Multiplatform Approach for Plasma Proteomics: Complementarity of Olink Proximity Extension Assay Technology to Mass Spectrometry-Based Protein Profiling

The plasma proteome is the ultimate target for biomarker discovery. It stores an endless amount of information on the pathophysiological status of a living organism, which is, however, still difficult to comprehensively access. The high complexity of the plasma proteome can be addressed by either a system-wide and unbiased tool such as mass spectrometry (LC–MS/MS) or a highly sensitive targeted immunoassay such as the proximity extension assay (PEA). To address relevant differences and important shared characteristics, we tested the performance of LC–MS/MS in the data-dependent and data-independent acquisition modes and Olink PEA to measure circulating plasma proteins in 173 human plasma samples from a Southern German population-based cohort. We demonstrated the measurement of more than 300 proteins with both LC–MS/MS approaches applied, mainly including high-abundance plasma proteins. By the use of the PEA technology, we measured 728 plasma proteins, covering a broad dynamic range with high sensitivity down to pg/mL concentrations. Then, we quantified 35 overlapping proteins with all three analytical platforms, verifying the reproducibility of data distributions, measurement correlation, and gender-based differential expression. Our work highlights the limitations and the advantages of both targeted and untargeted approaches and proves their complementary strengths. We demonstrated a significant gain in proteome coverage depth and subsequent biological insight by a combination of platformsa promising approach for future biomarker and mechanistic studies.血浆蛋白质组是生物标志物发现的终极靶标。它承载着活体生物病理生理状态的海量信息，然而目前仍难以实现全面获取。血浆蛋白质组的高复杂性可通过两类工具应对：一类是系统级无偏性工具，如液相色谱-串联质谱（LC–MS/MS）；另一类是高灵敏度靶向免疫分析技术，如邻近延伸分析法（proximity extension assay, PEA）。为探究相关差异与重要共通特征，我们测试了数据依赖采集（data-dependent acquisition, DDA）与数据非依赖采集（data-independent acquisition, DIA）模式下的LC–MS/MS技术，以及Olink PEA平台的性能，以测量德国南部人群队列中173份人体血浆样本的循环血浆蛋白水平。 我们通过两种LC–MS/MS分析方法，共实现了300余种蛋白质的定量检测，检测对象以高丰度血浆蛋白为主。借助PEA技术，我们完成了728种血浆蛋白的定量，该技术覆盖宽动态范围，且具备皮克每毫升（pg/mL）级的高检测灵敏度。随后，我们通过全部三种分析平台对35种重叠蛋白进行了定量，验证了数据分布、测量相关性以及基于性别的差异表达的重现性。 本研究阐明了靶向与非靶向分析策略各自的局限与优势，证实了二者的互补价值。我们证明，通过整合多种分析平台，可显著提升蛋白质组覆盖深度并获得更深入的生物学认知——这为未来的生物标志物与机制研究提供了极具前景的研究思路。