Nanosecond photochemically promoted click chemistry for enhanced neuropeptide visualization and rapid protein labeling

Abstract

Comprehensive protein identification and concomitant structural probing of proteins are of great biological significance. However, this is challenging to accomplish simultaneously in one confined space. Here, we develop a nanosecond photochemical reaction (nsPCR)-based click chemistry, capable of structural probing of proteins and enhancing their identifications through on-demand removal of surrounding matrices within nanoseconds. The nsPCR is initiated using a photoactive compound, 2-nitrobenzaldehyde (NBA), and is examined by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Benefiting from the on-demand matrix-removal effect, this nsPCR strategy enables enhanced neuropeptide identification and visualization from complex tissue samples such as mouse brain tissue. The design shows great promise for structural probing of proteins up to 155 kDa due to the exclusive accessibility of nsPCR to primary amine groups, as demonstrated by its general applicability using a series of proteins with various lysine residues from multiple sample sources, with accumulated labeling efficiencies greater than 90%.

Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI

“…With NBA and the nanosecond laser irradiation Li and coworkers [64] introduced the photoactive compound, 2nitrobenzaldehyde (NBA) in combination with the nanosecond laser irradiation at a wavelength of 355 nm for the derivatization of primary amine groups in peptides and proteins. The nanosecond laser irradiation enables the generation of the reactive 2-nitrosobenzoic anion (NS − ) which can rapidly react with primary amines in peptides and proteins, resulting in a mass shift of 133 Da.…”

Advances in High‐resolution Maldi Mass Spectrometry for Neurobiology

“…This highlights the importance of considering the potential chemical and biochemical reactions introduced during the extraction procedure. Additionally, a nanosecond photochemical reaction-based click chemistry was recently developed for the removal of matrix interference upon ionization, which enhanced neuropeptide identification by serving as a simplified cleanup step (Li et al, 2019c). Demonstrating the importance of proper sample preparation, Ly et al (2019) tested different approaches to create an optimized MSI protocol, from dissection to analysis, to determine neuropeptide distributions in the retrocerebral complex of the American cockroach, Periplaneta Americana.…”

Insight to Functional Conformation and Noncovalent Interactions of Protein-Protein Assembly Using MALDI Mass Spectrometry

“…Promising for future applications in structural proteomics and imaging is the development of a derivatization method based on nanosecond photoreactions of neuropeptides using nitrobenzaldehyde giving the reactive 2-nitrosobenzoic anion able to react with the primary group of a protein [132]. By combining the use of bisthiopropionic acid N-succinimidyl ester (BipS), a photocleavable, isotopically coded, fluorescent cross-linker, the first direct evidence was provided for the docking site of a phosphorylated G-protein-coupled receptor C terminus on the multifunctional adaptor protein beta-arrestin, clearly demonstrating the broad potential and application in structural and cellular biology [133]…”