RESUMO
Background: The wide dynamic range of circulating proteins coupled with the diversity of proteoforms present in plasma has historically impeded comprehensive and quantitative characterization of the plasma proteome at scale. Automated nanoparticle (NP) protein corona-based proteomics workflows can efficiently compress the dynamic range of protein abundances into a mass spectrometry (MS)-accessible detection range. This enhances the depth and scalability of quantitative MS-based methods, which can elucidate the molecular mechanisms of biological processes, discover new protein biomarkers, and improve comprehensiveness of MS-based diagnostics. Methods: Investigating multi-species spike-in experiments and a cohort, we investigated fold-change accuracy, linearity, precision, and statistical power for the using the Proteograph™ Product Suite, a deep plasma proteomics workflow, in conjunction with multiple MS instruments. Results: We show that NP-based workflows enable accurate identification (false discovery rate of 1%) of more than 6,000 proteins from plasma (Orbitrap Astral) and, compared to a gold standard neat plasma workflow that is limited to the detection of hundreds of plasma proteins, facilitate quantification of more proteins with accurate fold-changes, high linearity, and precision. Furthermore, we demonstrate high statistical power for the discovery of biomarkers in small- and large-scale cohorts. Conclusions: The automated NP workflow enables high-throughput, deep, and quantitative plasma proteomics investigation with sufficient power to discover new biomarker signatures with a peptide level resolution.
RESUMO
Data-independent acquisition (DIA) mass spectrometry methods provide systematic and comprehensive quantification of the proteome; yet, relatively few open-source tools are available to analyze DIA proteomics experiments. Fewer still are tools that can leverage gas phase fractionated (GPF) chromatogram libraries to enhance the detection and quantification of peptides in these experiments. Here, we present nf-encyclopedia, an open-source NextFlow pipeline that connects three open-source tools, MSConvert, EncyclopeDIA, and MSstats, to analyze DIA proteomics experiments with or without chromatogram libraries. We demonstrate that nf-encyclopedia is reproducible when run on either a cloud platform or a local workstation and provides robust peptide and protein quantification. Additionally, we found that MSstats enhances protein-level quantitative performance over EncyclopeDIA alone. Finally, we benchmarked the ability of nf-encyclopedia to scale to large experiments in the cloud by leveraging the parallelization of compute resources. The nf-encyclopedia pipeline is available under a permissive Apache 2.0 license; run it on your desktop, cluster, or in the cloud: https://github.com/TalusBio/nf-encyclopedia.
Assuntos
Proteômica , Software , Proteômica/métodos , Fluxo de Trabalho , Peptídeos/análise , Proteoma/análiseRESUMO
Data independent acquisition (DIA) is an attractive alternative to standard shotgun proteomics methods for quantitative experiments. However, most DIA methods require collecting exhaustive, sample-specific spectrum libraries with data dependent acquisition (DDA) to detect and quantify peptides. In addition to working with non-human samples, studies of splice junctions, sequence variants, or simply working with small sample yields can make developing DDA-based spectrum libraries impractical. Here we illustrate how to acquire, queue, and validate DIA data without spectrum libraries, and provide a workflow to efficiently generate DIA-only chromatogram libraries using gas-phase fractionation (GPF). We present best-practice methods for collecting DIA data using Orbitrap-based instruments and develop an understanding for why DIA using an Orbitrap mass spectrometer should be approached differently than when using time-of-flight instruments. Finally, we discuss several methods for analyzing DIA data without libraries.