原生質譜法主要應用於表徵蛋白質及其複合物，並提供其結構、化學計量及相互作用的資訊，是當前研究蛋白質結構和功能的有力工具。本研究回顧了原生質譜法相關的技術背景與應用，發展基於四極桿飛行時間質譜儀的原生質譜平台，也建立完整的分析流程，包含樣品製備、質譜分析及數據處理，並且進一步探討平台的優化，例如離子源的奈米電噴灑發射器尺寸、質譜儀的理論解析度和靈敏度。我們使用數種標準蛋白及抗體作為原生質譜平台測試對象，如肌紅蛋白、血紅蛋白、牛血清白蛋白、β-半乳糖苷酶、曲妥珠單抗及伴侶蛋白（GroEL）。目前，此平台已經可以量測到分子量約801千道爾頓的GroEL，顯示其對於分析大型蛋白質複合物的潛力。此外，我們也整合了原生質譜法與自上而下質譜法，可以透過原生質譜法獲得蛋白質複合物的完整質量，再藉由串聯質譜逐步將複合物分離成亞基及胺基酸片段，以獲得詳細的結構資訊。最後，我們利用此平台表徵了抗體藥物複合物，這是一類由細胞毒性藥物與抗體組成的大分子，其平均藥物抗體比是品質管控的重要指標。原生質譜法能保留抗體藥物複合體的完整結構，並根據質量推測平均藥物抗體比。總而言之，本研究建立了一個原生質譜平台，並且成功應用在蛋白質複合物的結構研究中。

Native mass spectrometry (native MS) is a powerful tool for characterizing proteins and their complexes, providing insights into their structure, stoichiometry, and interactions. This study reviews the technical background and applications of native MS, developing a native MS platform based on a quadrupole time-of-flight (Q-TOF) mass spectrometer. We established a comprehensive analysis workflow, including sample preparation, mass spectrometry analysis, and data processing, and explored platform optimization, such as the size of the nano-electrospray emitter and the theoretical resolution and sensitivity of the mass spectrometer. We tested the platform using standard proteins and antibodies, including myoglobin, hemoglobin, bovine serum albumin, β-galactosidase, trastuzumab, and chaperone protein (GroEL). The platform successfully measured GroEL with a molecular weight of approximately 801 kDa, demonstrating its potential for analyzing large protein complexes. Additionally, we integrated native MS with top-down MS (TDMS) to obtain the intact mass of protein complexes via native MS and then used tandem MS to fragment the complexes into subunits and amino acid sequences for detailed structural information. We characterized antibody-drug conjugates (ADCs) using this platform, preserving the intact structure of ADCs and estimating the average drug-to-antibody ratio, a crucial quality control metric. In summary, this study established a native MS platform successfully applied to structural studies of protein complexes.

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