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.

血浆蛋白组是生物标志物发现之终极目标。它蕴藏着关于生物体病理生理状态的无限信息，然而，全面获取这些信息仍然充满挑战。血浆蛋白组的高度复杂性可通过系统性的无偏见工具，如质谱分析（LC-MS/MS），或高度灵敏的靶向免疫分析，如邻近延伸分析（PEA）来应对。为了探究相关差异和重要的共有特征，我们测试了LC-MS/MS在数据依赖性和数据独立获取模式下的性能，以及Olink PEA在测量来自南德人群队列的173份人血浆样本中的循环血浆蛋白。我们展示了通过LC-MS/MS方法测量了超过300种蛋白质，主要包括高丰度血浆蛋白。通过PEA技术，我们测量了728种血浆蛋白，覆盖了广泛的动态范围，灵敏度高达pg/mL浓度。随后，我们使用三种分析平台对35种重叠蛋白进行了定量，验证了数据分布、测量相关性和基于性别的差异表达的再现性。我们的研究突出了靶向和非靶向方法各自的局限性和优势，并证明了它们互补的强大力量。我们通过平台的组合展示了蛋白质组覆盖深度和随后生物洞察力的显著提升，这为未来的生物标志物和机制研究提供了一种有前景的方法。