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

• Published in: Journal of proteome research Vol. 20; no. 10; pp. 4787 - 4800
• Main Authors: Biedka, Stephanie, Schmidt, Brigitte F., Frey, Nolan M., Boothman, Sarah M., Minden, Jonathan S., Wilson, Amber Lee
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.10.2021
• Subjects: Click Chemistry - methods; Mass Spectrometry; Peptides; Proteome; Proteomics - methods; proteomics; sample cleanup; protein mass spectrometry; protein; protein modification; reversible chemistry; click chemistry; protein chemistry; sample preparation; two-dimensional gel electrophoresis
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.1c00443

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.