[Progress in enrichment methods for protein N-phosphorylation].

Protein phosphorylation is one of the most common and important post-translational modifications that regulates almost all life processes. In particular, protein phosphorylation regulates the development of major diseases such as tumors, neurodegenerative diseases, and diabetes. For example, excessive phosphorylation of Tau protein can cause neurofibrillary tangles, leading to Alzheimer's disease. Therefore, large-scale methods for identifying protein phosphorylation must be developed. Rapid developmentin efficient enrichment methods and biological mass spectrometry technologies have enabled the large-scale identification of low-abundance protein O-phosphorylation modifications in, allowing for a more thorough study of their biological functions. The N-phosphorylation modifications that occur on the side-chain amino groups of histidine, arginine, and lysine have recently received increased attention. For example, the biological function of histidine phosphorylation in prokaryotes has been well studied; this type of modification regulates signal transduction and sugar metabolism. Two mammalian pHis kinases (NME1 and NME2) and three pHis phosphatases (PHPT1, LHPP, and PGAM5) have been successfully identified using various biological methods. N-Phosphorylation is involved in multiple biological processes, and its functions cannot be ignored. However, N-phosphorylation is unstable under acidic and thermal conditions owing to the poor chemical stability of the P-N bond. Unfortunately, the current O-phosphorylation enrichment method, which relies on acidic conditions, is unsuitable for N-phosphorylation enrichment, resulting in a serious lag in the large-scale identification of protein N-phosphorylation. The lack of enrichment methods has also seriously hindered studies on the biological functions of N-phosphorylation. Therefore, the development of efficient enrichment methods that target protein N-phosphorylation is an urgent undertaking. Research on N-phosphorylation proteome enrichment methods is limited, hindering functional research. Thus, summarizing such methods is necessary to promote further functional research. This article introduces the structural characteristics and reported biological functions of protein N-phosphorylation, reviews the protein N-phosphorylation modification enrichment methods developed over the past two decades, and analyzes the advantages and disadvantages of each method. In this study, both antibody-based and nonantibody-dependent methods are described in detail. Owing to the stability of the molecular structure of histidine, the antibody method is currently limited to histidine phosphorylation enrichment research. Future studies will focus on the development of new enrichment ligands. Moreover, research on ligands will promote studies on other nonconventional phosphorylation targets, such as two acyl-phosphates (pAsp, pGlu) and S-phosphate (pCys). In summary, this review provides a detailed analysis of the history and development directions of N-phosphorylation enrichment methods.

Site-specific identification and quantitation of endogenous SUMOylation based on SUMO-specific protease and strong anion exchange chromatography.

Small ubiquitin-like modifier (SUMO) modification regulates various eukaryotic cellular processes and plays a pivotal role in interferon (IFN)-mediated antiviral defense. While immunoprecipitation enrichment method is widely used for proteome-wide analysis of endogenous SUMOylation, the inability to target all SUMO forms and high cost of antibodies limited its further application. Herein, we proposed an antibody-free enrichment method based on SUMO-specific protease and strong anion exchange chromatography (SPAX) to globally profile the endogenous SUMOylation. The SUMO1/2/3-modified peptides could be simultaneously enriched by SAX chromatography by utilizing its electrostatic interaction with SUMO1/2/3 remnants, which contained multiple aspartic acids (D) and glutamic acids (E). To remove the co-enriched D/E-containing peptides which might interfere with the detection of low-abundance SUMOylated peptides, SUMO-specific protease was used to cleave the SUMO1/2/3 remnants from enriched SUMOylated peptides. As the deSUMOylated peptides lost SUMO remnants, their interaction with SAX materials became weaker, and the D/E-containing peptides could thus be depleted through the second SAX separation. The SPAX method identified over twice the SUMOylated sites than using SAX method only, greatly improving the identification coverage of endogenous SUMOylated sites. Our strategy was then applied to the site-specific identification and quantification of endogenous SUMOylation in A549 cells stimulated by IFN-γ for the first time. A total of 226 SUMOylated sites on 146 proteins were confidently identified, among which multiple up-regulated sites were involved in IFN-mediated antiviral defense, demonstrating the great promise of SPAX to globally profile and discover endogenous SUMOylation with significant biological functions.

A dual-response ratiometric fluorescent sensor by europium-doped silicon nanoparticles for fluorescent and smartphone imaging detection of tetracycline.

Given the threat to human health posed by the abuse of tetracycline (TC), the development of a portable, on-site methods for highly sensitive and rapid TC detection is crucial. In this work, we initially synthesized europium-doped silicon nanoparticles (SiEuNPs) through a facile one-pot microwave-assisted method. Due to its blue-red dual fluorescence emission (465 nm/627 nm), which was respectively attributed to the silicon nanoparticles and Eu3+, SiEuNPs were designed as a ratiometric fluorescent sensor for TC detection. For the dual-signal reverse response mechanism: TC quenched the blue emission from silicon nanoparticles through inner filter effect (IFE), and enhanced the red emission through "antenna effect" (AE) between TC and Eu3+, the nanoprobe was able to detect TC within a range of 0.2-10 µM with a limit of detection (LOD) of 10.7 nM. Notably, the equilibrium detection time was only 1 min, achieving rapid TC detection. Furthermore, TC was also measured in real samples (tap water, milk and honey) with recoveries ranging from 95.7 % to 117.0 %. More importantly, a portable smartphone-assisted on-site detection platform was developed, enabling real-time qualitative identification and semi-quantitative analysis of TC based on fluorescence color changes. This work not only provided a novel doped silicon nanoparticles strategy, but also constructed a ratiometric sensing platform with dual-signal reverse response for intuitive and real-time TC detection.

Textural Precursor Compositions Harvested for Independent Signal Generators: Scaling Micron-Sized Flower-Like Metal-Organic Frameworks as Amplifying Units for Dual-Mode Glycoprotein Assay.

In this work, a micron-sized flower-like metal-organic frameworks (MOFs)-based boronate-affinity sandwich-type immunoassay was fabricated for the dual-mode glycoprotein assay. For proof of concept, the flower-like MOFs were synthesized from transition Cu nodes and tetrakis (4-carboxyphenyl) porphyrin (TCPP) ligands by spontaneous standing assembly. In addition, the specificity toward glycoprotein involved the antigen recognition as well as covalent bonding via the boronate-glycan affinity, and the immediate signal responses were initiated by textural decomposition of the flower-like MOFs. Intriguingly, Cu nodes, of which the valence state is dominant by CuI species, can endow the Fenton-like catalytic reaction of the fluorogenic substrate for generating fluorescence signals. For benefits, TCPP ligands, in which each TCPP molecule has four guest donors, can provide multiple valences for the assembly of cyclodextrin-capped gold nanoparticles via host-guest interaction for colorimetry output. Albeit important, the scaling micrometer patterns for the flower-like MOFs carrying numerous Cu nodes and TCPP ligands can also function as amplifying units, signifying the output signal. The detection limit of the dual-mode glycoprotein assay can reach 10.5 nM for the fluorescence mode and 18.7 nM for the colorimetry mode, respectively. Furthermore, the merits of harvesting different signal generators toward the multimodal readout patterns can allow the mutual verification and make the analytical results more reliable. Collectively, our proposed assay may offer a new idea in combining the inherent textural merits from MOFs for dual signal generators, which can also emphasize accurate detection capability for glycoprotein assay.

A self-calibrating ratiometric fluorescence sensor with photonic crystal-based signal amplification for the detection of tetracycline in food.

A dual-color ratiometric fluorescence sensor based on photonic crystals (PCs) was developed to detect tetracycline (TC) in food. PC was fabricated via self-assembly of carbon dots (CDs)-loaded SiO2 nanoparticles. Gold nanoclusters (AuNCs) and copper ions (Cu2+) were then adsorbed onto the PC for sensor fabrication. The fluorescence of AuNCs was amplified by the PC with an enhancement ratio of 7.6, providing higher sensitivity. The fluorescence of AuNCs was quenched by Cu2+, whereas that of CDs remained unchanged as an internal reference. TC restored the fluorescence of AuNCs owing to its complexation with Cu2+, resulting in a change in the fluorescence intensity ratio. The sensor exhibited a good linear relationship with TC concentrations ranging from 0.1 to 10 µM, with a detection limit of 34 nM. Furthermore, the sensor was applied for TC detection in food with satisfactory recoveries and relative standard deviations, revealing great potential in practical application.

Proteomic analysis reveals that the co-ordination of cytosolic and mitochondrial pathways is beneficial for sabinene biosynthesis in engineered Saccharomyces cerevisiae.

Reconstruction and optimization of biosynthetic pathways can help to overproduce target chemicals in microbial cell factories based on genetic engineering. However, the perturbation of biosynthetic pathways on cellular metabolism is not well investigated and profiling the engineered microbes remains challenging. The rapid development of omics tools has the potential to characterize the engineered microbial cell factory. Here, we performed label-free quantitative proteomic analysis and metabolomic analysis of engineered sabinene overproducing Saccharomyces cerevisiae strains. Combined metabolic analysis andproteomic analysis of targeted mevalonate (MVA) pathway showed that co-ordination of cytosolic and mitochondrial pathways had balanced metabolism, and genome integration of biosynthetic genes had higher sabinene production with less MVA enzymes. Furthermore, comparative proteomic analysis showed that compartmentalized mitochondria pathway had perturbation on central cellular metabolism. This study provided an omics analysis example for characterizing engineered cell factory, which can guide future regulation of the cellular metabolism and maintaining optimal protein expression levels for the synthesis of target products.

Tracking the effects of PLGA-based nanoparticles on protein expression in living cells through quantitative proteomics.

Mass spectrometry (MS)-based proteomics can identify and quantify the differential abundance of expressed proteins in parallel, and bottom-up proteomic approaches are even approaching comprehensive coverage of the complex eukaryotic proteome. Protein-nanoparticle (NP) interactions have been extensively studied owing to their importance in biological applications and nanotoxicology. However, the proteome-level effects of NPs on cells have received little attention, although changes in protein abundance can reflect the direct effects of nanocarriers on protein expression. Herein, we investigated the effect of PLGA-based NPs on protein expression in HepG2 cells using a label-free quantitative proteomics approach with data independent acquisition (DIA). The percentage of two-fold change in the protein expression of cells treated with PLGA-based NPs was less than 10.15% during a 6 hour observation period. Among the changed proteins, we found that dynamic proteins involved in cell division, localization, and transport are more likely to be more susceptible to PLGA-based NPs.

Electrospun Fiber Membrane with the Dual Affinity of Chelation and Covalent Interactions for the Efficient Enrichment of Glycoproteins.

As important biomarkers of many diseases, glycoproteins are of great significance to biomedical science. It is essential to develop efficient glycoprotein enrichment platforms and investigate their adsorption mechanism. In this work, a conspicuous enrichment strategy for glycoproteins was developed by using an electrospun fiber membrane wrapped with polydopamine (PDA) and modified with 3-aminophenylboronic acid and nickel ions, named PAN/DA@PDA@APBA/Ni. The enrichment characteristics of PAN/DA@PDA@APBA/Ni toward glycoproteins were explored through adsorption behavior. Thanks to the existence of two sites of interaction (metal ion chelation and boronate affinity), PAN/DA@PDA@APBA/Ni exhibited significant enrichment capacity for glycoproteins, ovalbumin (604.6 mg/g), and human immunoglobulin G (331.0 mg/g). The adsorption kinetic results of glycoprotein ovalbumin on PAN/DA@PDA@APBA/Ni conform to the pseudo-first-order kinetic model in the first adsorption stage, while the second half adsorption stage is more in line with the pseudo-second-order kinetic model. Moreover, the physical characteristics of PAN/DA@PDA@APBA/Ni and subsequent adsorption experiments on electrospun fiber modified with only phenylboronic acid or nickel ions both confirmed two sites of interaction (metal ion chelation and boronate affinity, respectively). Furthermore, a stepwise elution method with dual-affinity interaction was designed and successfully applied to enrich glycoproteins in real biological samples. This work provides an idea for sample pretreatment, especially for the design of dual-affinity materials in glycoproteins enrichment.

Progress, Challenges and Opportunities of NMR and XL-MS for Cellular Structural Biology.

The validity of protein structures and interactions, whether determined under ideal laboratory conditions or predicted by AI tools such as Alphafold2, to precisely reflect those found in living cells remains to be examined. Moreover, understanding the changes in protein structures and interactions in response to stimuli within living cells, under both normal and disease conditions, is key to grasping proteins' functionality and cellular processes. Nevertheless, achieving high-resolution identification of these protein structures and interactions within living cells presents a technical challenge. In this Perspective, we summarize the recent advancements in in-cell nuclear magnetic resonance (NMR) and in vivo cross-linking mass spectrometry (XL-MS) for studying protein structures and interactions within a cellular context. Additionally, we discuss the challenges, opportunities, and potential benefits of integrating in-cell NMR and in vivo XL-MS in future research to offer an exhaustive approach to studying proteins in their natural habitat.

Enhancing protein dynamics analysis with hydrophilic polyethylene glycol cross-linkers.

Cross-linkers play a critical role in capturing protein dynamics in chemical cross-linking mass spectrometry techniques. Various types of cross-linkers with different backbone features are widely used in the study of proteins. However, it is still not clear how the cross-linkers' backbone affect their own structure and their interactions with proteins. In this study, we systematically characterized and compared methylene backbone and polyethylene glycol (PEG) backbone cross-linkers in terms of capturing protein structure and dynamics. The results indicate the cross-linker with PEG backbone have a better ability to capture the inter-domain dynamics of calmodulin, adenylate kinase, maltodextrin binding protein and dual-specificity protein phosphatase. We further conducted quantum chemical calculations and all-atom molecular dynamics simulations to analyze thermodynamic and kinetic properties of PEG backbone and methylene backbone cross-linkers. Solution nuclear magnetic resonance was employed to validate the interaction interface between proteins and cross-linkers. Our findings suggest that the polarity distribution of PEG backbone enhances the accessibility of the cross-linker to the protein surface, facilitating the capture of sites located in dynamic regions. By comprehensively benchmarking with disuccinimidyl suberate (DSS)/bis-sulfosuccinimidyl-suberate(BS3), bis-succinimidyl-(PEG)2 revealed superior advantages in protein dynamic conformation analysis in vitro and in vivo, enabling the capture of a greater number of cross-linking sites and better modeling of protein dynamics. Furthermore, our study provides valuable guidance for the development and application of PEG backbone cross-linkers.

Metal organic layers enabled cell surface engineering coupling biomembrane fusion for dynamic membrane proteome profiling.

Systematically dissecting the highly dynamic and tightly communicating membrane proteome of living cells is essential for the system-level understanding of fundamental cellular processes and intricate relationship between membrane-bound organelles constructed through membrane traffic. While extensive efforts have been made to enrich membrane proteins, their comprehensive analysis with high selectivity and deep coverage remains a challenge, especially at the living cell state. To address this problem, we developed the cell surface engineering coupling biomembrane fusion method to map the whole membrane proteome from the plasma membrane to various organelle membranes taking advantage of the exquisite interaction between two-dimensional metal-organic layers and phospholipid bilayers on the membrane. This approach, which bypassed conventional biochemical fractionation and ultracentrifugation, facilitated the enrichment of membrane proteins in their native phospholipid bilayer environment, helping to map the membrane proteome with a specificity of 77% and realizing the deep coverage of the HeLa membrane proteome (5087 membrane proteins). Furthermore, membrane N-phosphoproteome was profiled by integrating the N-phosphoproteome analysis strategy, and the dynamic membrane proteome during apoptosis was deciphered in combination with quantitative proteomics. The features of membrane protein N-phosphorylation modifications and many differential proteins during apoptosis associated with mitochondrial dynamics and ER homeostasis were found. The method provided a simple and robust strategy for efficient analysis of membrane proteome, offered a reliable platform for research on membrane-related cell dynamic events and expanded the application of metal-organic layers.

Pyrylium-based derivatization for rapid labeling and enhanced detection of thiol in mass spectrometry imaging.

Many endogenous antioxidants, including glutathione (GSH), cysteine (Cys), cysteinyl-glycine (Cys-Gly) and homocysteine (Hcy) possess free thiol functional groups. In most cases, matrix-assisted laser desorption ionization (MALDI) analyses of trace amounts of thiol compounds are challenging because of their instability and poor ionization properties. We present a mass spectrometry imaging (MSI) approach for mapping of thiol compounds on brain tissue sections. Our derivatization reagents 1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-2,4,6-trimethylpyridinium (MTMP) and 1-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-2,4,5-triphenylpyridinium (MTPP) facilitate the covalent charge-tagging of molecules containing free thiol group for the selective and rapid detection of GSH synthesis and metabolic pathway related metabolites by MALDI-MSI. The developed thiol-specific mass spectrometry imaging method realizes the quantitative detection of exogenous N-acetylcysteine tissue sections, and the detection limit in mass spectrometry imaging could reach 0.05 ng. We illustrate the capabilities of the developed method to mapping of thiol compounds on brain tissue from the chronic social defeat stress (CSDS) depression model mice.

On-capillary alkylation micro-reactor: a facile strategy for proteo-metabolome profiling in the same single cells.

Single-cell multi-omics analysis can provide comprehensive insights to study cell-to-cell heterogeneity in normal and disease physiology. However, due to the lack of amplification technique, the measurement of proteome and metabolome in the same cell is challenging. Herein, a novel on-capillary alkylation micro-reactor (OCAM) was developed to achieve proteo-metabolome profiling in the same single cells, by which proteins were first covalently bound to an iodoacetic acid functionalized open-tubular capillary micro-reactor via sulfhydryl alkylation reaction, and metabolites were rapidly eluted, followed by on-column digestion of captured proteins. Compared with existing methods for low-input proteome sample preparation, OCAM exhibited improved efficiency, anti-interference ability and recovery, enabling the identification of an average of 1509 protein groups in single HeLa cells. This strategy was applied to single-cell proteo-metabolome analysis of mouse oocytes at different stages, 3457 protein groups and 171 metabolites were identified in single oocytes, which is the deepest coverage of proteome and metabolome from single mouse oocytes to date, achieving complementary characterization of metabolic patterns during oocyte maturation.

Simultaneous Mapping of Amino Neurotransmitters and Nucleoside Neuromodulators on Brain Tissue Sections by On-Tissue Chemoselective Derivatization and MALDI-MSI.

Neurotransmitters (NTs) and neuromodulators (NMs) are two of the most important neurochemicals in the brain, and their imbalances in specific brain regions are thought to underlie certain neurological disorders. We present an on-tissue chemoselective derivatization mass spectrometry imaging (OTCD-MSI) method for the simultaneous mapping of NTs and NMs. Our derivatization system consists of a pyridiniumyl-benzylboronic acid based derivatization reagent and pyrylium salt, which facilitate covalent charge labeling of molecules containing cis-diol and primary amino, respectively. These derivatization systems improved the detection sensitivity of matrix-assisted laser desorption/ionization (MALDI)-MSI and simplified the identification of amino NTs and nucleoside NMs by the innate chemoselectivity of derivatization reagents and the unique isotopic pattern of boron-derivative reagents. We demonstrated the ability of the developed method on brain sections from a hypoxia mouse model and control. The simultaneous imaging of NTs and NMs provided a method for exploring how hypoxic stress and drugs affect specific brain regions through neurotransmitter modulation.

Decoding Protein Dynamics in Cells Using Chemical Cross-Linking and Hierarchical Analysis.

Protein dynamics play a crucial role in their diverse functions. The intracellular environment significantly influences protein dynamics, particularly for intrinsically disordered proteins (IDPs). To comprehensively capture structural information from various proteins within cells and characterize protein dynamics, chemical cross-linking mass spectrometry was employed. In this study, we introduce a hierarchical decoding strategy that enables the investigation of protein dynamics in vivo. Computational analysis based on distance restraints derived from cross-links is used to infer protein dynamics in cells. To facilitate this analysis, we leverage the prior structure obtained from AlphaFold2. By employing this strategy, we can characterize the full-length structure of multi-domain proteins taking into account their distinct dynamic features. Furthermore, by combining restraint sampling with an unbiased sampling and evaluation approach, we can provide a comprehensive description of the intrinsic motion of IDPs. Consequently, the hierarchical strategy we propose holds significant potential in advancing our understanding of the molecular mechanisms that undelie protein functions in cells.

Targeted cross-linker delivery for the in situ mapping of protein conformations and interactions in mitochondria.

Current methods for intracellular protein analysis mostly require the separation of specific organelles or changes to the intracellular environment. However, the functions of proteins are determined by their native microenvironment as they usually form complexes with ions, nucleic acids, and other proteins. Here, we show a method for in situ cross-linking and analysis of mitochondrial proteins in living cells. By using the poly(lactic-co-glycolic acid) (PLGA) nanoparticles functionalized with dimethyldioctadecylammonium bromide (DDAB) to deliver protein cross-linkers into mitochondria, we subsequently analyze the cross-linked proteins using mass spectrometry. With this method, we identify a total of 74 pairs of protein-protein interactions that do not exist in the STRING database. Interestingly, our data on mitochondrial respiratory chain proteins ( ~ 94%) are also consistent with the experimental or predicted structural analysis of these proteins. Thus, we provide a promising technology platform for in situ defining protein analysis in cellular organelles under their native microenvironment.

Crosslinker Nanocarriers Delivery to Chloroplasts for In Vivo Mapping of Photosynthetic Membrane Protein Complexes in Living Chlamydomonas reinhardtii Cells.

Photosynthesis, as the core of solar energy biotransformation, is driven by photosynthetic membrane protein complexes in plants and algae. Current methods for intracellular photosynthetic membrane protein complex analysis mostly require the separation of specific chloroplasts or the change of the intracellular environment, which causes the missing of real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membrane protein complexes in the chloroplasts of living Chlamydomonas reinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricated to deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO) into the chloroplasts to crosslink photosynthetic membrane protein complexes. After the in vivo crosslinked protein complexes were extracted and digested, mass spectrometry was employed to detect lysine-specific crosslinked peptides for further elucidating the protein conformations and interactions. With this method, the weak interactions between extrinsic proteins in the luminal side (PsbL and PsbH) and the core subunits (CP47 and CP43) in photosynthetic protein complexes were directly captured in living cells. Additionally, the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related to the biosynthesis of light-harvesting antennae. These results indicated that in vivo analysis of photosynthetic protein complexes based on crosslinker nanocarriers was expected to not only figure out the difficulty in the study of photosynthetic protein complexes in living cells but also provide an approach to explore transient and weak interactions and the function of uncharacterized proteins.

ON-OFF Fluorescent Cyanine Dye Based on a Benzothiophenyl Rotor Enables Selective Illumination of G-Quadruplexes in Mitochondria.

Conventional cyanine dyes exist as "always-on" fluorescent probes leading to inevitable background signals which often limit their performance and scope of applications. To develop specific fluorescent probes with high sensitivity and robust OFF/ON switching for targeting G4s, we introduced aromatic heterocycles through conjugation with polymethine chains to construct a rotor-π system. Here, a universal strategy is presented to synthesize pentamethine cyanines with different aromatic heterocycle substituents on the meso-polymethine chain. In these probes, SN-Cy5-S is self-quenched in aqueous solution due to H-aggregation. The structure indicates that SN-Cy5-S with a flexible meso-benzothiophenyl rotor conjugated to the cyanine backbone matches adaptively with G-tetrad planes, enhancing π-π stacking and resulting in triggered fluorescence. This allows recognition of G-quadruplexes due to the synergy of disaggregation-induced emission (DIE) and inhibited twisted intramolecular charge-transfer effects. This combination leads to a robust lighting-up fluorescence response for c-myc G4 with superior fluorescence enhancement (98-fold), allowing for a low detection limit of 1.51 nM, which is much more sensitive than the previously reported DIE-based G4 probes (22-83.5 nM). In addition, the superior imaging properties and rapid internalization time (5 min) in mitochondria allow SN-Cy5-S to also have a high potential for mitochondrially targeting anti-cancer therapy.

Improved Cross-Linking Coverage for Protein Complexes Containing Low Levels of Lysine by Using an Enrichable Photo-Cross-Linker.

Chemical cross-linking coupled with mass spectrometry (XL-MS) is an important technique for the structural analysis of protein complexes where the coverage of amino acids and the identification of cross-linked sites are crucial. Photo-cross-linking has multisite reactivity and is valuable for the structural analysis of chemical cross-linking. However, a high degree of heterogeneity results from this multisite reactivity, which results in samples with higher complexity and lower abundance. Additionally, the applicability of photo-cross-linking is limited to purified protein complexes. In this work, we demonstrate a photo-cross-linker, alkynyl-succinimidyl-diazirine (ASD) with the reactive groups of N-hydroxysuccinimide ester and diazirine, as well as the click-enrichable alkyne group. Photo-cross-linkers can provide higher site reactivity for proteins that contain only a small number of lysine residues, thereby complementing the more commonly used lysine-targeting cross-linkers. By systematically analyzing proteins with differing lysine contents and differing flexibilities, we demonstrated clear enhancement in structure elucidation for proteins containing less lysine and with high flexibility. In addition, enrichment approaches of alkynyl-azide click chemistry conjugated with biotin-streptavidin purification (coinciding with parallel orthogonal digestion) improved the identification coverage of cross-links. We show that this photo-cross-linking approach can be used for membrane proteome-wide complex analysis. This method led to the identification of a total of 14066 lysine-X cross-linked site pairs from a total of 2784 proteins. Thus, this cross-linker is a valuable addition to a photo-cross-linking toolkit and improves the identification coverage of XL-MS in functional structure analysis.

Mitochondria-Targeted Thymidylate Synthase Inhibitor Based on Fluorescent Molecularly Imprinted Polymers for Tumor Antimetabolic Therapy.

Antimetabolites targeting thymidylate synthase (TS), such as 5-fluorouracil and capecitabine, have been widely used in tumor therapy in the past decades. Here, we present a strategy to construct mitochondria-targeted antimetabolic therapeutic nanomedicines based on fluorescent molecularly imprinted polymers (FMIP), and the nanomedicine was denoted as Mito-FMIP. Mito-FMIP, synthesized using fluorescent dye-doped silica as the carrier and amino acid sequence containing the active center of TS as the template peptide, could specifically recognize and bind to the active site of TS, thus inhibiting the catalytic activity of TS, and therefore hindering subsequent DNA biosynthesis, ultimately inhibiting tumor growth. The imprinting factor of FMIP reached 2.9, and the modification of CTPB endowed Mito-FMIP with the ability to target mitochondria. In vitro experiments demonstrated that Mito-FMIP was able to efficiently aggregate in mitochondria and inhibit CT26 cell proliferation by 59.9%. The results of flow cytometric analysis showed that the relative mean fluorescence intensity of Mito-FMIP accumulated in the mitochondria was 3.4-fold that of FMIP. In vivo experiments showed that the tumor volume of the Mito-FMIP-treated group was only one third of that of the untreated group. In addition, Mito-FMIP exibited the maximum emission wavelength at 682 nm, which allowed it to be used for fluorescence imaging of tumors. Taken together, this study provides a new strategy for the construction of nanomedicines with antimetabolic functions based on molecularly imprinted polymers.