Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5425_Библиотеки_им_академика_М_И_Перельмана

Released glycans can then undergo chemical derivatization at their reducing end, or be directly analyzed in their native form[157]. Released glycan species contain a reactive carbonyl group at their reducing end that can be readily derivatized. Derivatization methods equalize the chemical properties of glycans and are espe­cially suitable for: glycan labeling with fluorescent tags to allow sensitive analyte detection; stabilization of the most labile groups of glycan chains, in particular Neu5Ac moieties, which can be easily lost during sample ionization; and providing every released glycan species with a uniform charge, which improves their ioniza­tion efficiency. This is of particular importance since carbohydrates are not as easily transferred to the gas phase as proteins or peptides, which are ubiquitously proto­nated, and do not ionize as efficiently, being particularly susceptible to ion suppres­sion when using positive ion mode analysis[155]. Moreover, during the ionization process of an analyte mixture, more hydrophobic biomolecules will cause the ion suppression of less hydrophobic ones. It is, thus, advisable to separate glycans according to their acidity prior to MS analysis. The most widely used derivatization strategies include reductive amination and glycan permethylation[157]. The latter method precludes the formation of intermolecular hydrogen bonds, which increase glycan hydrophobicity, volatility, and, consequently, ion signal intensity. Besides, it favors the formation of diagnostic fragment ions, i.e. monosaccharide oxonium ions, which provide invaluable structural information. Additionally, glycan per­methylation prevents intramolecular rearrangements, such as fucose transfer between the N‐glycan core and antennae, which preclude unequivocal glycan struc­tural assignment[158]. Finally, by eliminating the negative charge from Neu5Ac moieties, glycan derivatization stabilizes sialylated groups for positive ion mode analysis[159]. Recently, a novel method for linkage‐specific Neu5Ac derivatization was developed based on the ethyl esterification of α2,6Neu5Ac groups, followed by the lactonization and sequential stable amidation of α2,3Neu5Ac motifs[160]. This Neu5Ac derivatization method has been applied to confirm the abrogation of α2,6‐ sialylated N‐glycan species from the ErbB2 glycome in ST6GAL1 KO gastric cancer cells[48].

consequently, the concentration of suppressing molecules through the use of nanoscale ESI setups[160]. Moreover, when compared with MALDI, ESI produces better resolved peaks for glycoconjugates due to the absence of matrix‐derived adduct peaks.

In the case of glycoproteins, site‐specific mapping and structural characterization of attached glycan species are usually warranted. This type of structural analysis can be performed on intact glycoproteins (top‐down glycoproteomics) or glycopeptides resulting from proteolytic digestion (bottom‐up glycoproteomics). Although trypsin remains the gold standard enzyme for the digestion of glycoproteins into glycosylated and nonglycosylated peptides, tryptic glycopeptides may harbor multiple glycosyla­tion sites or be too long to be readily analyzed by MS[174]. Indeed, post-translational glycosylation often protects the peptidic backbone from enzymatic digestion (miscle­avage), yielding longer glycopeptides that are not as easily ionizable[175]. Thus, the use of other proteases bearing distinct cleavage specificities or the combination of multiple digesting enzymes may allow for wider and more comprehensive glycopro­teome coverage[176, 177]. However, the use of less specific proteases may generate glycopeptides that are not unique and that produce poorly understood fragmentation patterns, thus hampering precise glycopeptide assignment.

both the glycan and peptide portions can be obtained through distinct yet comple­mentary tandem fragmentation strategies. Indeed, hybrid fragmentation methods

homeostatic functions of tumor‐suppressive elements or by enhancing the onco­genic properties of proteins controlling cell adhesion, programmed cell death, mito­genic signaling, and extracellular communication. However, the mechanistic basis underlying such alterations, as well as the functional impact that these may have on tumor onset, development, and metastatic spread, warrant further investigation. Indeed, several biosynthetic and structural features of glycans pose unique chal­lenges to their compositional characterization, structural elucidation, carrier assign­ment, and functional study. The vast array of monosaccharide substrate donors and transporters and the partial redundant and overlapping specificities depicted by GTs and glycosidases exponentially increase the structural complexity of the human gly­cosylation landscape. Moreover, the nontemplate‐driven nature of glycosylation greatly hinders the unequivocal analytical‐based structural elucidation of complex glycan chains. In recent years, we have witnessed the exponential technological development of cellular‐, imaging‐, and analytical‐based tools and platforms that have been instrumental in the unprecedented association between a glycan struc­ture and its function within complex biological systems. The rapidly expanding tool­box of gene editing technologies has allowed the generation of cellular and animal models depicting cleaner and well‐defined glycosylation backgrounds in which the study of glycan biosynthesis and function can be performed in the absence of con­founding factors such as GT redundant and compensatory activity. Increasingly complex imaging systems have prompted the comprehensive characterization of glycan expression patterns and distribution across histologically defined regions, as well as the identification and mapping of tumor‐relevant glycan–protein interac­tions at the single complex level, in tissue specimens from oncological patients. The emerging glycan microarray technology has fueled the high‐throughput interroga­tion and dissection of the glycan‐binding specificities of disease‐relevant carbohy­drate‐based molecular interactions actively underpinning numerous features of malignant cells, including evasion to immune recognition and surveillance. The MS‐based elucidation of the composition, 3D structure, and site of attachment of cancer‐associated glycan antigens has the potential to revolutionize the structure‐ guided design and optimization of cancer therapeutics. In conclusion, glycans undeniably represent a novel set of tools that will become indispensable in the field of modern‐era precision oncology, either as robust molecular biomarkers bearing diagnostic, prognostic, predictive, and stratifying value, or as promising targets for the delivery of personalized anticancer therapeutic agents.