Global profiling of functional histidines in live cells using small-molecule photosensitizer and chemical probe relay labelling.

Recent advances in chemical proteomics have focused on developing chemical probes that react with nucleophilic amino acid residues. Although histidine is an attractive candidate due to its importance in enzymatic catalysis, metal binding and protein-protein interaction, its moderate nucleophilicity poses challenges. Its modification is frequently influenced by cysteine and lysine, which results in poor selectivity and narrow proteome coverage. Here we report a singlet oxygen and chemical probe relay labelling method that achieves high selectivity towards histidine. Libraries of small-molecule photosensitizers and chemical probes were screened to optimize histidine labelling, enabling histidine profiling in live cells with around 7,200 unique sites. Using NMR spectroscopy and X-ray crystallography, we characterized the reaction mechanism and the structures of the resulting products. We then applied this method to discover unannotated histidine sites key to enzymatic activity and metal binding in select metalloproteins. This method also revealed the accessibility change of histidine mediated by protein-protein interaction that influences select protein subcellular localization, underscoring its capability in discovering functional histidines.

ACR-Based Probe for the Quantitative Profiling of Histidine Reactivity in the Human Proteome.

The quantitative profiling of residue reactivity in proteins promotes the discovery of covalent druggable targets for precise therapy. Histidine (His) residues, accounting for more than 20% of the active sites in enzymes, have not been systematically characterized for their reactivity, due to lack of labeling probes. Herein, we report a chemical proteomics platform for the site-specific quantitative analysis of His reactivity by combination of acrolein (ACR) labeling and reversible hydrazine chemistry enrichment. Based on this platform, in-depth characterization of His residues was conducted for the human proteome, in which the rich content of His residues (>8200) was quantified, including 317 His hyper-reactive residues. Intriguingly, it was observed that the hyper-reactive residues were less likely to be the sites for phosphorylation, and the possible mechanism of this antagonistic effect still needs to be evaluated in further research. Based on the first comprehensive map of His residue reactivity, many more residues could be adopted as the bindable sites to disrupt the activities of a diverse number of proteins; meanwhile, ACR derivatives could also be used as a novel reactive warhead in the development of covalent inhibitors.

Highly Efficient Enrichment of O-GalNAc Glycopeptides by Using Immobilized Metal Ion Affinity Chromatography.

Proteomics analysis of O-GalNAc glycosylation is important for the screening of biomarkers and the assessment of therapeutic responses. However, its analysis still faces challenges due to the poor performance of currently available enrichment methods. In this study, an enrichment method was established on the basis of Ti-IMAC(IV) materials, which could enrich the intact O-GalNAc glycopeptides via both the hydrophilic interaction and affinity interaction. This method enabled nearly 200 intact O-GalNAc glycopeptides identified from only 0.1 µL of human serum. This was nearly 2-fold different from that of the HILIC method. An in-depth analysis of the O-GalNAc glycosylation was performed, and 2093 intact glycopeptides were identified from 7.2 µL of human serum samples. This is the largest O-GalNAc glycosylation database of human serum from a trace amount of sample. Furthermore, 52 significantly changed intact O-GalNAc glycopeptides were determined by the quantitative analysis of hepatocellular carcinoma (HCC) and control serum samples, indicating the potential applications of this enrichment method in biomarker discovery.

Superhydrophobic conjugated microporous polymers grafted silica microspheres for liquid chromatographic separation.

Vigorously developing new high performance liquid chromatography (HPLC) stationary phases to meet the versatile separation requirements is still an important issue in the field of analytical chemistry. Conjugated microporous polymers (CMPs) are a new type of three-dimensional network porous material with high specific surface area, good chemical stability and superhydrophobicity. Herein, we firstly report the synthesis and applications of CMPs@SiO2 material for HPLC stationary phase. The CMPs@SiO2 material can be in situ fabricated via Sonogashira coupling of 1,3,5-triethynylbenzene and 1,4-diiodobenzene on the surface of spherical silica. The morphology and physicochemical properties of the synthesized stationary phase material were investigated by a series of characterization methods. Due to the superhydrophobic nature of the CMPs@SiO2 material, the packed CMPs@SiO2 HPLC column displays ultrastrong chromatographic retention and can be used for separation of both hydrophobic and hydrophilic compounds with good selectivity. Significantly, CMPs@SiO2 column can be performed for separation with pure acetonitrile as the eluent. Thus, the new column was successfully exploited for monitor and analysis of the hydrolysis of silane coupling agents. Furthermore, based on its oleophilicity, this report firstly utilized the CMPs@SiO2 material to identify and analyze the quality of cooking oils through one-step enrichment and subsequent HPLC separation. We will further exploit to fabricate versatile CMPs-based stationary phases, highlighting their potential applications in different separation scopes.