Nanopipettes Enable Native Mass Spectrometry Studies of the Intrinsically Disordered Protein α-Synuclein in Biochemical Buffers

Buffer and electrolyte conditions in vivo and in vitro are known to influence protein structure and function. Intrinsically disordered proteins (IDPs) are particularly sensitive to their solution conditions such as ionic strength and molecular crowding, and their dynamic structural ensembles rapidly respond to the cellular environment. While structural mass spectrometry (MS) techniques are uniquely able to capture aspects of this structural diversity, technical limitations have largely precluded the use of native MS and ion mobility (IM) to interrogate the conformations of IDPs in the presence of different buffers and biologically relevant concentrations of nonvolatile salts. Here, we overcome this challenge by employing sub-100 nm nanopipette electrospray ionization emitters that enable gentle and salt-tolerant analysis to study the conformational distribution of α-Synuclein (αS) monomers using native IM-MS in varied solution conditions, including in phosphate-buffered saline. We show using native MS that it is possible to capture salt- and buffer-induced changes in the αS conformational ensemble in commonly used biochemical buffers, which reflect structural changes from in silico predictions and in-solution measurements. This work demonstrates the power of nanopipette emitters for the study of IDPs and establishes native MS as a method that can be routinely used to study the conformational landscape of IDPs in nonvolatile and biologically relevant buffers.