磷酸化介導的信號傳遞調節異常，導致許多疾病的發生與進程。受質激酶測定法，因能夠量化其隨著表型受質而改變的特定胺基酸位點磷酸化，而提供了更好的專一性來監測疾病的狀態。本研究結合特定胺基酸位點胜肽和多反應監測質譜(MRM-MS) 技術平台，靈敏且快速地相對定量和絕對定量奈米莫耳濃度的激酶與奈米細胞層級中受質激酶的活性。我們使用非小細胞肺癌作為概念驗證，設計了三種選自腫瘤組成型磷酸化的受質胜肽（HDGF-S165、RALY-S135和NRD1-S94）來證明此構想的可行性。在PC9非小細胞肺癌中，測得的HDGF-S165活性為3.2±0.2 fmol μg-1 min-1，而RALY-S135和NRD1-S94在25 ng和5 ng細胞裂解液的分別顯示出高於4倍和20倍的活性。實驗結果除了顯示此方法具有良好的準確度(小於15%的標準偏差) 以及再現性(小於15%的變異係數) 之外，且也暗示著不同受質胜肽具有不同的內生性激酶。另外，此實驗方法有別於以往散彈槍式蛋白質體學的流程，從細胞裂解至質譜儀的數據採集流程僅需3個小時。在多重分析實驗結果揭示了六種非小細胞肺癌細胞株之中，隨著表型受質而改變的磷酸化表現之差異性，並也暗示HDGF-S165和NRD1-S94與酪氨酸激酶抑制劑抗藥性的潛在關聯性。

Dysregulation of phosphorylation-mediated signaling drives the initiation and progression of many diseases. Though the mass spectrometry (MS)-based phosphoproteomics provided large-scale identification and quantification of altered phosphorylation associated with disease mechanisms, the complex sample preparation limits its general applicability in clinical specimens. A substrate-specific kinase assay capable of quantifying the altered site-specific phosphorylation of its phenotype-dependent substrates provides better specificity to monitor a disease state. To address the unmet need, we report a sensitive and rapid substrate-specific kinase assay by integrating site-specific peptide reporter and multiple reaction monitoring (MRM)-MS platform for relative and absolute quantification of substrate-specific kinase activity at the sensitivity of nanomolar kinase and nanogram cell lysate. Using non-small cell lung cancer (NSCLC) as a proof-of-concept, in the first part of thesis, three substrate peptides were selected from overly constitutive phosphorylation in tumors (HDGF-S165, RALY-S135, and NRD1-S94) and designed to demonstrate the feasibility. All three proteins were found to be either associated with poor prognosis and advanced phenotypes of NSCLC or highly expressed in other cancer. The kinase specificity were validated and kinase reactions were optimized. In the second part of the thesis, the assay was developed and showed good accuracy (<15% nominal deviation) and reproducibility (<15% CV). In PC9 cells, the measured activity for HDGF-S165 was 3.2±0.2 fmol μg-1 min-1, while RALY-S135 and NRD1-S94 showed 4- and 20-fold higher activity at the sensitivity of 25 ng and 5 ng lysate, respectively, these results suggest different endogenous kinases for each substrate peptide. In contrast to conventional shotgun phosphoproteomics workflow, the overall pipeline from cell lysate to MS data acquisition only takes 3 hours. The multiplexed analysis revealed differences in the phenotype-dependent substrate phosphorylation profiles across six NSCLC cell lines and suggested a potential association of HDGF-S165 and NRD1-S94 with TKI resistance. Encouraged by the ease of design, sensitivity, accuracy, and reproducibility, in the third part of the thesis, we further implemented the assay to potentially enable analysis of candidate phosphosite biomarkers in clinical samples such as serum. We performed a preliminary experiment adapting our assay into the MALDI-MS platform without the IMAC enrichment step. Using PKA and NRD1-S94 as model kinase-substrate pair, we were able to detect kinase activity in a gastric cancer serum pool, but further optimization is needed to boost the phosphopeptide signal for reliable quantitation. These new results open the possibility of a rapid and non-invasive kinase assay to target specific diseases with aberrant kinase activity such as cancer. Nevertheless, some key issues need to be improved. On the analytical perspective, substrates with multiple phosphorylation sites, and the variability of endogenous kinases in cells and samples may introduce challenges. In summary, the developed assay is generic to apply to other candidate phosphosite biomarkers as an efficient validation platform.

Contents

Cover Page 1
ACKNOWLEDGEMENTS 2
中文摘要: 3
ABSTRACT 4
LIST OF FIGURES 8
LIST OF TABLES 11
LIST OF ABBREVIATIONS 12
Chapter 1: Introduction 15
1.1 Introduction to Protein Phosphorylation 15
1.2 Analysis of Aberrant Phosphorylation by MS-based Phosphoproteomics 15
1.3 Kinase Activity Assays and Their Traditional Platforms 20
1.4 Mass Spectrometry-based Kinase Activity Assays 23
1.5 Thesis Objectives 27
Chapter 2: Experimental Section 31
2.1 Chemicals and Reagents 31
2.2 Cell Culture and Patient Tissues 32
2.3 Kinase Prediction 33
2.4 Kinase Reaction 33
2.5 Phosphopeptide Enrichment 34
2.6 MALDI-TOF-MS 34
2.7 LC-MS/MS Analysis 35
2.8 Multiple Reaction Monitoring Mass Spectrometry (MRM-MS) 36
2.9 Calculation of Kinase Activity 37
Chapter 3 Results 38
3.1 Design of the Targeted Substrate-specific MRM Assay 38
3.2 Validation of Peptide Substrate Phosphorylation 41
3.2.1 Selection of Target Peptides 41
3.2.2 Validation of HDGF-S165 Phosphorylation 43
3.2.3 Validation of RALY-S135 and NRD1-S94 Phosphorylation 45
3.2.4 Evaluation of the Specificities of the Three Substrate Peptides 46
3.3 Optimization and Selection of Kinase Reaction Conditions 54
3.4 Analytical Performance of the Substrate-specific Assay 57
3.4.1Assay Linear Working Range Using ERK2 and HDGF-S165 57
3.4.2 Assay Accuracy and Precision 58
3.4.3 Assay Sensitivity 59
3.4.4 Potential Utility in Phosphosite Validation 59
3.5 Multiplexed Substrate-specific Profiling in NSCLC Non-small Cell Lung Cancer Cells 68
3.5.1 Absolute Substrate-specific Profiles =Across Various NSCLC cells 68
3.5.2 Hierarchical Clustering Analysis Potential Biological Associations 69
3.5.3 Possible Sources of the Kinase Activities in NSCLC cells. 70
3.5.4 Summary of the Biological Analysis and Future Outlook 71
3.6 Other Potential Phosphosite Targets and Challenges 73
3.7 Adapting the Assay to the MALDI-MS Platform 80
3.8 Substrate- specific MALDI-MS Assay for Serum Analysis 82
3.9 Discussion 88
Chapter 4 Conclusions 91
Appendices 94

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