Simultaneous quantitation and discovery of cancer-related proteins in plasma using a fast, high-resolution accurate mass spectrometry-based assay provides insight into precision oncology and drug discovery

The inherent heterogeneity of cancer has spurred significant technological advancements to enhance the detailed study required to characterize this complex disease, predominantly defined by uncontrolled changes. Efforts to address and treat this multi-faceted variability are executed as investigation of plasma - a readily accessible biological fluid rich in information regarding physiological states. This work seeks to present robust and standardized methods for the quantitation and discovery of known cancer-related proteins found in plasma. Specifically, our methods aim to reduce variability and labor across workflows and spur the adoption of next-generation plasma proteomic and mass spectrometry methods in discovery and development research. PQ500 peptides were prepared per manufacturer instructions, and human plasma samples from BioIVT Inc were digested using the AccelerOme automated platform and pooled as the sample matrix. Two experiments evaluated hybrid-DIA performance. The first tested different targeted peptide numbers (30-300) in SureQuant and tMS2 hybrid-DIA methods using PQ500 peptides spiked into pooled plasma. The second assessed linearity, limit of detection, and limit of quantification using serial dilutions analyzed on the Orbitrap Astral Zoom MS. LC-MS analysis was performed on a Vanquish Neo UHPLC with an EASY-Spray column and optimized gradients, with samples analyzed using both hybrid-DIA methods on the Orbitrap Astral Zoom mass spectrometer. This analysis spans 144 proteins identified as cancer-associated. Key tumor suppressors include TP53 and PTEN, while major oncogenic drivers such as EGFR, ERBB2, KIT, and SHH promote cell proliferation and survival. Proteins like VEGFA, MMP2, and MMP9 regulate angiogenesis and metastasis, and immune modulators such as PVR, VTCN1, and B2M support tumor immune evasion. Metabolic and redox enzymes including PKM2, LDHA, and TXN drive cancer cell metabolism and stress adaptation. Clinically relevant biomarkers-CEACAM5 (CEA), KLK3 (PSA), MUC16 (CA125), WFDC2 (HE4), and MSLN-aid in diagnosis and monitoring. Overall, these proteins cover major cancer hallmarks including proliferation, angiogenesis, immune escape, invasion, and metabolic reprogramming, representing both established and emerging therapeutic targets. Future directions will leverage the unique capabilities of mass spectrometry-based proteomics to investigate post-translational modifications, including phosphorylation and glycosylation, which are crucial in signaling and cancer. By integrating plasma proteomics studies, quantifiable reference peptides, and the Orbitrap Astral Zoom mass spectrometer, we aim to streamline precision oncology and drug discovery by minimizing variability in these dynamic models.