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.

Molecular Sieving on the Surface of a Nano-Armored Protein.

The molecular sieving properties of protein surface-attached polymers are the central features in how polymers extend therapeutic protein lifetimes in vivo. Yet, even after 30 years of research, permeation rates of molecules through polymer-surrounded protein surfaces are largely unknown. As a result, the generation of protein-polymer conjugates remains a stochastic process, unfacilitated by knowledge of structure-function-polymer architecture relationships. In this work, polymers are grown from the surface of avidin using atom transfer radical polymerization (ATRP) and used to determine how polymer length and density influence the binding kinetics of ligands as a function of ligand size and shape. The rate of binding is strongly dependent on the grafting density of polymers and the size of the ligand but interestingly, far less dependent on the length of the polymer. This study unveils a deeper understanding of relationship between polymer characteristics and binding kinetics, discovering important steps in rational design of protein-polymer conjugates.