Endo-M Mediated Chemoenzymatic Approach Enables Reversible Glycopeptide Labeling for O-GlcNAcylation Analysis.

To selectively enrich O-linked ß-N-acetylglucosamine (O-GlcNAc) peptides in their original form from complex samples, we report the first reversible chemoenzymatic labeling approach for proteomic analysis. In this strategy, the O-GlcNAc moieties are ligated with long N-glycans using an Endo-M mutant, which enables the enrichment of the labeled glycopeptides by hydrophilic interaction liquid chromatography (HILIC). The attached glycans on the enriched glycopeptides are removed by wild-type Endo-M/S to restore the O-GlcNAc moiety. Compared with classic chemoenzymatic labeling, this approach enables the tag-free identification, and eliminates the interference of bulky tags in glycopeptide detection. This approach presents a unique avenue for the proteome-wide analysis of protein O-GlcNAcylation to promote its mechanism research.

Highly Efficient Enrichment of O-GlcNAc Glycopeptides Based on Chemical Oxidation and Reversible Hydrazide Chemistry.

Protein O-GlcNAcylation has been implicated in a broad range of cellular processes, while the functional research is still lagging behind other post-translational modification (PTMs), as a result of the low stoichiometry and limited enrichment efficiency. Herein, a strategy, named CHO-GlcNAc, was developed for O-GlcNAc glycopeptide enrichment. In this strategy, the O-GlcNAc glycopeptides were first enzymatically labeled with a Gal moiety, followed by chemical oxidation to efficiently introduce the aldehyde groups. The labeled O-GlcNAc glycopeptides could be efficiently enriched based on the equilibrium between the hydrazine and oxime bonds. Good specificity of the glycopeptide enrichment was observed from the mixtures of glycopeptide and non-glycopeptides using the CHO-GlcNAc method. Then, it was applied to analyze O-GlcNAcylation in the nucleus of HeLa cells, and 829 potential O-GlcNAcylation sites on 274 glycoproteins were identified, including the two readers of m6A (YTHDF1 and YTHDF3), which could provide clues for the mechanism of crosstalk between O-GlcNAcylation and other PTMs of proteins and RNA. Thus, this method could be a versatile tool for the proteomic analysis of O-GlcNAcylation.

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.

D-2-allylglycine embedded imidazolium-bridged polyhedral oligomeric silsesquioxane hybrid monolithic column for efficient separation of both small molecules and macromolecules.

The development of multifarious stationary phases is still a growing demand so as to solve the tasks of ever evolving actual applications. Herein, with D-2-allylglycine hydrochloride (AG·HCl) as the hydrophilic monomer, diene ionic liquid 1-allyl-3-vinylimidazolium bromide (AVI·Br) and polyhedral oligomeric silsesquioxane methacryl substituted (POSS-MA) as the dual crosslinkers, the highly cross-linked imidazolium-bridged POSS-AVI-AG hybrid monolithic column was fabricated via the "one-pot" free radical copolymerization. The AG·HCl embedded POSS-AVI-AG column displays typical reversed-phase liquid chromatography/hydrophilic interaction liquid chromatography mixed-mode retention mechanisms. Both hydrophobic phenols, alkylbenzenes, aromatic amines and hydrophilic nucleosides/nucleic acid bases, amides and thioureas were successfully separated with high column efficiencies (up to 571,000 plates/m for amides), outperforming our previously reported AVI·Br modified POSS-AVI column. Moreover, the column was also explored for the separation of cytochrome c tryptic digests and egg white protein extraction. All these results demonstrate that the POSS-AVI-AG column has a good potential in separation of both small molecules and complex biological samples with multiple mechanisms.