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点。其存储着活生物体病理生理状态的海量信息，但这些信息仍难以被全面获取。血浆蛋白质组的高复杂性可通过两种策略应对：一是基于全系统、无偏倚的技术，如质谱法（LC–MS/MS）；二是高灵敏度的靶向免疫分析技术，如邻近延伸分析法（PEA）。为探究不同技术间的差异与共通特征，我们分别测试了数据依赖采集和数据非依赖采集模式下的LC–MS/MS技术，以及Olink PEA技术，对来自德国南部人群队列的173份人体血浆样本中的循环血浆蛋白进行检测。两种LC–MS/MS检测模式均实现了对300余种蛋白质的定量，其中以高丰度血浆蛋白为主。通过PEA技术，我们共检测到728种血浆蛋白，其覆盖范围广、动态跨度大，且灵敏度极高，可低至pg/mL级浓度。随后，我们对三种分析平台均能检测到的35种重叠蛋白进行定量分析，验证了数据分布、检测相关性以及基于性别的差异表达的可重复性。本研究阐明了靶向与非靶向检测方法各自的局限与优势，并证实二者具备互补性。我们证实，联合使用多种检测平台可显著提升蛋白质组覆盖深度，进而深化生物学认知，这为未来生物标志物研究与机制探究提供了极具前景的研究策略。