Reversible Click Chemistry Tag for Universal Proteome Sample Preparation for Top-Down and Bottom-Up Analysis

Successful proteome analysis requires reliable sample preparation beginning with protein solubilization and ending with a sample free of contaminants, ready for downstream analysis. Most proteome sample preparation technologies utilize precipitation or filter-based separation, both of which have significant disadvantages. None of the current technologies are able to prepare both intact proteins or digested Peptides. Here, we introduce a reversible protein tag, ProMTag, that enables whole proteome capture, cleanup, and release of intact proteins for top-down analysis. Alternatively, the addition of a novel Trypsin derivative to the workflow generates Peptides for bottom-up analysis. We show that the ProMTag workflow yields >90% for intact proteins and >85% for proteome digests. For top-down analysis, ProMTag cleanup improves resolution on 2D gels; for bottom-up exploration, this methodology produced reproducible mass spectrometry results, demonstrating that the ProMTag method is a truly universal approach that produces high-quality proteome samples compatible with multiple downstream analytical techniques. Data are available via ProteomeXchange with identifier PXD027799.

click chemistry; protein; protein chemistry; protein mass spectrometry; protein modification; proteomics; reversible chemistry; sample cleanup; sample preparation; two-dimensional gel electrophoresis.