Protein Footprinting Comes of Age: Mass Spectrometry for Biophysical Structure Assessment

Abstract

Protein footprinting mediated by mass spectrometry has evolved over the last 30 years from proof of concept to commonplace biophysics tool, with unique capabilities for assessing structure and dynamics of purified proteins in physiological states in solution. This review outlines the history and current capabilities of two major methods of protein footprinting: reversible hydrogen-deuterium exchange (HDX) and hydroxyl radical footprinting (HRF), an irreversible covalent labeling approach. Technological advances in both approaches now permit high-resolution assessments of protein structure including secondary and tertiary structure stability mediated by backbone interactions (measured via HDX) and solvent accessibility of side chains (measured via HRF). Applications across many academic fields and in biotechnology drug development are illustrated including: detection of protein interfaces, identification of ligand/drug binding sites, and monitoring dynamics of protein conformational changes along with future prospects for advancement of protein footprinting in structural biology and biophysics research.

Surface modification of chitosan films with a fibronectin fragment–DNA aptamer complex to enhance osteoblastic cell activity: A mass spectrometry approach probing evidence on protein behavior

“…The multi‐charge distribution of proteins was preserved, but the m/z value was centered on 1933.78, indicating an average exchange of 75 H atoms (±10). It is well known that the ESI study of protein–DNA interactions can be challenging: source parameters suitable to retain non‐covalent interactions should be selected, an adequate in‐solution protein/aptamer ratio should be evaluated to avoid non‐specific binding or ESI protein signal suppression. For this reason, different initial tests were performed on the protein/aptamer ratio to establish a suitable 1:5 ratio (data not shown).…”

“…The multi-charge distribution of proteins was preserved, but the m/z value was centered on 1933.78, indicating an average exchange of 75 H atoms (±10). It is well known 21 that the ESI study of protein-DNA interactions can be challenging: [22][23][24] whereas all the four peptides contain polar residues. These findings could suggest that the aptamer-protein interactions are not driven only by electrostatic binding, but even by polar groups all along the FN structure.…”

Laser‐Initiated Radical Trifluoromethylation of Peptides and Proteins: Application to Mass‐Spectrometry‐Based Protein Footprinting

“…Protein footprinting with reactive species (•OH, [1] carbenes [2] ) is becoming effective in structural proteomics because it modifies proteins before they undergo conformational change. Although •OH reacts with all amino acids, [3] rate constants span nearly three orders of magnitude, indicating that less-reactive residues (e.g., Gly, Ala, Asp, Asn, Ser, Thr) are not measurably footprinted whereas reactive sulfur and aromatic amino acids are preferentially modified.…”

Fluorierte Reagenzien in der Strukturproteomik

“…Footprinting charakterisiert Proteinstrukturen durch chemische Reaktionen, die die Solvenszugänglichkeit des Rückgrats oder der Seitenketten eines Proteins bestimmen . Protein‐Footprinting umfasst verschiedene Markierungsstrategien, je nach der Art der zu verwendenden chemischen Reaktion.…”