N-linked glycosylation (N-glycosylation) is a common protein post-translational modification, occurring on more than half of mammalian proteins; in striking contract with small molecule modifications (such as methylation, phosphorylation) with only single structures, N-glycosylation has multiple dimensional structural features (monosaccharide composition, sequence, linkage, anomer), which generates enormous N-glycan structures; and these structures widely regulate protein structure and functions. For the modification site, N-glycosylation occurs on the Asn residue among the consensus N-X-S/T/C (X≠P) motif; mutation-originated amino acid change may lead to loss of such an original motif and thus loss-of-glycosylation (LoG) or gain of such a new motif and thus gain-of-glycosylation (GoG). Both LoG and GoG generates new structures and functions of glycoproteins, which has been observed in the S protein of SARS-Cov-2 as well as malignant diseases. Here we report our glycoproteome-wide qualitative N-glycoproteomics characterization of GoGs in breast cancer Adriamycin drug resistance (ADR) cells (MCF-7/ADR) and cancer stem cells (MCF-7/ADR CSCs); comprehensive N-glycosite and N-glycan structure information at the intact N-glycopeptide level were reported.

N-连接糖基化（N-glycosylation）是一种常见的蛋白质翻译后修饰，发生在超过一半的哺乳动物蛋白质上；与仅具有单一结构的小分子修饰（如甲基化、磷酸化）形成鲜明对比的是，N-糖基化具有多维结构特征（单糖组成、序列、连接、端基异构体），从而产生巨大的 N-聚糖结构；这些结构广泛调节蛋白质的结构和功能。对于修饰位点，N-糖基化发生在共有 NXS/T/C (X≠P) 基序中的 Asn 残基上；突变引起的氨基酸变化可能导致这种原始基序的丢失，从而导致糖基化丢失 (LoG) 或这种新基序的获得，从而导致糖基化获得 (GoG)。LoG 和 GoG 都会产生糖蛋白的新结构和功能，这已在 SARS-Cov-2 的 S 蛋白以及恶性疾病中观察到。在这里，我们报告了乳腺癌阿霉素耐药 (ADR) 细胞 (MCF-7/ADR) 和癌症干细胞 (MCF-7/ADR CSC) 中 GoG 的糖蛋白组学范围的定性 N-糖蛋白组学特征；报道了完整 N-糖肽水平上的综合 N-糖苷和 N-聚糖结构信息。