FUTURE WORK - 藉由定量磷酸化蛋白質體學分析探討MCM2在肺癌細胞中的調控網絡

First, we attempt to validate the phosphorylation status of HMGA1 induced by MCM2 in lung cancer cells. Since the phospho-specific antibody for HMGA1are not available in market so far, we could not detect the phosphorylation of HMGA1 by western blot.

Both the MCM2-overexpressed A549 cells and MCM2-silenced H1299 cells showed the differential phosphorylation level of the phosphosites of Ser99 on HMGA1, indicating the important link between MCM2 and HMGA1 phosphorylation.

Phosphorylation of HMGA1 and its relevance to MCM2 needs substantial evidences and should be investigated in detail.

Second, it is still unclear that which specific pathway is induced by MCM2 to affect the biological processes such as cell proliferation, cell migration and cell cycle in human lung cancer cells. Future studies are needed to elucidate the molecular pathway regulating these biological processes in order to clarify the role of MCM2 in lung cancer cells.

Third, the western blot analysis showed that p53 is correlated with MCM2 but we did not know how exactly the p53 regulates MCM2. More experimental validations are needed to prove the relationship between p53 and MCM2 at transcriptional level.

ABBREVIATION

13CD2O 20% formaldehyde-¹³C, d2 solution ACN acetonitrile

CH₂O 37% formaldehyde solution DMSO dimethyl sulfoxide

DTT dithiothreitol FDR false discovery rate

FDR false discovery rate

GO gene ontology

HAMMOC hydroxy acid-modified metal oxide chromatography IAM iodoacetamide

iTRAQ isobaric tags for relative and absolute quantitation LC-MS/MS liquid chromatography-tandem mass spectrometry LTQ linear Trap Quadrupole

LysC Lysyl endopeptidase

MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt

MTT 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide NaBH3CN sodium cyanoborohydride

PBS phosphate-buffered saline PI propidium iodide

PMS phenazine methosulfate

PTS phase-transfer surfactants PVDF polyvinylidene difluoride

SCX strong cation exchange chromatography SDB-XC polyStyrenedivinyl- benzene

SDC sodium deoxycholate

SDS-PAGE sodium dodecyl sulfatepolyacrylamide gel electrophoresis Ser serine

shRNA short hairpin RNA siRNA small interfering RNA SLS sodium lauroyl sarcosine StageTip Stop-and-go-extraction tip TEABC triethylammonium bicarbonate TFA trifluoroacetic Acid

Thr threonine TiO2 titanium dioxide Tyr tyrosine

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Figure 1. Schematic illustration of the experimental design of this study. For

MCM2 overexpression in A549 cells, the cells were transfected with 1 ug of MCM2-overexprssing plasmid and harvested after 24 hours. For silencing MCM2 in H1299 cells, the cells were transfected with siRNA at final concentration of 10 nmol and harvested after 48 hours. The extracted protein was subjected to the phosphoproteomics workflow and nanoLC−MS/MS analysis. The differential regulation of phosphorylated proteins in these two phosphoproteomes was compared and the results were validated.

Figure 2. Schematic workflow of phosphoproteomics. Protein was extracted from

cell lysates. The protein was then subjected to reduction, alkylation and protein digestion for overnight. The digested peptides from the control and treatment samples were subjected to dimethy labelling, CH₂O (light) for control samples and ¹³CD₂O (heavy) for treated samples. The light-labeled and heavy-labeled samples were mixed and proceed to HAMMOC-TiO₂ phosphopeptide enrichment and finally nanoLC−MS/MS.

Figure 3. Endogenous expression of MCM2 in lung cancer cells. Protein samples (30

ug) were electrophoresed on a 10% SDS polyacrylamide gel. The membrane was incubated with the following primary antibody diluted in blocking buffer at 4°C overnight: rabbit anti-MCM2 (1:1000), mouse anti-P53 (1:1000) and mouse anti-Actin (1:5000) followed by incubation with appropriate horseradish peroxidase-labeled secondary antibody (1:100000) for 2 hours at room temperature. Signal was developed with Clarity Western ECL Substrate Kit. The result showed that H1299 (p53 null) show higher endogenous expression of MCM2 compared to A549 (p53 wild-type).

Figure 4. Overexpression of MCM2 in A549 increased cell growth in lung cancer

cells. (A) and (B) The cell viability of MCM2-overexpressing cells. After 24 hours of

post-transfection, 5000 cells were seeded onto the 96-well plate and incubated for 24 or 48 hours (C) and (D) The colony formation assay of MCM2-overexpressing cells. After 24 hours of post-transfection, 500 cells were seeded onto the 6-well plates and incubated for 8 days. The number of colonies in MCM2-overexpressing and control cells was counted manually. MCM2 overexpressed A549 cells exhibited a significant increase in cell proliferation (increased 65.0% and 38.2% of cell viability at 24 and 48 hours) and higher number of colonies formed as compared to control vector. (*, P<0.05)

Figure 5. Silencing MCM2 in H1299 cells decreased cell proliferation. (A) and (B)

The cell viability of MCM2-silenced cells. After 24 hours of post-transfection, 5000 cells were seeded onto the 96-well plate and incubated for 24 or 48 hours. The cell viability was determined using the MTS assay. (C) and (D) The colony formation assay of MCM2-silenced cells. After 24 hours of post-transfection, 500 cells were seeded onto the 6-well plates and incubated for 8 days. The number of colonies in MCM2-silenced and control cells was counted manually. MCM2 silenced H1299 cells (both si-2 and si-3) showed a significant decrease in cell proliferation and lower number of colonies formed as compared to control siRNA. Result showed that si-2 reduced 18.8% and 14.2% of cell viability while si-3 reduced 20.7% and 16.6% of cell viability at 24 and 48 hours. (*,

P<0.05)

Figure 6. The relationship between P53 and MCM2. (A) and (B) P53 overexpression

in H1299 cells. Cells were transfected with either control plasmid or pcDNA3.1(-)/TP53 for 24 hrs. Overexpression of P53 in H1299 resulted in downregulation of MCM2 expression. (C) and (D) P53 knockdown in A549 cells. Plasmid shRNA-TP53 was used to knockdown p53 expression in A549 cells (knockdown efficiency=44%). MCM2 protein expression was increased in p53-silencing cells.

Figure 7. Confirmation of the MCM2 protein expression upon MCM2 overexpression in A549 cells and silencing MCM2 in H1299 cells. (A) A549 cells

were transfected with 1 ug pcDNA3.1(+)/MCM2 or control vector. Cells were harvested at 12, 24 and 48 hours and the protein levels were analyzed by Western blotting.

Overexpression level of MCM2 was highest at 24 hours post-transfection. (B) and (C) H1299 cells were treated with three siMCM2 (si-1,si-2 and si-3) or control siRNA at final concentration of 10nM. Cells were harvested at 24 and 48 hours and the protein levels were analyzed by Western blotting. Two siRNAs for MCM2 (si-1 and si-2) showed high knockdown efficiency at 48 hours.

Figure 8. Distribution of single, doubly, triply and quadruply phosphorylated

peptides. In the phosphoproteome of MCM2 overexpression, a majority of

phosphopeptides was either single (48.3%) or doubly (45.1%) phosphorylated, followed by a small portion of triply (6.2%) and quadruply (0.4%) phosphorylated. In the phosphoproteome of silencing MCM2, a majority of phosphopeptides was either single (41.6%) or doubly (48.2%) phosphorylated, followed by a small portion of triply (9.5%) and quadruply (0.7%) phosphorylated.

Figure 9. Distribution of phosphorylated serine, threonine, and tyrosine sites. Pie

chart showed the percentages of serine, threonine and tyrosine phosphorylation sites identified in phosphoproteome of (A) MCM2 overexpression and (B) silencing MCM2.

In the phosphoproteome of MCM2 overexpression, the distribution of phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr) was 85%, 14% and 1%

respectively. In the phosphoproteome of silencing MCM2, The distribution of phosphoserine (pSer), phosphothreonine (pThr) and phosphotyrosine (pTyr) was 84%, 15% and 1% respectively.

Figure 10. Phosphoproteome of MCM2 in lung cancer cells. (A) and (B) Venn

diagram showed the overlap of differentially regulated phosphoproteins and phosphosites in MCM2 overexpression and silencing MCM2. (C) and (D) Overlap of phosphosites that showed the opposing phosphorylation change pattern in phosphoproteome of MCM2 overexpression and silencing MCM2. Phosphoproteins that change phosphorylation level significantly are selected by ratio H/L normalized <= 0.67 (1.5-fold reduced) or >= 1.5 (1.5-fold increased). Localization of PTM probabilities are required to be at least of 0.75.

Figure 11. List of GO-term of regulated phosphoproteins in phosphoproteome of MCM2 overexpression in A549 cells. The Functional Annotation Tool on DAVID

v6.7 was used to facilitate the annotation of up- (>1.5 fold) and down-regulated (>1.5 fold) phosphoproteins. MCM2-overexpressed proteins were involved in RNA splicing, protein folding, regulation of protein complex assembly, regulation of cytoskeleton organization, regulation of actin filament polymerization.

0.00E+00 1.00E-02 2.00E-02 3.00E-02 4.00E-02 negative regulation of protein complex assembly

negative regulation of protein complex…

0.00E+00 1.00E-02 2.00E-02 3.00E-02 4.00E-02 5.00E-02 6.00E-02 cellular macromolecular complex subunit organization regulation of transcription from RNA polymerase II promoter cellular protein complex assembly negative regulation of programmed cell death negative regulation of cell death

Figure 12. List of GO-term of regulated phosphoproteins in phosphoproteome of

silencing MCM2 in H1299 cells. The Functional Annotation Tool on DAVID v6.7 was

used to facilitate the annotation of up- (>1.5 fold) and down-regulated (>1.5 fold) phosphoproteins. MCM2-silenced proteins were mainly involved in macromolecular complex subunit organization, RNA processing, microtubule-based movement, DNA replication and cell cycle.

Figure 13. Protein enrichment analysis. Visualization of enriched gene ontology term

of (A) MCM2 overexpression and (B) silencing MCM2 phosphoproteome using

Enrichment Map Cytoscape Plugin. Differentially up- and down-regulated

phosphoproteins were analyzed for enrichment using DAVID database.

A

B

mRNA processing, RNA splicing

Microtubule-based process,

macromolecular complex assembly

mRNA processing

Figure 14. Overexpression of MCM2 in A549 increased cell migration. (A) The

ability of cell migration was examined by transwell migration assay. 3x10⁴ cells were seeded onto the upper chamber of a transwell and incubated for 6 hrs. (B) The number of migrated cells in MCM2-overexpressing and control cells. MCM2 overexpressed cells show significant increase in migrated cells. (*, P<0.05)

Figure 15. Silencing MCM2 in H1299 cells decreased cell migration. (A) The ability

of cell migration was examined by transwell migration assay. 3x10⁴ cells were seeded onto the upper chamber of a transwell and incubated for 6 hrs. (B) The number of migrated cells in MCM2-silenced and control cells. MCM2-silenced cells show significant decrease in migrated cells. (*, P<0.05)

Figure 16. Silencing MCM2 in H1299 cells induced cell cycle arrest. Cells were

stained with 5 μg/ml propidium iodide for 15 minutes in the dark. The DNA content of the cells was analyzed using a FACSCanto instrument. At least 10,000 cells were collected for each measurement in a triplicate experiment. The percentage of cells in different phases of the cell cycle was analyzed using ModFit LT. MCM2 knockdown with 10nM siRNA (si-2 and si-3) resulted in a increase in G1 phase (54.46% and 55.32%) and a decrease in S phase (38.13% and 36.55%) at 48 hours siRNA post-transfection. The accumulation of cells in G2/M phase was also observed in MCM2-silenced cells (5.41% and 6.9%) as compared to control cells (3.13%). (*, P<0.05;**,

P<0.01)

Uniprot: P17096 Symbol: HMGA1 Protein names: High mobility group protein HMG-I/HMG-Y Best score evidence ID: 4767 Best score MS/MS ID: 5684

Figure 17. The MS/MS spectrum of the phosphopeptide KLEKEEEEGISQESSEEEQ from HMGA1 (high mobility group protein

HMG-I/HMG-Y).

Figure 18. Phosphorylation of HMGA1 at S99 is essential for viability. (A) The

amino acid sequences showing the position to be mutated by substitution. The Serine residues at position of 99 within HMGA1 protein were substituted with non-phosphorylatable alanine (Ala) and phosphomimetic glutamic acid (Glu) by site‐directed mutagenesis. (B) The overexpression level of HMGA1 mutants was assessed by measuring the total HMGA1 protein. Western blot analysis showed the HMGA1 wild type and HMGA1 mutants (S99E and S99A) have similar total HMGA1 protein level, indicating that their overexpression efficiency are of the same magnitude.

(C) A549 cells were transfected with pcDNA3.1(+) without insert, HMGA1 wild-type or

HMGA1 mutants (S99E and S99A). At 24 hours of post-transfection, 5000 cells were seeded

onto the 96-well plate and incubated for 24 or 48 hrs.

Mutation of Ser99 to alanine significantly reduced (*,

P<0.05)

the cell viability of A549 cell

Table 1. PCR primer sequences for plasmid construction

Gene The sequence of primers

MCM2 (Forward)

5’-GCTAGCGCCACCATGGCGGAATCATCGGAA 3’

MCM2 (Reverse)

5’ CGCACGCGTACAAGCTTTCAGAACTGCTGCAGGAT 3’

HMGA1(Forward)

5’- AAAGGATCCGCCACCATGAGTGAGTCGAGCTCG 3’

HMGA1(Reverse)

5’ AAATTGGGCCCTCACTGCTCCTCCTCCGAGGACT 3’

HMGA1-S99A

(Forward)

5' GGAGGGCATCGCGCAGGAGTC 3'

HMGA1-S99A

(Reverse)

5' GACTCCTGCGCGATGCCCTCC 3'

HMGA1-S99E

(Forward)

5' GGAGGGCATCCAGCAGGAGTCC 3'

HMGA1-S99E

(Reverse)

5' GGACTCCTGCTGGATGCCCTCC 3'

Table 2. Number of phosphopeptides, phosphosites and phosphoproteins

Spectra were analyzed using MaxQuant version 1.5.0.30 and searched against SwissProt Database (version 2014-09). In MCM2 overexpressing A549 cells, a total 1409 unique phosphopeptides on 593 phosphoproteins were identified. In MCM2 silencing H1299 cells, a total 1347 unique phosphopeptides on 592 phosphoproteins were identified.

Experiment MCM2 overexpression in A549 cells

MCM2 Silencing in H1299 cells

No. of phosphopeptides

1409 1347

No. of phosphosites

1484 1599

No. of phosphoproteins

593 592

Table 3. List of differentially regulated phosphosites that response to both MCM2 overexpression in A549 cells and silencing MCM2 in H1299 cells

a The official protein Uniprot ID given to the identified protein.

b The official gene symbol given to identified protein.

c The official protein name obtained from Uniprot

d The amino acid position sites to be phosphorylated within the identified phosphosites

e The amino acid to be phosphorylated in the identified phosphosites

f The normalized ratio between two medium and light label partners of the identified phosphosites in MCM2 overexpression experiment

g The normalized ratio between two medium and light label partners of the identified phosphosites in silencing MCM2 experiment

h The localization probability of the identified phosphosites in MCM2 overexpression experiment

i The localization probability of the identified phosphosites in silencing MCM2 experiment

No. Uniprot^a Symbol^b Protein name^c Positions within 2 P17096 HMGA1 High mobility group protein

HMG-I/HMG-Y

8 P49736 MCM2 DNA replication licensing factor MCM2

139 S 13.38 0.188 1 1

9 P49736 MCM2 DNA replication licensing factor MCM2

13 O15173 PGRC2 Membrane-associated progesterone receptor component 2

211 T 0.637 0.543 0.999378 0.973173

Table 4. List of differentially regulated phosphosites

No. Uniprot^a Symbol^b Protein names^c Sequence window^d Positions

within

Up-regulated phosphosites upon MCM2 overexpression in A549 cells

1 Q07157 ZO1 Tight junction protein ZO-1 SRPDPEPVSDNEEDSYDEEIHDPRSGRSGVV 132 Y 26.391 0.7808 VQIPVSRPDPEPVS(0.998)DNEEDS(0.221)Y(0.781 )DEEIHDPR

2 Q07157 ZO1 Tight junction protein ZO-1 KKVQIPVSRPDPEPVSDNEEDSYDEEIHDPR 125 S 16.028 0.9992 VQIPVSRPDPEPVS(1)DNEEDS(0.553)Y(0.447)DE EIHDPR

3 P49736 MCM2 DNA replication licensing factor MCM2

SSPAQRRRGNDPLTSSPGRSSRRTDALTSSP 27 S 13.627 0.9946 RGNDPLTS(0.001)S(0.999)PGR 4 P49736 MCM2 DNA replication licensing

factor MCM2

AGRGLGRMRRGLLYDSDEEDEERPARKRRQV 139 S 13.379 1 GLLYDS(1)DEEDEERPAR

5 Q8IZM8 ZN654 Zinc finger protein 654 SSSNEKQTISLPVSTSKSRKESTEPKTCIES 368 S 10.792 0.992 QT(0.422)IS(0.581)LPVS(0.842)T(0.163)S(0.992)K 6 P62995;

DRDQIYRRRSPSPYYSRGGYRSRSRSRSYSP 270;266 S 10.792 0.9299 RRS(0.055)PS(0.719)PY(0.234)Y(0.062)S(0.93)R

7 Q8IZM8 ZN654 Zinc finger protein 654 KDSSSNEKQTISLPVSTSKSRKESTEPKTCI 366 S 10.792 0.8421 QT(0.422)IS(0.581)LPVS(0.842)T(0.163)S(0.992)K 8 Q15149 PLEC Plectin AQSTKGYYSPYSVSGSGSTAGSRTGSRTGSR 4620 S 8.9892 0.9051 GYYSPYSVS(0.006)GS(0.905)GS(0.136)T(0.464)A

GS(0.072)RT(0.106)GS(0.31)R 9 P49736 MCM2 DNA replication licensing

factor MCM2

EGLALDDEDVEELTASQREAAERAMRQRDRE 108 S 7.4536 0.9653 AIPELDAYEAEGLALDDEDVEELT(0.035)AS(0.9 65)QR

10 Q96I25 SPF45 Splicing factor 45 DRHEASGFARRPDPDSDEDEDYERERRKRSM 155 S 5.6277 1 RPDPDS(1)DEDEDYERER

11 Q15149 PLEC Plectin STKGYYSPYSVSGSGSTAGSRTGSRTGSRAG 4622 S 4.7957 0.9218 GYYSPY(0.001)S(0.012)VS(0.012)GS(0.012)GS(0.9 22)T(0.765)AGS(0.276)R

12 Q9H7N4 SFR19 Splicing factor, arginine/serine-rich 19

RLDKSDPRGPSPAPASSPKREVLYDSEGLSG 724 S 4.5167 0.8156 GPS(0.004)PAPAS(0.816)S(0.18)PKREVLY(0.269) DS(0.818)EGLS(0.914)GEER

13 Q9NYF8 BCLF1 Bcl-2-associated transcription factor 1

KESGKQKFNDSEGDDTEETEDYRQFRKSVLA 402 T 4.4777 1 QKFNDS(1)EGDDT(1)EETEDYR

14 Q92882 OSTF1 Osteoclast-stimulating factor 1

VRTLSNAEDYLDDEDSD______________ 213 S 3.8796 1 TLSNAEDYLDDEDS(1)D

15 Q9NYF8 BCLF1 Bcl-2-associated transcription factor 1

DYFSDKESGKQKFNDSEGDDTEETEDYRQFR 397 S 3.768 1 QKFNDS(1)EGDDT(1)EETEDYR

16 P24534 EF1B Elongation factor 1-beta GPADVEDTTGSGATDSKDDDDIDLFGSDDEE 95 S 3.4117 0.8466 YGPADVEDT(0.006)T(0.019)GS(0.064)GAT(0.064) DS(0.847)KDDDDIDLFGS(0.963)DDEEES(0.037)E EAKR

17 Q13442 HAP28 28 kDa heat- and acid-stable phosphoprotein

AGDPKKEKKSLDSDESEDEEDDYQQKRKGVE 63 S 3.1474 1 SLDS(1)DES(1)EDEEDDYQQK

18 Q13442 HAP28 28 kDa heat- and acid-stable phosphoprotein

DGAAGDPKKEKKSLDSDESEDEEDDYQQKRK 60 S 3.1474 0.9996 SLDS(1)DES(1)EDEEDDYQQK

Table 4. Continued

No. Uniprot^a Symbol^b Protein names^c Sequence window^d Positions

within

19 P46821 MAP1B Microtubule-associated protein 1B

TEPVEAYVIQKEREVTKGPAESPDEGITTTE 885 T 2.8707 0.8142 EREVT(0.814)KGPAES(0.183)PDEGIT(0.001)T(0.0 01)T(0.001)EGEGECEQT(1)PEELEPVEK 20 Q96SB4 SRPK1 SRSF protein kinase 1 HSESDLPEQEEEILGSDDDEQEDPNDYCKGG 51 S 2.8304 1 GSAPHSESDLPEQEEEILGS(1)DDDEQEDPNDYC

K 21 P17096 HMGA1 High mobility group protein

HMG-I/HMG-Y

RGRPKKLEKEEEEGISQESSEEEQ_______ 99 S 2.7934 1 KLEKEEEEGIS(1)QES(1)S(1)EEEQ

22 Q5JSH3 WDR44 WD repeat-containing protein 44

IMRRTKEYVSNDAAQSDDEEKLQSQPTDTDG 403 S 2.6827 0.9134 EYVS(0.009)NDAAQS(0.913)DDEEKLQS(0.053)Q PT(0.021)DT(0.004)DGGR

23 Q12789 TF3C1 General transcription factor 3C polypeptide 1

VRCPRVRKNSSTDQGSDEEGSLQKEQESAMD 1068 S 2.5159 0.981 VRKNS(0.427)S(0.427)T(0.145)DQGS(0.981)DEEG S(0.019)LQK

24 Q9H7N4 SFR19 Splicing factor, arginine/serine-rich 19

ASSPKREVLYDSEGLSGEERGGKSSQKDRRR 738 S 2.373 0.9135 REVLYDS(1)EGLS(1)GEER 25 Q9H7N4 SFR19 Splicing factor,

arginine/serine-rich 19

SPAPASSPKREVLYDSEGLSGEERGGKSSQK 734 S 2.373 0.818 REVLYDS(1)EGLS(1)GEER 26 P04792 HSPB1 Heat shock protein beta-1 IESPAVAAPAYSRALSRQLSSGVSEIRHTAD 78 S 2.3684 1 ALS(1)RQLS(0.792)S(0.208)GVSEIR

27 P35659 DEK Protein DEK DSSTTKKNQNSSKKESESEDSSDDEPLIKKL 301 S 2.2119 1 KES(1)ES(1)EDS(1)S(1)DDEPLIKK

28 P35659 DEK Protein DEK STTKKNQNSSKKESESEDSSDDEPLIKKLKK 303 S 2.2119 1 KES(1)ES(1)EDS(1)S(1)DDEPLIKK

29 P35659 DEK Protein DEK KKNQNSSKKESESEDSSDDEPLIKKLKKPPT 306 S 2.2119 1 KES(1)ES(1)EDS(1)S(1)DDEPLIKK

30 P35659 DEK Protein DEK KNQNSSKKESESEDSSDDEPLIKKLKKPPTD 307 S 2.2119 1 KES(1)ES(1)EDS(1)S(1)DDEPLIKK

31 Q9Y618 NCOR2 Nuclear receptor corepressor 2

GNTSQPPAFFSKLTESNSAMVKSKKQEINKK 2286 S 2.18 0.9761 LT(0.994)ES(0.976)NS(0.023)AMVKS(0.007)K 32 P13611 CSPG2 Versican core protein QEVNPVRQEIESETTSEEQIQEEKSFESPQN 2116 S 2.1637 0.9643 QEIES(0.196)ET(0.876)T(0.964)S(0.964)EEQIQEE

33 P13611 CSPG2 Versican core protein RQEVNPVRQEIESETTSEEQIQEEKSFESPQ 2115 T 2.1637 0.9643 QEIES(0.196)ET(0.876)T(0.964)S(0.964)EEQIQEE K

34 P13611 CSPG2 Versican core protein SRQEVNPVRQEIESETTSEEQIQEEKSFESP 2114 T 2.1637 0.8758 QEIES(0.196)ET(0.876)T(0.964)S(0.964)EEQIQEE

在文檔中藉由定量磷酸化蛋白質體學分析探討MCM2在肺癌細胞中的調控網絡 (頁 40-92)