Membrane Protein Structures and Interactions From Covalent Labeling Coupled With Mass Spectrometry

Abstract

Membrane proteins are incredibly important biomolecules because they mediate interactions between a cell's external and internal environment. Obtaining information about membrane protein structure and interactions is thus important for understanding these essential biomolecules. Compared with the analyses of water-soluble proteins, the structural analysis of membrane proteins is more challenging owing to their unique chemical properties and the presence of lipid components that are necessary to solubilize them. The combination of covalent labeling (CL) and mass spectrometry (MS) has recently been applied with great success to study membrane protein structure and interactions. These studies have demonstrated the many advantages that CL-MS methods have over other traditional biophysical techniques. In this review, we discuss both amino acid-specific and non-specific labeling approaches and the special considerations needed to address the unique challenges associated with interrogating membrane proteins. This review highlights the aspects of this approach that require special care to be applied correctly and provides a comprehensive review of the membrane protein systems that have been studied by CL-MS.

Advances in mass spectrometry‐based footprinting of membrane proteins

“…In this section, we will summarize very recent advances on residue specific labeling of MPs. Earlier studies of residue specific reagents on MPs were in a review by Pan and Vachet [38]. Residue‐specific footprinters modify one or a few residues [90]; those residues are often located on the extra‐ or intracellular segment of MPs, and they often are involved in protein–ligand and protein–protein interactions.…”

“…We will primarily focus on developments that have occurred recently (mostly in recent 10 years). Earlier studies were reviewed elsewhere [38,39].…”

Hydroxyl Radical Protein Footprinting: A Mass Spectrometry-Based Structural Method for Studying the Higher Order Structure of Proteins

“…Most recently, HRPF has been assessed as part of a larger review on protein footprinting . In light of these as well as other HRPF reviews in recent years, − this review will mainly highlight contemporary innovations in the methodology and the most recent applications in the HRPF field.…”

Free‐Radical Membrane Protein Footprinting by Photolysis of Perfluoroisopropyl Iodide Partitioned to Detergent Micelle by Sonication

“…[15] Although residue-specific reagents (e.g.,E DC, [16] NEM [17] )c an also be applied to IMPs to footprint single, accessible residues (e.g., Cys,Glu, and Asp), the reactions run the risk of perturbing protein high order structure. [18] We reasoned that photolyzable reagents with high partition coefficients (P) into nonpolar solvents would be wellsuited for transmembrane labeling.W echose PFIPI because it has ah igh positive logP (Figure 1), small size,r eady availability,and suitability to form radicals upon 248 nm laser photolysis on the fast photochemical oxidation of proteins (FPOP) platform. [19] FPOP,i ni ts original conception, is ah ydroxyl radical (COH) protein footprinting approach that utilizes ap ulsed KrF laser (248 nm) to homolyze hydrogen peroxide in solution to COH.…”

“…Although residue‐specific reagents (e.g., EDC, [16] NEM [17] ) can also be applied to IMPs to footprint single, accessible residues (e.g., Cys, Glu, and Asp), the reactions run the risk of perturbing protein high order structure [18] …”