Investigation of protein folding by mass spectrometry

Abstract

Mass spectrometry is emerging as one of the most exciting new techniques being applied to studies of protein folding. Recent developments in soft ionization techniques enable intact proteins to be generated in the gas phase from aqueous solution, and fragmentation methods are providing a means of obtaining sequence-specific information. These techniques, particularly in combination with established methods such as NMR spectroscopy, allow the investigation of both covalent and noncovalent events that occur during refolding processes. One important type of application involves mass spectrometry in an analytical role, for example, in characterizing the products of oxidative refolding experiments. Mass spectrometry can also be used to reveal fundamentally new information about the conformational properties of folding intermediates, both in isolation and in complexes with molecular chaperones, through the exploitation of hydrogen-deuterium exchange phenomena. Of particular interest is the insight this approach can provide into the cooperativity of structure formation and the distribution of intermediates at different steps along folding pathways. In this paper we describe recent insights into protein folding resulting from the application of mass spectrometry and discuss the future potential of the method for studies in this area.

Mass Spectrometric Approaches Using Electrospray Ionization Charge States and Hydrogen-Deuterium Exchange for Determining Protein Structures and Their Conformational Changes

“…Thus, significant hydrogen scrambling was observed during H/D analysis on cytochrome c after activation by sustained off-resonance irradiation in the ion cyclotron resonance cell of a mass spectrometer (66). However, other studies (49,66,67) suggested that scrambling was minimal for short helical peptides under typical CID conditions in triple quadrupole instruments. Similarly, there appeared to be no evidence for hydrogen scrambling during fragmentation of transmembrane peptides during CID in a hybrid (Q-TOF) mass spectrometer, but subsequent model studies of transmembrane peptides incorporated in a lipid bilayer indicated that the extent of scrambling was dependent on the nature of the charge carrier and amino acid sequence (67,68).…”

Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation

“…To analyze the deuterium incorporation in the aggregates, the latter are dissociated into monomers by transfer to a DMSO solution (9), and analyzed by a combination of electrospray ionization mass spectrometry (ESI-MS) (8,16) and NMR (9,10,16). MS has the unique ability to detect and characterize populations of molecules with different degrees of exchange (20). NMR analysis complements the MS analysis by defining the average proton occupancy over the distribution of protein molecules on a residue-specific basis.…”

High-frequency AC electrospray ionization source for mass spectrometry of biomolecules

“…in mass spectrometry (MS) has revolutionized the study of proteins and peptides and contributed significantly to biological and biomedical research. In contrast to ionization techniques such as fast atom bombardment (FAB), electron impact (EI), and thermal ionization that tend to fragment large molecules, ESI and MALDI minimize fragmentation, hence making them appropriate for analysis of biomolecules [3,4], polymeric macromolecules [5], and the study of the environmental implications of hazardous materials [6]. The operating principle of ESI is that it generates charged droplets from the tip of a static conical liquid structure [7] known as Taylor's cone [8].…”