Recalibrating Nanoparticle Protein Corona Analysis for Accurate Biological Identity and Soluble Plasma Proteome Profiling

The nanoparticle (NP) protein corona is considered the biological identity that determines NP fate, safety, targeting, and therapeutic effectiveness in biofluids. Nonetheless, standard corona isolation workflows assume the recovered protein signature originates primarily from plasma proteins adsorbed directly onto NP surface, while largely overlooking co-isolation of endogenous nanoscale biological structures such as extracellular vesicles (EVs). This oversight can distort the apparent “biological identity” of the NP. Here, we show that EVs are a major hidden contributor to the perceived protein corona composition in human plasma. Using highly monodispersed polystyrene NPs (50-1000 nm) and superparamagnetic beads, we compared corona formation in standard human plasma and plasma depleted of EVs by immunoaffinity capture against 37 EV surface epitopes. Mass spectrometry revealed that EV depletion reduced the number of proteins identified on polystyrene NPs by 60-75% and on magnetic beads by 45-50%, demonstrating a substantial fraction of the conventionally assigned corona proteome arises from EV-associated carryover. EV removal also restructured the apparent abundance hierarchy, restoring the expected prominence of soluble plasma proteins such as albumin and shifted dominant signals away from intracellular cytoskeletal component proteins that are characteristic of EV carryover towards genuine soluble plasma adsorbates including apolipoproteins and complement factors. These results highlight that standard corona workflows can inadvertently co-isolate a vast array of EV-associated proteins, yielding inaccurate proteomic profiles. Discriminating genuine soluble plasma corona proteins from EV-associated material is essential for accurate interpretation of NP-biofluid interactions and is critical for advancing nanomedicine, ensuring predictive safety and efficacy profiles, and enhancing the precision of NP-based biomarker discovery. Significance Statement The nanoparticle (NP) “protein corona” defines how engineered nanomaterials interact with living systems, dictating their therapeutic safety, efficacy, and diagnostic utility. Conventionally, corona isolation workflows assume recovered proteins are adsorbed directly from the fluid phase. This study reveals a major, previously overlooked artifact: standard separation techniques routinely co-isolate endogenous extracellular vesicles (EVs), drastically distorting the perceived biological identity of NPs. Eliminating EVs via immunoaffinity depletion reduces identified corona proteins by up to 75% and restructures the apparent proteomic hierarchy. Distinguishing true soluble adsorbates from vesicular carryover is essential for accurately predicting NP behavior in vivo and prevents false positives in nano-diagnostics, establishing a critical new standard for high-fidelity biomarker discovery and nanomedicine.