在本實驗中，我們收集了十八個鼻咽癌病人與十個健康對照組之血漿來
進行蛋白質比較性分析。我們將收集的檢體分為三組：治療組、治療前組
以及復發組。並利用二維電泳法與質譜分析技術來進行實驗，再以統計分
析定義出有明顯差異性的蛋白質，本實驗在鼻咽癌病人血漿中共分析出了
30 個蛋白質。結果顯示有十種在血漿中會表現量下降的蛋白質，分別是
Serotransferrin、Vitamin D-binding protein、alpha-1 antitrypsin、Haptoglobin、
Apolipoprotein B fragment、Syntaxin-7、Apolipoprotein A-I 、 PRO1779、
Transthyrin、MDN1 protein。在這些蛋白質裡，同時我們建立了去除高含量
血漿蛋白質的方法，另外我們選定了兩個有興趣研究的蛋白質：alpha-1
antitrypsin (AAT)及Vitamin D-binding protein (VDB)來進行以後的實驗。首
先以西方墨點法來確認此兩種蛋白質的真實身分是否與質譜分析結果相
符，並以酵素免疫分析方法來定量病人血漿中這兩種蛋白質之含量，以得
到病人血液中此兩種蛋白質的絕對濃度。我們藉由以上的數據與其他臨床
上的影響因子(如性別、年齡等等) 進行交叉比對並找出與鼻咽癌之關連
性。目前在臨床上針對鼻咽癌所做的相關蛋白質表現方面的研究仍為少
數，相信相關的研究將可以增加對鼻咽癌病人治療上的評估與癌化過程的
認識。這些表現量降低的蛋白質也具有進一步研究的價值。

In this study, we collected 18 plasma samples from NPC patients, and 10 plasma samples
from healthy person for plasma proteomic analysis. We classified these samples into 3 groups:
treatment, pretreatment and recurrent. Two-dimensional electrophoresis (2-DE) and
MALDI-TOF were performed followed by comparative and statistic analysis. In conclusions,
we totally identified 30 proteins in Nasopharyngeal carcinoma (NPC) plasma, and found 10
proteins with expression level down regulated (p<0.001). These proteins were characterized to
be Serotransferrin, Vitamin D-binding protein (VDB), alpha-1 antitrypsin (AAT), Haptoglobin,
Apolipoprotein B fragment, Syntaxin-7, Apolipoprotein, A-I, PRO1779, Transthyrin, MDN1
protein, respectively. Consequently, we established a protocol to remove high abundant
proteins (e.g., albumin, immunoglobin etc.) in plasma. We are especially interested in ATT
and VDB. Western blotting assay was performed to confirm ATT and VDB expression.
Furthermore, the quantity of ATT and VDB were measured by ELISA to obtain the threshold
value of these proteins. Finally, we want to realize the relationship between these down
regulation proteins and clinical parameters in NPC malignancy and tumor progression. Since
there are few protein expression research of NPC in clinical studies, our works will provide
insights in NPC studies for tumor progression with potential to elevate treatment efficiency.

Abstract in Chinese……………………………………………………………................ 1
Abstract in English…………………………...………………………………………..... 2
Abbreviation……………………………………………………………………………... 3
1. Introduction……………………………………………………………........................ 5
1.1 Importance for protein analysis in Nasopharyngeal carcinoma (NPC)……............. 5
1.2 Nasopharyngeal carcinoma (NPC)…….…..………………………..……................ 6
1.2.1 Anatomy……………………..……..…………………..…………………............ 6
1.2.2 Classification……………………......………………………………..................... 6
1.2.3 Epidemiology…………………,.....………………………………………............ 6
1.2.4 Etiology…………,.…………………………………………………..................... 7
1.2.5 Prognosis model………………………………..…………………………............ 7
1.2.6 Diagnosis……………………..……………………………………………........... 8
1.2.7 Treatment……………….…………………………………………………........... 9
1.3 Proteome……………………….……………………………………………........... 9
1.3.1 Mass spectrometry…………………………….……………………………......... 11
1.3.1.1 Matrix-assisted laser desorption/ionization (MALDI)…………………............ 11
1.3.1.2 Time of flight (TOF) analyzer……..................................................................... 11
1.3.2 Two-dimensional electrophoresis (2-DE)…………………………..………......... 12
1.4 Plasma proteome in clinical approach…………………….………………….......... 13
1.5 Proteomic studies in Nasopharyngeal carcinoma………………….…………......... 14
1.6 Specific aims…………………………………..……………………………............ 15
2. Experimental flowcharts………………………….………………….......................... 16
2.1 Differential protein expression analysis procedures……………………….............. 16
2.2 Differential expression protein identification procedures…………………….......... 17
2.3 Procedures for the quantification of protein of interest…......................................... 18
3. Materials and Methods………………………………….............................................. 19
3.1 Sources of the NPC specimens……………………………………………….......... 19
3.2 Sample preparing………………………….………………………………….......... 19
3.2.1 Protein extraction of NPC plasma…………………………………..………......... 19
3.3 Two-dimensional electrophoresis (2-DE)………………..…………………............ 20
3.3.1 First dimension-isoelectric focusing (IEF)……………………..…………........... 20
3.3.2 Immobiline DryStrip Equilibration………..………………………………........... 22
3.3.3 Sodiumdodecylsulfate polyacrylamide gel electrophoresis...…..…………........... 22
3.3.4 Zinc imidazole negative staining............................................................................ 24
3.3.5 Coomassie Blue staining……………………………..…………………............... 24
3.4 Gel imaging analysis.................................................................................................. 24
3.5 In gel digestion…………….....……………..……………………………………… 25
3.5.1 Washing………….....…….……………………………………………………… 25
3.5.2 Coomassie brilliant blue R-250 de-staining………….....……………..…………. 25
3.5.3 Reduction/alkylation……......………..…………………………………………... 25
3.5.4 Digestion/extraction……….....….………….……………………………………. 25
3.6 MALDI-TOF operations……….....……………..…………………………………. 26
3.7 Data analysis……….....……………….…………………………………………… 26
3.8 Western blotting………….....………………..…………………………………….. 26
3.9 Enzyme-linked immunosorbent assay (ELISA)........................................................ 28
4. Results………………………...……...…………………………................................... 29
4.1 NPC patients………….....…………………….…………………………………… 29
4.2 Comparative analysis of normal and NPC plasma in 2-DE gels…….....………….. 29
4.2.1 MAIDI-TOF analysis in protein database….....………………………………….. 30
4.2.1.1 MS analysis criteria……….....……………………..…………………………... 30
4.2.1.2 Identification of differential expression proteins……….....……………..…….. 31
4.2.1.3 Quantification and statistical analysis of down expression proteins…….....…... 31
4.3 Analysis for AAT and VDB expression by western blotting…….....…………..….. 32
4.3.1 Precipitated sample………….....…….…………………………………………... 32
4.3.2 Plasma sample (unprecipitated sample)….....…………………………….....…… 32
4.4 Quantitatively analysis for AAT and VDB by ELISA…….....……………………. 33
5. Discussion………...………………………………………………………..................... 35
5.1 Discussion in two-dimensional electrophoresis……….....………………………… 35
5.2 Discussion in western blotting….....………………………..……………………… 36
5.3 Discussion in ELISA………...….....……………………………………………….. 37
6. Introduction for down regulation proteins………………...……............................... 38
6.1 Vitamin-D binding protein (VDB)………….....……………………...……………. 38
6.2 Alpha-1 antitrypsin (AAT)…….....…………………...……………………………. 39
6.3 Serotransferrin (transferrin)…….....………………......…………………………… 41
6.4 Haptoglobin…………........………………………………………………………… 42
6.5 Apolipoprotein B (apo B) and Apolipoprotein A-I (apo A-I)……….....…...……… 44
6.6 Syntaxin-7…………….......………………………………………………………... 44
7. Conclusions………………………………………………...……………...................... 46
8. References………………………………………………………………....................... 47
9. Figures and Tables……………………………………………………......................... 54
10. Mass analysis results.................................................................................................... 71
(Materials and Methods)
Table 1. List of materials were used in proteomics......................................................... 23
Table 2. List of devices used in proteomics..................................................................... 23
(Results)
Table 3. Clinical parameters list of 18 NPC patients....................................................... 54
Table 4. Demographics of 18 NPC patients..................................................................... 55
Table 5. Statistical analysis of down regulation proteins in NPC.................................... 56
Table 6. Illustration of down regulated proteins.............................................................. 57
Table 7. Illustration of identified proteins....................................................................... 58
Figure 1. 2-DE synthetic gels of NPC plasma proteins................................................... 60
Figure 2. Down regulated proteins in 2-DE gels (Spot 1-5)............................................ 61
Figure 3. Down regulated proteins in 2-DE gels (Spot 6-10).......................................... 62
Figure 4. Comparison of down-regulation proteins intensity between normal and
NPC.................................................................................................................................
63
Figure 5. Down-regulation proteins were compared between normal, treated and
untreated groups of NPC..................................................................................................
64
Figure 6. Comparison of down-regulation proteins in different NPC stages................... 65
Figure 7. Comparison of down-regulation proteins between controls, recurrent and
nun-recurrent groups of NPC...........................................................................................
66
Figure 8. Western blotting of AAT and VDB by 10% TCA/Acetone precipitation........ 67
Figure 9. Western blotting of AAT and VDB.................................................................. 68
Figure 10. Plasma levels of AAT in healthy control (n=10) and NPC patients
(n=18)..............................................................................................................................
69
Figure 11. Plasma levels of VDB in healthy control (n=10) and NPC patients
(n=18)..............................................................................................................................
70
(Mass analysis results)
Figure 12. Mass analysis result of serotransferrin........................................................... 71
Figure 13. Mass analysis result of Vitamin-D binding protein........................................ 73
Figure 14. Mass analysis result of alpha-1 antitrypsin..................................................... 74
Figure 15. Mass analysis result of haptoglobin................................................................ 76
Figure 16. Mass analysis result of apolipoprotein B fragment........................................ 77
Figure 17. Mass analysis result of Syntaxin-7................................................................. 78
Figure 18. Mass analysis result of Apolipoprotein A-I.................................................... 79
Figure 19. Mass analysis result of Transthyretin............................................................. 81
Figure 20. Mass analysis result of MDN1 protein........................................................... 82
Table 8. Comparison result of protein sequence of serotransferrin................................. 72
Table 9. Comparison result of protein sequence of Vitamin-D binding protein.............. 73
Table 10. Comparison result of protein sequence of alpha-1 antitrypsin........................ 75
Table 11. Comparison result of protein sequence of haptoglobin................................... 76
Table 12. Comparison result of protein sequence of apolipoprotein B fragment............ 77
Table 13. Comparison result of protein sequence of Syntaxin-7..................................... 78
Table 14. Comparison result of protein sequence of Apolipoprotein A-I........................ 80
Table 15. Comparison result of protein sequence of Transthyretin................................. 81
Table 16. Comparison result of protein sequence of MDN1 protein............................... 83
(Appendix)
(Literature Review)
Figure 1. Anatomy of the nasopharynx and lymph nodes in neck……...…………........ 85
Figure 2. Pathogenesis model of Nasopharyngeal carcinoma………………………..... 86
Figure 3. Structure of alpha-CHCA…………………………………..……………....... 86
Figure 4. Progression of typical MALDI-TOF analysis…………….………………..... 87
Figure 5. Illustrates a typical MALDI (matrix assisted laser desorption ionization)
instrument of linear mode and reflectron mode………………………….......................
88
Figure 6. Two dimensional electrophoresis approaches……………………..…............ 89
Table 1. Staging criteria: AJCC 1997 system……………………….…......………....... 93
Table 2. Age-adjusted incidence rate of nasopharyngeal cancer in selected Asian
countries, 1988–1992………………..………………………….…………....................
94
(Discussion)
Figure 7. Proposed DBP response in tissue injury or infection....................................... 90
Figure 8. Possible mechanisms of imbalance between neutrophil elastase and alpaha-1
antitrypsin in cancer development and progression.........................................................
91
Figure 9. The iron metabolism circulation pathway from bloodstream to cells.............. 92
Figure 10. The irons metabolism pathway by haptoglobin interacting to CD163........... 92

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