Protein Structural Analysis via Mass Spectrometry-Based Proteomics

Abstract

Modern mass spectrometry (MS) technologies have provided a versatile platform that can be combined with a large number of techniques to analyze protein structure and dynamics. These techniques include the three detailed in this chapter: 1) hydrogen/deuterium exchange (HDX), 2) limited proteolysis, and 3) chemical crosslinking (CX). HDX relies on the change in mass of a protein upon its dilution into deuterated buffer, which results in varied deuterium content within its backbone amides. Structural information on surface exposed, flexible or disordered linker regions of proteins can be achieved through limited proteolysis, using a variety of proteases and only small extents of digestion. CX refers to the covalent coupling of distinct chemical species and has been used to analyze the structure, function and interactions of proteins by identifying crosslinking sites that are formed by small multi-functional reagents, termed crosslinkers. Each of these MS applications is capable of revealing structural information for proteins when used either with or without other typical high resolution techniques, including NMR and X-ray crystallography.

Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)*

“…By contrast, the relatively low amounts of samples required (19) and the rapidity (6) by which mass spectrometric epitope mapping is executed is of great advantage in this respect (20). Chemical cross-linking mass spectrometry (21,22), hydrogen/deuterium exchange (HDX) 1 mass spectrometry (23) and mass spectrometric methods that employ chemical modification on proteins, such as Fast Photochemical Oxidation of Proteins (FPOP) (24,25) or chemical modification of surface exposed residues (26,27) have been applied in epitope mapping experiments (28) and in determinations of protein -protein interaction sites in general (29), but their application may be limited when rather demanding chemistries are involved, or when performing such experiments becomes laborious, and/or requires sophisticated laboratory equipment (20,30). Significant advances in epitope mapping protocols/methods have been reached with the two most commonly used mass spectrometric methods: epitope extraction and epitope excision (20,31,32).…”

The structure of the large regulatory α subunit of phosphorylase kinase examined by modeling and hydrogen‐deuterium exchange

“…In HDX, amide backbone exchange of protons with deuterons provides information on a protein's secondary and tertiary structure, as well as dynamics, by measuring the number of hydrogens that exchange for deuterons on the amide backbone of the protein when incubated in D 2 O buffer. Exchange of these hydrogens depends on solvent exposure, dynamics, and secondary structure, as hydrogen‐bonds and burial of residues inhibit exchange . We have measured HDX for the α subunit as part of the non‐activated (αβγδ) 4 PhK complex to gain insight into its structure.…”

Emerging Role of Mass Spectrometry‐Based Structural Proteomics in Elucidating Intrinsic Disorder in Proteins

“…However with the advent of MS, LiP is now routinely used in combination with MS, through SDS‐PAGE, ESI‐MS, or MALDI. [ 49 ] After brief exposure to low concentration of protease, the digestion is quenched, and peptides are analyzed by MS. In case of trypsin, reaction is quenched at low pH, as trypsin is active only at slightly alkaline pH.…”