Improving the Identification of Lysine-Acetylated Peptides Using a Molecularly Imprinted Monolith Prepared by a Deep Eutectic Solvent Monomer.

To overcome the identification challenge of low-abundance lysine acetylation (Kac), a novel approach based on a molecularly imprinted polymer (MIP) was developed to improve the extraction capacity of Kac peptides in real samples. Green deep eutectic solvents (DESs) were introduced and used as one of the synergistic functional monomers with zinc acrylate (ZnA). Glycine-glycine-alanine-lysine(ac)-arginine (GGAKacR) was chosen as a template and N,N'-methylenbisacrylamide (MBAA) was used as a cross-linker. The obtained GGAKacR-MIP had excellent selectivity for the template with an imprinting factor (IF) of up to 21.4. The histone digest addition experiment demonstrated that GGAKacR-MIP could successfully extract GGAKacR from a complex sample. Finally, the application to the extraction of Kac peptides from mouse liver protein digestion was studied in detail. The number of Kac peptides and Kac proteins identified was 130 and 110, which were 3.71-fold and 3.93-fold higher than those of the untreated sample. In addition, the number of peptides and proteins identified after treatment increased from 5535 and 1092 to 17 149 and 4037 (3.10-fold and 3.70-fold, respectively). The results showed that the obtained MIP may provide an effective technical tool for the identification of Kac-modification and peptide fractionation, as well as a potential approach for simultaneously identifying post-translational-modified proteomic and proteomic information.

Improving sorption performance of a molecularly imprinted monolithic column by doping mesoporous molecular sieve SBA-15.

For the first time a hybrid molecularly imprinted polymer (MIP) doped with 3-(trimethoxysilyl) propyl methacrylate (γ-MPS)-modified mesoporous molecular sieve SBA-15 for target peptide recognition has been developed. Zinc acrylate and methacrylic acid were used as binary functional monomers, and ethylene dimethacrylate was used as cross-linking agent to prepare an imprinted monolith against Val-Tyr-Ala-Leu-Lys(glutarylation) (VYALKglu). The morphology of the polymers was characterized by scanning electron microscopy, FT-IR spectroscopy, energy dispersive spectroscopy, and 1H NMR. The SBA-15-MPS MIP showed high recovery of 87.1% and the IF of 12.9 for the enrichment of the template peptide. When the template peptide concentration ranged from 5 to 90 µg mL-1, the correlation coefficients (R2) for the calibration function obtained was better 0.999. The limit of detection (LOD, 0.30 µg mL-1) and limit of quantification (LOQ, 1.0 µg mL-1) were achieved for signal-to-noise ratios of 3:1 and 10:1, respectively. When other kinds of synthetic peptides were used as analogs, the selectivity of the SBA-15-MPS MIP was much better than the SBA-15-MPS NIP (without template peptides) with relative selectivity coefficients of 52.8-265. In contrast, little quinolones and biogenic amines are adsorbed with the SBA-15-MPS MIP. The SBA-15-MPS MIP could enrich VYALKglu from spiked histone digestion with the average recovery of 87.8% and the relative standard deviation (RSD) of 0.99%. As a conclusion, doping of SBA-15 is an effective approach to the improvement of performance of molecularly imprinted monolith.

Improving identification of molecularly imprinted monolith to benzoylation modified peptides by a deep eutectic solvents monomer-induced cooperation.

In this study, a strategy of improving imprinting performance was developed using an enhanced cooperation effect of functional monomers based on deep eutectic solvents (DESs) monomer for the specific enrichment of benzoylation modified peptides. Zinc acrylate and DESs monomers were used as binary functional monomers, and ethylene glycol dimethacrylate was used as the cross-linking agent with SGRGKbz as template to prepare an imprinted monolith. It was observed that the use of DESs monomer significantly improveed the affinity of benzoylation imprinted monolith and increased the adsorption capacity. Under optimal conditions, the recovery and imprinting factor (IF) of the imprinted monolith for SGRGKbz can reach 93.0% and 10.58, respectively. The average recovery of SGRGKbz extracted from the spiked histone digestion solution can reach 88.4% (n = 5, RSD = 3.4%). After treatment with the benzoylation imprinted monolith, 12 benzoylation modified peptides, 13 benzoylation modified sites and 12 benzoylation proteins could be identified in the digestion of mouse liver protein, while only one of each benzoylation modified peptide, benzoylation modified site and benzoylation protein could be identified in the untreated digestion of mouse liver protein. The results indicated that the prepared imprinted monolith using DESs-based functional monomer was an effective method to increase the affinity of the resulting MIP.

Dual-function monolithic enzyme reactor based on dopamine/graphene oxide coating for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment.

A new dual-function enzyme reactor was prepared based on a dopamine/graphene oxide coated boron affinity monolithic column, which can be used for simultaneous protein enzymatic hydrolysis and glycopeptide enrichment. Firstly, a boron affinity monolithic column was prepared as the carrier for enzyme reactor. Secondly, the monolithic column was coated with dopamine/graphene oxide to provide higher specific surface area for the increase in the amount of trypsin bound. Then, dopamine can self-polymerize under alkaline conditions to produce multiple reaction sites. By the Schiff base reaction and Michael addition reaction with amino, sulfhydryl groups to trypsin, enzyme were immobilized on the boron affinity monolithic carrier. The enzyme activity was characterized by kinetic parameters maximum rate (Vmax) of the enzyme reaction and Michaelis constant (Km). Km of the dual-function enzyme reactors doped with PDA/GO and without PDA/GO were 34.37 and 120.93 mM, Vmax were 1.35 and 3.35 mM/min, respectively. The performance of the dual-function enzyme reactor was evaluated by protein extraction of mouse liver. After digested by the dual-function enzyme reactor, the number of peptides was 4,801, which was 960 more than the number of peptides in the solution digestion. At the same time, the dual-function enzyme reactor displayed the ability to capture cis-dihydroxy compounds specificly. A total of 55 glycopeptides were enriched in the dual-functional enzyme reactor, corresponding to 33 glycoproteins. The dual-function enzyme reactor provided repeatable performance and robust with long-term storage.

Morphological and Phylogenetic Evidences Reveal Four New Species of Cantharellus Subgenus Cantharellus (Hydnaceae, Cantharellales) From China.

Species of Cantharellus subgenus Cantharellus are interesting and important for their mycorrhizal properties, medicinal values, and edibility. In China, there are many undescribed species of the subgenus. In this study, four new species of subg. Cantharellus, viz. Cantharellus albopileatus, Cantharellus chuiweifanii, Cantharellus pinetorus, and Cantharellus ravus from Hainan and Hunan Provinces, respectively, were described based on morphological and phylogenetic evidence as a contribution to the knowledge of the species diversity in China. Detailed descriptions, color photographs of fresh basidiomata, and line drawings of microstructures of these four new species are presented as well as comparisons with related species.