynthesis II;
P12, Epithelial Adherens Junction Signalling; P13, Role of IL-17A in psoriasis; P14, Aryl
Hydrocarbon Receptor Signalling; P15, Arsenate Detoxification I (Glutaredoxin). The y-axis
displays the percentage of the number of molecules identified in a given pathway, relative to
the total number of molecules that make up that pathway. The line graph shows -log (pvalue), indicating the overlap of the analysis with the canonical pathway. The colours of the
bars display the number of up-regulated (red), down-regulated (green), and unchanged
molecules (grey) in each canonical pathway for the TaxR vs Sensitive and CarbR vs
Sensitive datasets, respectively(5). Pathway analysis was performed using IPA software.
Figure S6 TaxR top 5 pathways
Figure S7 CarbR Top 5 pathways
Five human epithelial ovarian tumours (adenocarcinomas) tissue samples (SOV-1/2/3/4/5)
were collected and analyzed.These tissue samples were analyzed by 2D-GE and spots were
selected according to the following criteria: exclusive presence in only one gel, 2-fold and 5fold change in intensity in at least one of the gels when compared to the others. Spot
presence and intensity analysis were performed using PDQuest Advanced 8.0. Spots
located in the same location on different gels were assigned the same SSP number, which is
a unique sample spot protein number assigned by the PDQuest software. A total of 114
different spots, along with replicates of the same spot in different gels, were cut and
independently analysed by mass spectrometry.
Figure S8 Representative 2D-PAGE silver stained gels of the ovarian tissue biopsies SOV-1/2/3/4/5.
Proteins (approximately 50 µg) were separated on pH 3-10 non-linear 11 cm IPG strips in the
1
first dimension (IEF) and by precast gels (Criterion
TGX Precast Gels, any kD, IPG + 1 well
comb, 11 cm IPG strip) in the second dimension (SDS-PAGE). Resulting gels were silver
stained. Differentially expressed proteins were determined by 2D image analysis using
PDQuest Advanced 8.0.1.
SOV-1
SOV-2
SOV-3
SOV-4
SOV-5
Figure S9. Spots in the ovarian tissue gels that were selected for excision and further analysis by LC-MS/MS. Spot
intensity analysis was performed for spot selection and only the spots that followed the selection criteria
(exclusive presence, 2-fold and 5-fold change in intensity) were excised.
1
Perkins DN, Pappin DJ, Creasy DM, and Cottrell JS: Probability-based protein identification by
searching sequence databases using mass spectrometry data. Electrophoresis 20: 3551-3567, 1999.
2
Craig R and Beavis RC: TANDEM: matching proteins with tandem mass spectra.
Bioinformatics 20: 1466-1467, 2004.
3
Keller A, Nesvizhskii AI, Kolker E, and Aebersold R: Empirical statistical model to estimate the
accuracy of peptide identifications made by MS/MS and database search. Analytical chemistry 74:
5383-5392, 2002.
4
Nesvizhskii AI, Keller A, Kolker E, and Aebersold R: A statistical model for identifying proteins
by tandem mass spectrometry. Analytical chemistry 75: 4646-4658, 2003.
5
Ingenuity: Ingenuity Pathway Analysis (IPA). Ingenuity Systems, 2013.