Abstract
A sizeable proportion of active protein sequences lack structural motifs making them irresolvable by NMR and crystallography. Such intrinsically disordered proteins (IDPs) or regions (IDRs) play a major role in biological mechanisms. They are often involved in cell regulation processes, and by extension can be the perpetrator or signifier of disease. In light of their importance and the shortcomings of conventional methods of biophysical analysis to identify them and to describe their conformational variance, IDPs and IDRs have been termed "the dark proteome." In this chapter we describe the use of ion mobility-mass spectrometry (IM-MS) coupled with electrospray ionization to analyze the conformational diversity of IDPs. Using the LEA protein COR15A as an exemplar system and contrasting it with the behavior of myoglobin, we outline the methods for analyzing an IDP using nanoelectrospray ionization coupled with IM-MS, covering sample preparation, purification; optimization of mass spectrometry conditions and tuning parameters; data collection and analysis. Following this, we detail the use of a "toy" model that provides a predictive framework for the study of all proteins with ESI-IM-MS.
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