Addition of Unstructured Proline/Alanine/Serine (PAS) Tails to Folded Proteins Induces Chain Ejection Model Ionization in Native ESI-MS

Abstract

Denatured and unstructured proteins have been observed to take up high charge densities in native ESI-MS through the chain ejection model (CEM). This behavior is hypothesized to originate from exposed interior hydrophobic residues upon denaturation with nearly complete unfolding upon ejection of the protein. In this work, we demonstrate that natively unstructured, strongly hydrophilic proline/alanine/serine (PAS) sequences and even folded proteins derivatized with PAS tails can also ionize at least partly through CEM. To determine the mechanistic origin of hydrophilic CEM, PAS tails of different lengths were analyzed and conjugated to bovine serum albumin. The solution-phase sizes of the species correlate well with the size of droplets that efficiently emit small ions through ion evaporation, thus offering a mechanism for CEM in the absence of hydrophobicity. The charge-state distributions and folding state of these structured/unstructured hybrids upon ionization were monitored by IMS-MS, showing that CEM ionization can induce partial as well as complete unfolding of folded proteins. This results in CEM-like charge acquisition and signal enhancement to an extent that is dependent on the relative size of the natively unfolded region.