Proteomics

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Mass Spectrometry-Based Top-Down Proteomics for Proteoform Profiling of Protein Corona

ABSTRACT: The protein corona, a layer of biomolecules—primarily proteins—that adsorb to nanoparticle (NP) surfaces in biological fluids, critically affects NP safety and their therapeutic and diagnostic functions. Additionally, the protein corona effectively reduces proteome complexity and the dynamic range of protein concentrations in blood plasma and other complex fluids. By suppressing highly abundant proteins, the protein corona enhances the potential for biomarker discovery across diverse health conditions, thereby advancing diagnostic and therapeutic applications. Traditionally, protein corona studies have relied on mass spectrometry (MS)-based bottom-up proteomics (BUP) to analyze corona composition. However, BUP approaches do not accurately identify specific proteoforms—distinct molecular variants of proteins—within the corona. This limitation impedes the nanomedicine field’s ability to precisely predict the biological fate and pharmacokinetics of nanomedicines, as well as their effectiveness in early-stage biomarker discovery and disease detection. Here, we present protocols utilizing capillary zone electrophoresis (CZE)-MS-based top-down proteomics (TDP) to characterize the proteoform landscape of the protein corona. Our procedures detail the recovery of intact proteoforms from NP surfaces and the measurement of these proteoforms using CZE-MS/MS and CZE-high-field asymmetric waveform ion mobility spectrometry (FAIMS)-MS/MS. The entire workflow is completed within three to four days. Implementing this protocol offers mechanistic insights into how proteoforms modulate NP–bio interactions, enabling the nanomedicine community to acquire more comprehensive protein corona profiles. This advancement has the potential to significantly enhance the diagnostic and therapeutic efficacy of nanomedicine technologies.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Blood Plasma

SUBMITTER: Liangliang Sun  

LAB HEAD: Liangliang Sun

PROVIDER: PXD060784 | Pride | 2025-09-08

REPOSITORIES: Pride

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100nl-HP-Amine-TDP-08312024-run001.raw Raw
100nl-HP-Amine-TDP-08312024-run001_ms2_toppic_proteoform_single.tsv Tabular
100nl-HP-Amine-TDP-08312024-run002.raw Raw
100nl-HP-Amine-TDP-08312024-run002_ms2_toppic_proteoform_single.tsv Tabular
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Mass spectrometry-based top-down proteomics for proteoform profiling of protein coronas.

Sadeghi Seyed Amirhossein SA   Fang Fei F   Tabatabaeian Nimavard Reyhane R   Wang Qianyi Q   Zhu Guijie G   Saei Amir Ata AA   Sun Liangliang L   Mahmoudi Morteza M  

Nature protocols 20250805

The protein corona is a layer of biomolecules-primarily proteins-that adsorbs to nanoparticle (NP) surfaces in biological fluids. If the purpose of the NP is therapeutic, this can have a profound effect on its biological activity and function in vivo. Protein corona formation can also be exploited for diagnostic purposes and to differentially enrich proteins for biomarker discovery. For all of these applications, it is useful to determine which proteins, and which specific proteoforms, bind to d  ...[more]

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