Abnormal sialylation of proteins is highly associated with many major diseases, such as cancers and neurodegenerative diseases. However, this study is challenging owing to the difficulty in enriching trace sialylated glycopeptides (SGs) from highly complex biosamples. The key to solving this problem relies strongly on the design of novel SG receptors to capture the sialic acid (SA) moieties in a specific and tunable manner. Inspired by the saccharide-saccharide interactions in life systems, here we introduce saccharide-based SG receptors into this study. Allose (a monosaccharide) displays specific and pH-sensitive binding toward SAs. Integrating allose units into a polyacrylamide chain generates a saccharide-responsive smart copolymer (SRSC). Such design significantly improves the selectivity of SA binding; meanwhile, this binding can be intelligently triggered in a large extent by solution polarity and pH. As a result, SRSC exhibits high-performance enrichment capacity toward SGs, even under 500-fold interference of bovine serum albumins digests, which is notably higher than conventional materials. In real biosamples of HeLa cell lysates, 180 sialylated glycosylation sites (SGSs) have been identified using SRSC. This is apparently superior to those obtained by SA-binding lectins including WGA (18 SGSs) and SNA (22 SGSs). Furthermore, lactose displays good chemoselectivity toward diverse disaccharides, which indicated the good potential of lactose-based material in glycan discrimination. Subsequently, the lactose-based SRSC facilitates the stepwise isolation of O-linked or N-linked SGs with the same peptide sequence but varied glycans by CH3CN/H2O gradients. This study opens a new avenue for next generation of glycopeptide enrichment materials.
An arginine-functionalized stationary phase for hydrophilic interaction liquid chromatography (HILIC) has been prepared for the first time by clicking arginine onto silica gel. It offers an efficient separation of organic acids, nucleotides and sugars. More interestingly, it exhibited excellent selectivity and enrichment toward acidic glycopeptides, even at a ratio of 1 : 150 to non-glycopeptides.
Acceptor stem is an essential region in the recognition of tRNAs by their cognate aminoacyl-tRNA synthetase. In this study, a library containing 20 nt random region and tryptophanyl-tRNA synthetase (TrpRS) from Bacillus subtilis were used for in vitro selection to find a new structural feature in the tRNA(Trp) acceptor stem sequence that is required for B. subtilis TrpRS recognition. After three rounds of selection, the TrpRS binding RNAs dominate the RNA pool. The aptamers share a common structure of three G.C base pairs, which was also found in the acceptor stem of wild-type B. subtilis tRNA(Trp). A series of tRNA(Trp) variants was prepared by in vitro transcription, and their efficiencies of tryptophanylation (k(cat)/K(M)) were measured with the aid of TrpRS from B. subtilis. The mutants that possess the three G.C base pairs and G73 discriminator base exhibit almost the same aminoacylation efficiencies as B. subtilis tRNA(Trp), while the G73 discriminator base itself cannot confer efficient aminoacylation to the tRNA(Trp) molecule. Thus, these three base pairs (G2.C71, G3.C70, and G4.C69) in the B. subtilis tRNA(Trp) acceptor stem were established to be new identity elements, and their importance was between the previously characterized major element G73 and minor elements A1/U72 and G5/C68. The minimum set of identity elements that is required to confer efficient aminoacylation by B. subtilis TrpRS included G73, G2.C71, G3.C70, and G4.C69.
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