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%.

Fig. 1

Nanosecond photochemical reaction (nsPCR). a Nanosecond photochemistry on 2-nitrobenzaldehyde (NBA) creating reactive 2-nitrosobenzoic anion (NS⁻) for the establishment of a localized micro-electric field and the labeling of primary amines in a protein. Tagging one NS⁻ results in a mass shift of 133 Da for proteins of interest. b On-demand three-stage matrix removal regulated by laser ON/OFF switch: Stage 1, laser irradiation establishing micro-electric field thermal gradient; Stage 2, molecular movement driven by localized PME and MST; Stage 3, the charged proteins stay near the center of laser spot while small matrices travel outside—they are thus separated within 3–5 ns and 20–50 μm. Furthermore, the electroneutral biomolecules tend to re-distribute along the thermal gradient upon laser irradiation. Both types of biomolecules can also be cleaned-up by a proton-matrix competition-related potential mechanism. c Schematic illustration of 2-nitrobenzaldehyde (NBA)-based nsPCR for large protein chemical labeling through nanosecond tagging surface accessible amine groups at the N termini and lysine residues

Fig. 2

Enhanced neuropeptide visualization via nsPCR. a Venn diagrams showing two sets of comparative data for both lipid and neuropeptide identifications with and without NBA treatments. b Optical images of mouse brain for control and experimental group with NBA. c Direct comparison of putative neuropeptide ADKNFLRFamide ([M + H]⁺, m/z 1009.568) without (left panel) and with NBA treatment (right panel); d APQRNFLRFamide ([M + H]⁺, m/z 1147.650); e RKPPFNGSIFamide ([M + K]⁺, m/z 1199.610); f RSAEGLGRMGRL ([M + K]⁺, m/z 1340.660). g Direct comparison of lipid (triacylglycerol, [TAG(52:9) + Na]⁺, m/z 867.651) distributions in mouse brain between control and labeled group with NBA. h Typical distribution of lipid (monomethyl-phosphatidylethanolamine, [MMPE(44:10) + 34 + Li]⁺, m/z 878.645) with NBA. i Overlay of lipid (phosphatidylcholine, [PC(36:1) + H]⁺, m/z 788.615, red) and neuropeptide ([SKNYLRFamide + H]⁺, m/z 926.522, green) ion images. All images were obtained with m/z tolerance of 5 ppm. Scale bar, 2 mm. Step size, 50 μm

Fig. 3

Highly efficient peptide labeling via nsPCR. A wide range of peptides (a–d, with increasing masses) with various K-residue numbers were tested using nsPCR labeling. Isotopic distributions of peptide 11 in e and insert in d confirmed the accuracy of the labeling results. w/o, without, w/, with. Individual peptide sequence information are listed in Table 1. Source data is provided in a Source Data file

Fig. 4

Large protein labeling via nsPCR. a Correlation between average number of nsPCR-crosslinked NBA and the molecular weight of large proteins. b Correlation between average number of nsPCR-crosslinked NBA and the total number of free amine groups on large proteins. Cyt c, cytochrome c; HSA, human serum albumin; bTF, bovine transferrin. The proteins in control group are preserved with 100 mM ammonium acetate, while unfolded proteins are obtained through heating in 100 mM ammonium acetate at 95 °C for 10 min. The error bars represent S.D. with n = 3 biologically independent experiments. c The structural effects of terminal sialylation on glycoprotein mouse transferrin (TF) probed by nsPCR. As a control (de-sialylated), two sialic acids (Neu5Gc, mass shift between peaks 1 and 2) on mouse transferrin are removed with sialidase under the native condition. The number of surface accessible amine group for sialylated (mass shift between 1 and 3) and de-sialylated (mass shift between peaks 2 and 4) transferrin is probed as 14.4 ± 1.1 (n = 3) and 20.7 ± 1.3 (n = 3), respectively. W/O, without; W/, with. Source data is provided in a Source Data file