심혈관질환의 바이오마커 발굴 : Biomarker Discovery for Cardiovascular Disease

다국어 초록 (Multilingual Abstract)

Proteomics is a valuable approach to discover biomarkers in human plasma for early diagnosis. However, detection of biomarkers in the plasma is still challenging because of highly abundant proteins. In our study, we established albumin/IgG depletion methods for identification of low abundance proteins using two commercial kits with additional buffer conditions and various concentrations of cold acetone. Trypsin digestion, desalting, and data-dependent acquisition were also optimized. The optimized process was applied to plasma samples of healthy controls and AMI patients to discover new biomarkers for diagnosis of cardiovascular disease. More than 80% depletion of albumin/IgG was achieved with two commercial kits and 98% depletion of albumin was obtained with 70% cold acetone. Recovery of four reference proteins, BNP (47-76), insulin, cytochrome c, and ubiquitin was obtained in all optimized methods. The best recovery of reference proteins was obtained using the ProteoExtract albumin/IgG removal kit with buffer A (61%~106%). After cold acetone precipitation, three reference proteins were recovered more than 48% except ubiquitin (12%). The number of identified proteins by Mascot was 28, 35, 17, and 34 for ProteoExtract, ProteoPrep, 70%, and 50% cold acetone, respectively. Furthermore, the optimized methods detected MS/MS fragmentation patterns of elevated BNP in patient samples with cardiac disease. Four precursor ions, such as 625.30, 770.35, 789.06, and 1036.96 showed possibility as new biomarker candidates for AMI diagnosis. Alpha-1-acid glycoprotein 1, antithrombin Ⅲ, clusterin, obscurin, and titin were also discovered as novel biomarkers. Our study provides the methods for efficient biomarker discovery by minimal removal of high abundant proteins. Furthermore, we demonstrated that our established methods can be discovered meaningful biomarker candidates for cardiovascular disease in human plasma.

목차 (Table of Contents)

• 1. Introduction 9
• 1.1 Difficulty of plasma analysis for biomarker discovery 9
• 1.2 Depletion of highly abundant proteins for detection of low abundance proteins 10
• 1.3 Acute myocardial infarction in cardiovascular disease 11
• 1.4 Biomarkers of acute myocardial infarction 13
• 1.5 The aim of study 15
• 2. Materials and methods 16
• 2.1 Materials 16
• 2.2 Sample preparation 17
• 2.3 Albumin/IgG depletion 17
• 2.4 SDS-PAGE 18
• 2.5 In-solution digestion 19
• 2.6 Desalting 19
• 2.7 Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) analysis 20
• 2.8 Quantitative analysis and protein identification 22
• 2.9 Statistical analysis 22
• 3. Results 23
• 3.1 Albumin/IgG depletion efficiency 23
• 3.2 Use of four reference proteins as low abundance proteins 26
• 3.3 Identification of reference proteins after albumin/IgG depletion 28
• 3.4 Condition optimization for UHPLC-MS/MS analysis 35
• 3.5 Protein identification in human plasma 38
• 3.6 Application of optimized methods to AMI patient plasma 51
• 3.7 Screening of novel AMI biomarker candidates 54
• 3.8 Verification of novel AMI biomarker candidates 61
• 3.9 Validation of novel AMI biomarker candidates 64
• 4. Discussion 69
• 5. Conclusion 74
• References 75