Search

Rationale: A general N‐glycoproteomics analysis pipeline has been established for characterization of mutation‐related gain‐of‐glycosylation (GoG) at intact N‐glycopeptide molecular level, generating comprehensive site and structure information of N‐glycosylation. Methods: This study focused on mutation‐originated GoG using a mass spectrometry‐based N‐glycoproteomics analysis workflow. In brief, GoG intact N‐glycopeptide databases were built, consisting of 2701 proteins (potential GoG N‐glycosites and amino acids derived from MUTAGEN, VARIANT and VAR_SEQ in UniProt) and 6709 human N‐glycans (≤50 sequence isomers per monosaccharide composition). We employed the site‐ and structure‐specific N‐glycoproteomics workflow utilizing intact N‐glycopeptides search engine GPSeeker to identify GoG intact N‐glycopeptides from parental breast cancer stem cells (MCF‐7 CSCs) and adriamycin‐resistant breast cancer stem cells (MCF‐7/ADR CSCs). Results: With the criteria of spectrum‐level false discovery rate control of ≤1%, we identified 87 and 94 GoG intact N‐glycopeptides corresponding to 37 and 35 intact N‐glycoproteins from MCF‐7 CSCs and MCF‐7/ADR CSCs, respectively. Micro‐heterogeneity and macro‐heterogeneity of N‐glycosylation from GoG intact N‐glycoproteins with VAR_SEQ and VARIANT were found in both MCF‐7 CSCs and MCF‐7/ADR CSCs systems. Conclusions: The integration of site‐ and structure‐specific N‐glycoproteomics approach, conjugating with GoG characterization, provides a universal workflow for revealing comprehensive N‐glycosite and N‐glycan structure information of GoG. The analysis of mutation‐originated GoG can be extended to GoG characterization of other N‐glycoproteome systems including complex clinical tissues and body fluids. [ABSTRACT FROM AUTHOR]

Isotopic-labeling quantitative N-glycoproteomics characterization of cell-surface differentially expressed N-glycosylation in MCF-7/ADR cancer stem cells (CSCs) relative to MCF-7/ADR cells was carried out at the intact N-glycopeptide level with trypsin digestion, ZIC-HILIC enrichment, isotopic diethyl labeling, RPLC-MS/MS analysis of the 1:1 mixture, and GPSeeker DB search. With a spectrum-level false discovery rate of ≤ 1%, 1,336 intact N-glycopeptides from the combination of 301 unique peptide backbones and 169 putative N-glycan linkages (52 monosaccharide compositions) were identified; the corresponding intact N-glycoproteins and N-glycosites were 289 and 305, respectively, among which 176 N-glycosites were confirmed with GlcNAc-containing site-determining b/y fragment ion pairs. The N-glycan moieties in 546 intact N-glycopeptide IDs were identified with more than one structure-diagnostic fragment ions where multiple linkage structures exist for each of the monosaccharide compositions. With the criteria of ≥ 1.5-fold change and p value < 0.05, 72 cell-surface differentially expressed intact N-glycopeptides (DEGPs) were found in MCF-7/ADR CSCs relative to MCF-7/ADR cells, where 8 and 64 were downregulated and upregulated, respectively. [ABSTRACT FROM AUTHOR]

High-throughput proteome-level characterization of stemness markers of MCF-7 cancer stem cells was carried out using our recently developed site- and structure-specific isotopic-labeling quantitative N-glycoproteomics pipeline. 144 differentially expressed intact N-glycopeptides were quantified all at once yet with good accurate consistency with the individual biochemical measurements of proteins, RNA or DNA. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • The method of building the putative gain-of-glycosylation (GoG) protein database is established • The glycoproteome level characterization of GoGs is benchmarked with two cell lines • GoGs from common mutations including MUTAGEN, VARIANT and VAR_SEQ are demonstrated. [ABSTRACT FROM AUTHOR]

[Display omitted] • 2764 IDs distinguished from sequence isomers by structure-diagnostic fragment ions. • With fold change ≥1.5 and p value < 0.05, 104 DEGPs were determined. • ITGAV, ITGB5 and LAMA1 were associated with ECM-receptor interaction by KEGG analysis. • APC, DVL3 and INVS were associated with Wnt signaling pathway by KEGG analysis. • Down-regulated intact N-glycopeptide with bisecting GlcNAc from SPT20H were found. • Upregulated intact N-glycopeptide with β1,6-branching N-glycan from ITGB5 were found. Breast cancer is responsible for the highest mortality all over the world. Cancer stem cells (CSCs) along with epithelial mesenchymal transition (EMT) are identified as a driver of cancer which are responsible for cancer metastasis and drug resistance. Several signaling pathways are associated with drug resistance. Additionally, glycosyltransferases regulate different types of glycosylation which are involved in drug resistance. To the end, it is urgent to figure out the knowledge on cell-surface altered N-glycosylation and putative markers. Here, differential cell-surface intact N-glycopeptides in adriamycin (ADR)-resistant michigan breast cancer foundation-7 stem cells (MCF-7/ADR CSCs) relative to ADR-sensitive MCF-7 CSCs were analyzed with site- and structure-specific quantitative N-glycoproteomics. The intact N-glycopeptides and differentially expressed intact N-glycopeptides (DEGPs) were determined and quantified via intact N-glycopeptide search engine GPSeeker. Totally, 4777 intact N-glycopeptides were identified and N-glycan sequence structures among 2764 IDs were distinguished from their isomers by structure-diagnostic fragment ions. Among 1717 quantified intact N-glycopeptides, 104 DEGPs were determined (fold change ≥ 1.5 and p value < 0.05). Annotation of protein–protein interaction and biological processes among others of DEGPs were finally carried out; down-regulated intact N-glycopeptide with bisecting GlcNAc from p38-interacting protein and up-regulated intact N-glycopeptide with β1,6-branching N-glycan from integrin beta-5 were found. [ABSTRACT FROM AUTHOR]

Breast cancer is one of the most malignant diseases among females. N-glycoproteomics studies have shown that N-glycosylation alteration of tumor cells is the key player of cancer progression, multidrug resistance (MDR) and high mortality. Cancer stem cells (CSCs) have the remarkable potential of self-renewing and differentiation which leads to drug resistance and metastasis. To investigate the differentially expressed N-glycosylation in adriamycin-resistant breast cancer stem cells MCF-7/ADR CSCs (relative to MCF-7 CSCs) and find the putative biomarkers, 1:1 paired ZIC-HILIC-enriched and stable isotopic diethyl labelled (SIDE) intact N-glycopeptides from MCF-7/ADR CSCs and MCF-7 CSCs were analyzed with C18-RPLC-ESI-MS/MS (HCD with stepped NCE); differentially expressed intact N-glycopeptides (DEGPs) were identified and quantified via search engine GPSeeker. With control of spectrum-level FDR≤1%, 5515 intact N-glycopeptides were identified (1737 N-glycosites, 1705 peptide backbones and 1516 intact N-glycoproteins; 181 putative N-glycan linkages and 68 monosaccharide compositions). Among 5515 intact N-glycopeptide IDs, 3864 were identified with glycoform score≥1, i.e., one or more structure-diagnostic fragment ions were observed to distinguish sequence isomers. With the three technical replicates and the criteria of fold change≥1.5 and p value<0.05, 380 DEGPs (corresponding to 153 intact N-glycoproteins) were found along with 293 down-regulated and 87 up-regulated. For these 153 intact N-glycoproteins, the molecular functions and biological processes of were comprehensively discussed, and side-to-side comparison of differential expression results with other method were also made. • Among 5515 IDs, 380 DEGPs (fold change≥1.5 and p<0.05) were found as putative N-glycoprotein drug resistance biomarkers. • With control of no less than 1.5-fold change and p < 0.05. 380 DEGPs (corresponding to 153 intact N-glycoproteins) were found along with 293 down-regulated and 87 up-regulated. • These 153 intact N-glycoproteins were found as putative N-glycoprotein drug resistance biomarkers candidates for further biological validation and final understanding of the drug resistance mechanisms. [ABSTRACT FROM AUTHOR]