A comprehensive pipeline for translational top-down proteomics from a single blood draw

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Nature Protocols


Top-down proteomics (TDP) by mass spectrometry (MS) has become increasingly popular in translational spheres as researchers discern the value of proteoform-resolved measurements. The advent of robust prefractionation strategies for intact proteoforms and the emerging availability of high-resolution benchtop mass spectrometers have catalyzed the emergence of high-throughput TDP for the analysis of biological and clinical samples. Here, we provide a comprehensive protocol for TDP of a sample type often investigated in translational research: human peripheral blood mononuclear cells (PBMC). The workflow described herein comprises sample collection, cell lysis, proteoform prefractionation, instrument setup, liquid chromatography-tandem MS (LC-MS/MS) analysis, and data processing, for a minimum completion time of 48 hours. Importantly, this workflow is compatible with multiple PBMC processing strategies and existing biorepository practices, can be used for both small-scale targeted proteoform studies or large-scale multi-sample discovery studies, and has been successfully applied in quantitative contexts for biomarker discovery.



Top-down proteomics, Translational proteomics, Mass spectrometry, Proteoform, Proteomics





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