RESUMO
SignificanceDeep profiling of the plasma proteome at scale has been a challenge for traditional approaches. We achieve superior performance across the dimensions of precision, depth, and throughput using a panel of surface-functionalized superparamagnetic nanoparticles in comparison to conventional workflows for deep proteomics interrogation. Our automated workflow leverages competitive nanoparticle-protein binding equilibria that quantitatively compress the large dynamic range of proteomes to an accessible scale. Using machine learning, we dissect the contribution of individual physicochemical properties of nanoparticles to the composition of protein coronas. Our results suggest that nanoparticle functionalization can be tailored to protein sets. This work demonstrates the feasibility of deep, precise, unbiased plasma proteomics at a scale compatible with large-scale genomics enabling multiomic studies.
Assuntos
Proteínas Sanguíneas , Aprendizado Profundo , Nanopartículas , Proteômica , Proteínas Sanguíneas/química , Nanopartículas/química , Coroa de Proteína/química , Proteoma , Proteômica/métodosRESUMO
Large-scale, unbiased proteomics studies are constrained by the complexity of the plasma proteome. Here we report a highly parallel protein quantitation platform integrating nanoparticle (NP) protein coronas with liquid chromatography-mass spectrometry for efficient proteomic profiling. A protein corona is a protein layer adsorbed onto NPs upon contact with biofluids. Varying the physicochemical properties of engineered NPs translates to distinct protein corona patterns enabling differential and reproducible interrogation of biological samples, including deep sampling of the plasma proteome. Spike experiments confirm a linear signal response. The median coefficient of variation was 22%. We screened 43 NPs and selected a panel of 5, which detect more than 2,000 proteins from 141 plasma samples using a 96-well automated workflow in a pilot non-small cell lung cancer classification study. Our streamlined workflow combines depth of coverage and throughput with precise quantification based on unique interactions between proteins and NPs engineered for deep and scalable quantitative proteomic studies.
Assuntos
Proteínas Sanguíneas/análise , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Neoplasias Pulmonares/diagnóstico , Coroa de Proteína/análise , Proteômica/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Sanguíneas/química , Carcinoma Pulmonar de Células não Pequenas/sangue , Cromatografia Líquida de Alta Pressão/métodos , Diagnóstico Diferencial , Feminino , Voluntários Saudáveis , Humanos , Neoplasias Pulmonares/sangue , Masculino , Pessoa de Meia-Idade , Nanopartículas/química , Projetos Piloto , Coroa de Proteína/química , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodos , Fatores de TempoRESUMO
Flash chromatography is the preferred approach for small molecule purification in pharmaceutical discovery. This paper will discuss the potential for flash supercritical fluid chromatography (SFC) as an alternative technology for these purifications. It was shown that the high sample loadings seen with flash LC could also be achieved using flash SFC. The dry load injection technique greatly increases the amount of sample that can be applied to a flash SFC column while still achieving separation. Flash SFC has much lower solvent usage and higher purification productivities relative to flash LC. Product concentrations post purification were higher for flash SFC vs. flash LC, reducing the time required to isolate dry product. There still exist a number of technical details to be worked out with flash SFC, mainly around the equipment and column/cartridge technology.