臺灣博碩士論文加值系統

背景：惡性腫瘤在過去幾十年來一直是台灣死亡排行榜上的第一位。目前，仍未有血清標誌提供診斷口腔癌以及鼻咽癌，也未有高靈敏性及專一性的血清標誌提供診斷乳癌、肺癌、及攝護線癌。
方法：利用磁珠具有不同親和力的磁珠作純化及MALDI-TOF質譜儀作分析，我們針對乳癌、肺癌、攝護腺癌、口腔癌、及鼻咽癌篩選鑑定了具潛力的血清腫瘤標誌。每一種癌症統合的蛋白圖譜先與正常檢體的統合圖譜做比對，然後再利用統計軟體分析其間的差異。
結果：因為銅磁珠可以呈現最多正常及癌症檢體間的差異，所以初步利用銅磁珠作蛋白圖譜分析。除了乳癌，在五種分析的癌症中，我們找到了幾個具有高靈敏性(>80%)及高專一性(>90%)的血清腫瘤標誌，分別是4個分子量分別為2878±5、3809±5、4975±5、和 8610±5的肺癌標誌，2個分子量分別為1892±5和2022±5的攝護腺癌標誌，1個分子量為2664±5的口腔癌標誌，及1個分子量為2020±5的鼻咽癌標誌。若結合2個鼻煙癌標誌作分析，偵測到的靈敏性將大大的提高。最後，兩個最靈敏、最專一、分子量分別是2020 and 2658的腫瘤標誌被鑑定出，他們分別是補體C3前驅物(攝護腺癌及鼻煙癌標誌)及alpha-fibrinogen (口腔癌標誌)。
結論：高效能蛋白技術可以運用來發現新的且較好的腫瘤標誌。利用此技術，我們找到了4個肺癌、2個攝護腺癌、1個口腔癌、及一個鼻咽癌的腫瘤標誌。這些高靈敏性及高專一性的腫瘤標誌非常具有潛力可以作為惡性疾病的診斷工具。

Background: Malignant tumors are at the top leading cause of death in Taiwan in the past decades. Currently, there is no serum marker for detecting oral or nasopharyngeal cancers (ORC or NPC), neither available serum marker with high sensitivity and specificity for breast, lung, and prostate cancers (BC, LC, and PC).
Methods: Affinity purification with magnet beads and MALDI-TOF mass spectrometry analysis were used to screen and identify potential serum tumor markers for BC, LC, PC, ORC, and NPC. Compiled protein profile of each cancer group was compared to normal samples, and the differential spectra were statistically analyzed using bioinformatic softwares.
Results: Since Cu bead can discriminate most differential spectra between normal and tumor group, it was used for protein profiling. Among the five tested malignant diseases except breast cancer, several markers with high sensitivity (>80%) and specificity (>90%) were found. They are 4 LC-specific markers with molecular weight of 2878±5, 3809±5, 4975±5, and 8610±5; 2 PC-specific markers with molecular weight of 1892±5 and 2022±5; 1 ORC-specific marker with molecular weight of 2664±5; and 1 NPC-specific marker with molecular weight of 2020±5. After combination of two NPC-specific markers, the detecting sensitivity was greatly increased (94%0. Finally, two most sensitive and specific markers were identified with a PC and NPC marker (2020 Da) and an ORC marker (2658 Da). They are complement C3 precursor and alpha fibrinogen, respectively.
Conclusion: High-throughput proteomic approach could greatly facilitate the discovery of novel and better serum markers. 4 LC-specific markers, 2 PC-specific markers, 1 ORC-specific marker and 1 NPC-specific marker were found. The high specificity and sensitivity achieved by the use of these tumor-specific markers show great potential for the detection of the malignant diseases.

Contents
Introduction
A. Malignant disease and serum tumor markers………………………1
B. High-throughput proteomic technologies…………………………..1
1. Classical approach……………………………………………….….2
2. Surface-enhanced laser/desorption time-of-flight (SELDI-TOF) approach…………………………………………………………….2
3. Affinity-beads purification and MALDI-TOF MS approach……….3
Materials and Methods
Study populations and blood sample collection……………………….5
Proteomic analysis……………………………………………………..5
Statistical data analysis………………………………………………...6
Identification of protein markers………………………………………7
Results
A. Sample preparation and optimization………………………………8
1. Unnecessary removal of albumin…………………………………...8
2. Sufficient beads capacity……………………………………………8
3. Titration of the fractionated sera samples…………………………...8
B. Beads selection……………………………………………………..9
1. Hydrophobic series: C3, C8, C18…………………………………...9
2. Metal series: Cu, Fe………………………………………………..10
3. Cation beads……………………………………………………….10
C. Evaluation of protein profiling using Cu beads…………………...11
1. Enough detection range of 10 kDa……………………………...…11
2. High reproducibility of Cu-beads-purification and MALDI-TOF MS analysis…………………………………………………………….11
D. Search for specific tumor marker…………………………………12
1. Breast cancer (BC)…………………………………………………12
2. Lung cancer (LC)…………………………………………………..12
3. Prostate cancer (PC)……………………………………………….13
4. Oral cancer (ORC)…………………………………………………14
5. Nasopharyngeal cancer (NPC)…………………………………….15
E. Identification of the tumor markers……………………………….17
Discussion………………………………………………………………18
References………………………………………………………………22
Tables
Table 1 Top ten leading cause of death in Taiwan in 2003……………26
Table 2 Top fifteen leading cancers in Taiwan in 2003 and the
serum markers currently used in clinic now………………….27
Table 3 Reproducibility of mass spectra profiled by Cu-beads……….28
Table 4 Patient characteristics………………………………………...29
Table 5 Determination of the sensitivity and specificity for the
four LC-specific differential markers………………………...30
Table 6 Determination of the sensitivity and specificity for the
two PC-specific differential markers…………………………31
Table 7 Determination of the sensitivity and specificity for the
six ORC-specific differential markers………………………..32
Table 8 Determination of the sensitivity and specificity for the
twelve NPC-specific differential markers……………………33
Table 9 Statistical analysis of the combined markers in detection
of NPC………………………………………………………..34
Table10 MALDI-TOF mass spectra and database matching for
protein identification…………………………………………35
Figures
Figure 1 General steps of the used method in this study…………….36
Figure 2 Unnecessary removal of albumin for mass spectrometry….37
Figure 3 Sufficient beads capacity…………………………………..38
Figure 4 Sample dilution…………………………………………….39
Figure 5 Comparison of the protein profiles between C3, C8, and
C18 beads………………………………………………….40
Figure 6 Both Cu and Cation beads suitable for profiling…………..41
Figure 7 Sufficient detection range of 10 kDa………………………42
Figure 8 Reproducibility of mass spectra profiled by Cu-beads…….43
Figure 9 Search for specific tumor marker of breast cancer…………44
Figure 10 Search for specific tumor marker of lung cancer (LC)…….45
Figure 11 Distribution of LC-specific differential markers…………..46
Figure 12 Search for specific tumor marker of prostate cancer (PC)…47
Figure 13 Distribution of PC-specific differential markers…………...48
Figure 14 Search for specific tumor marker of oral cancer…………...49
Figure 15 Distribution of ORC-specific differential markers………...50
Figure 16 Search for specific tumor marker of nasopharyngeal cancer
(NPC)………………………………………………………51
Figure 17 Distribution of NPC-specific differential markers…………52
Figure 18 Cu-bead fractionated proteins were further purified by C8 beads for protein identification…………………………….53
Figure 19 Identification of proteins by TOF/TOF analysis…………...54

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