Top-down proteomics (TDP) by mass spectrometry (MS) is a technique by which intact proteins are analyzed. It has become increasingly popDesalting and concentrating GELFrEEular in translational research because of the value of characterizing distinct proteoforms of intact proteins. Compared to bottom-up proteomics (BUP) strategies, which measure digested peptide mixtures, TDP provides highly specific molecular information that avoids the bioinformatic challenge of protein inference. However, the technique has been difficult to implement widely because of inherent limitations of existing sample preparation methods and instrumentation. Recent improvements in proteoform pre-fractionation and the availability of high-resolution benchtop mass spectrometers have made it possible to use high-throughput TDP for the analysis of complex clinical samples. Here, we provide a comprehensive protocol for analysis of a common sample type in translational research: human peripheral blood mononuclear cells (PBMCs). The pipeline comprises multiple workflows that can be treated as modular by the reader and used for various applications. First, sample collection and cell preservation are described for two clinical biorepository storage schemes. Cell lysis and proteoform pre-fractionation by gel-eluted liquid fractionation entrapment electrophoresis are then described. Importantly, instrument setup and liquid chromatography–tandem MS are described for TDP analyses, which rely on high-resolution Fourier-transform MS. Finally, data processing and analysis are described using two different, application-dependent software tools: ProSight Lite for targeted analyses of one or a few proteoforms and TDPortal for high-throughput TDP in discovery mode. For a single sample, the minimum completion time of the entire experiment is 72 h.
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)