Native mass spectrometry for the investigation of protein structural (dis)order.

A central challenge in structural biology is represented by dynamic and heterogeneous systems, as typically represented by proteins in solution, with the extreme case of intrinsically disordered proteins (IDPs) [1-3]. These proteins lack a specific three-dimensional structure and have poorly organized secondary structure. For these reasons, they escape structural characterization by conventional biophysical methods. The investigation of these systems requires description of conformational ensembles, rather than of unique, defined structures or bundles of largely superimposable structures. Mass spectrometry (MS) has become a central tool in this field, offering a variety of complementary approaches to generate structural information on either folded or disordered proteins [4-6]. Two main categories of methods can be recognized. On one side, conformation-dependent reactions (such as cross-linking, covalent labeling, H/D exchange) are exploited to label molecules in solution, followed by the characterization of the labeling products by denaturing MS [7-11]. On the other side, non-denaturing ("native") MS can be used to directly explore the different conformational components in terms of geometry and structural compactness [12-16]. All these approaches have in common the capability to conjugate protein structure investigation with the peculiar analytical power of MS measurements, offering the possibility of assessing species distributions for folding and binding equilibria and the combination of both. These methods can be combined with characterization of noncovalent complexes [17, 18] and post-translational modifications [19-23]. This review focuses on the application of native MS to protein structure and dynamics investigation, with a general methodological section, followed by examples on specific proteins from our laboratory.