Goetze Sandra, van Drogen Audrey, Albinus Jonas B, Fort Kyle L, Gandhi Tejas, Robbiani Damiano, Laforte Véronique, Reiter Lukas, Levesque Mitchell P, Xuan Yue, Wollscheid Bernd
Institute of Translational Medicine (ITM), Department of Health Sciences and Technology (D-HEST), ETH Zurich, Zurich, Switzerland.
Swiss Institute of Bioinformatics (SIB), Lausanne, Switzerland.
Clin Proteomics. 2024 Apr 2;21(1):26. doi: 10.1186/s12014-024-09478-5.
Clinical samples are irreplaceable, and their transformation into searchable and reusable digital biobanks is critical for conducting statistically empowered retrospective and integrative research studies. Currently, mainly data-independent acquisition strategies are employed to digitize clinical sample cohorts comprehensively. However, the sensitivity of DIA is limited, which is why selected marker candidates are often additionally measured targeted by parallel reaction monitoring.
Here, we applied the recently co-developed hybrid-PRM/DIA technology as a new intelligent data acquisition strategy that allows for the comprehensive digitization of rare clinical samples at the proteotype level. Hybrid-PRM/DIA enables enhanced measurement sensitivity for a specific set of analytes of current clinical interest by the intelligent triggering of multiplexed parallel reaction monitoring (MSxPRM) in combination with the discovery-driven digitization of the clinical biospecimen using DIA. Heavy-labeled reference peptides were utilized as triggers for MSxPRM and monitoring of endogenous peptides.
We first evaluated hybrid-PRM/DIA in a clinical context on a pool of 185 selected proteotypic peptides for tumor-associated antigens derived from 64 annotated human protein groups. We demonstrated improved reproducibility and sensitivity for the detection of endogenous peptides, even at lower concentrations near the detection limit. Up to 179 MSxPRM scans were shown not to affect the overall DIA performance. Next, we applied hybrid-PRM/DIA for the integrated digitization of biobanked melanoma samples using a set of 30 AQUA peptides against 28 biomarker candidates with relevance in molecular tumor board evaluations of melanoma patients. Within the DIA-detected approximately 6500 protein groups, the selected marker candidates such as UFO, CDK4, NF1, and PMEL could be monitored consistently and quantitatively using MSxPRM scans, providing additional confidence for supporting future clinical decision-making.
Combining PRM and DIA measurements provides a new strategy for the sensitive and reproducible detection of protein markers from patients currently being discussed in molecular tumor boards in combination with the opportunity to discover new biomarker candidates.
临床样本无可替代，将其转化为可搜索且可重复使用的数字生物样本库对于开展具有统计学效力的回顾性和整合性研究至关重要。目前，主要采用数据非依赖采集策略来全面数字化临床样本队列。然而，数据非依赖采集（DIA）的灵敏度有限，这就是为什么通常会通过平行反应监测额外测量选定的标志物候选物。
在此，我们应用了最近共同开发的混合平行反应监测/数据非依赖采集（Hybrid-PRM/DIA）技术，作为一种新的智能数据采集策略，该策略能够在蛋白质原型水平上对罕见临床样本进行全面数字化。Hybrid-PRM/DIA通过智能触发多重平行反应监测（MSxPRM），结合使用DIA对临床生物样本进行发现驱动的数字化，提高了对当前临床感兴趣的特定一组分析物的测量灵敏度。重标记的参考肽被用作MSxPRM的触发物以及内源性肽的监测。
我们首先在临床环境中对来自64个注释人类蛋白质组的185种选定的肿瘤相关抗原蛋白质原型肽进行了Hybrid-PRM/DIA评估。我们证明了即使在接近检测限的较低浓度下，检测内源性肽的重现性和灵敏度也有所提高。多达179次MSxPRM扫描显示不会影响整体DIA性能。接下来，我们使用一组针对28种在黑色素瘤患者分子肿瘤委员会评估中有相关性的生物标志物候选物的30种绝对定量（AQUA）肽，将Hybrid-PRM/DIA应用于生物样本库中的黑色素瘤样本的整合数字化。在DIA检测到的大约6500个蛋白质组中，选定的标志物候选物如泛素融合蛋白（UFO）、细胞周期蛋白依赖性激酶4（CDK4）、神经纤维瘤病1型（NF1）和黑素小体蛋白（PMEL）可以通过MSxPRM扫描进行一致且定量的监测，为支持未来临床决策提供了额外的可信度。
将平行反应监测（PRM）和数据非依赖采集（DIA）测量相结合，为在分子肿瘤委员会中当前正在讨论的患者蛋白质标志物的灵敏且可重复检测提供了一种新策略，同时也为发现新的生物标志物候选物提供了机会。
