Simultaneous and site-specific profiling of heterogeneity and turnover in protein S-acylation by intact S-acylated peptide analysis with a cleavable bioorthogonal tag.

Protein S-acylation is an important lipid modification characteristic for heterogeneity in the acyl chain and dynamicity in the acylation/deacylation cycle. Most S-acylproteomic research has been limited by indirect identification of modified proteins/peptides without attached fatty acids, resulting in the failure to precisely characterize S-acylated sites with attached fatty acids. The study of S-acylation turnover is still limited at the protein level. Herein, aiming to site-specifically profile both the heterogeneity and the turnover of S-acylation, we first developed a site-specific strategy for intact S-acylated peptide analysis by introducing an acid cleavable bioorthogonal tag into a metabolic labelling method (ssMLCC). The cleavable bioorthogonal tag allowed for the selective enrichment and efficient MS analysis of intact S-acylated peptides so that S-acylated sites and their attached fatty acids could be directly analysed, enabling the precise mapping of S-acylated sites, as well as circumventing false positives from previous studies. Moreover, 606 S-palmitoylated (C16:0) sites of 441 proteins in HeLa cells were identified. All types of S-acylated peptides were further characterized by an open search, providing site-specific profiling of acyl chain heterogeneity, including S-myristoylation, S-palmitoylation, S-palmitoleylation, and S-oleylation. Furthermore, site-specific monitoring of S-palmitoylation turnover was achieved by coupling with pulse-chase methods, facilitating the detailed observation of the dynamic event at each site in multi-palmitoylated proteins, and 85 rapidly cycling palmitoylated sites in 79 proteins were identified. This study provided a strategy for the precise and comprehensive analysis of protein S-acylation based on intact S-acylated peptide analysis, contributing to the further understanding of its complexity and biological functions.

Global Analysis of Endogenously Intact S-Acylated Peptides Reveals Localization Differentiation of Heterogeneous Lipid Chains in Mammalian Cells.

S-acylation is a widespread lipidation form in eukaryotes in which various fatty acids can be covalently attached to specific cysteine residues. However, due to the low reactivity of the lipid moieties and lack of specific antibodies, purification of intact S-acylated peptides remains challenging. Here, we developed a pretreatment method for direct separation and global analysis of endogenously intact S-acylated peptides by nanographite fluoride-based solid-phase extraction (nGF-SPE), together with the investigation and optimization of the enrichment procedure as well as the LC-MS/MS analysis process. Consequently, we performed the first global profiling of endogenously intact S-acylated peptides, with 701 S-palmitoylated peptides from HeLa cell lysates in a restricted search. Furthermore, coupling the nGF-SPE method with open search mode, altogether 1119 intact S-acylated peptides were identified with the attached palmitate, palmitoleate, myristate, and octanoate chain, respectively, providing a global insight into the endogenously heterogeneous modification state. Notably, we found and validated that S-palmitoleoylation (C16:1) provided less affinity toward lipid rafts compared with S-palmitoylation (C16:0). This study developed the first straightforward way to characterize endogenously intact S-acylated peptides on a proteome-wide scale, providing the modified residues together with their attached lipid moieties simultaneously, which paves the way for further understanding of protein S-acylation.

A neutral polysaccharide from green tea: Structure, effect on α-amylase activity and hydrolysis property.

A neutral tea polysaccharide (TPSN) was isolated from green tea. Gas chromatography analysis showed that TPSN was composed of d-glucose, l-arabinose and d-galactose residues at a molar ratio of 90.0: 9.1: 0.9. The weight-averaged molecular weight of TPSN was determined as about 2.0 × 105 g mol-1 using static light scattering analysis. The result of nuclear magnetic resonance (NMR) spectroscopy indicated that TPSN and water-soluble starch had similar structures. TPSN exhibited inhibitory activity towards α-amylase through the noncompetitive inhibition mechanism, but the tertiary structure of α-amylase related to enzymatic activity, analyzed using circular dichroism spectroscopy, was not affected by TPSN. Meanwhile, TPSN exhibited hydrolysis properties catalyzed by α-amylase. Molecular docking analysis revealed that the various behaviors of TPSN to α-amylase could be attributed to that the different chain segments of TPSN combined with different amino acid residues of α-amylase.

Chain conformation of an acidic polysaccharide from green tea and related mechanism of α-amylase inhibitory activity.

An acidic tea polysaccharide (TPSA) isolated from green tea was fractionated using a precipitation-fractionation method into seven fractions with different molecular weights. TPSA was characterized as a hyperbranched polysaccharide with a globular homogeneous conformation by analysis of solution parameters of each fraction using static light scattering and viscosity analyses. Observation by transmission electron microscopy confirmed that TPSA occurred as globular homogeneous particles with size in the range of 20-40 nm. To simulate the branched chain segments of TPSA, four model molecules were designed based on chemical structure of TPSA. Molecular docking analysis indicated that the branched chain segments of TPSA similar to the TPSA-4 model molecule showed preferential binding to α-amylase to form the TPSA/α-amylase complex through hydrogen bonding interactions. Circular dichroism spectroscopy showed that the structure of α-amylase was not significantly affected by TPSA. The mechanism of α-amylase inhibitory activity of TPSA was simulated by molecular docking analysis. The branched chain segments of TPSA similar to the TPSA-4 model molecule likely act as a potential competitor to the starch substrate to inhibit the activity of α-amylase.