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Glycation is a non-enzymatic posttranslational modification coming from the reaction between reducing sugars and free amino groups in proteins, where early glycation products (fructosyl-lysine, FL) and advanced glycation end products (AGEs) are formed. The occurrence of glycation and accumulation of AGEs have been closely associated with hepatocellular carcinoma (HCC). Here, we reported the characterization of differential glycation in HCC using tissue proteomics with stable isotopic labeling; early glycation-modified peptides were enriched with boronate affinity chromatography (BAC), and AGEs-modified peptides were fractionated with basic reversed-phase separation. By this integrated approach, 3717 and 1137 early and advanced glycated peptides corresponding to 4007 sites on 1484 proteins were identified with a false discovery rate (FDR) of no more than 1%. One hundred fifty-five sites were modified with both early and advanced end glycation products. Five early and 7 advanced glycated peptides were quantified to be differentially expressed in HCC tissues relative to paired adjacent tissues. Most (8 out of 10) of the proteins corresponding to the differential glycated peptides have previously been reported with dysregulation in HCC. The results together may deepen our knowledge of glycation as well as provide insights for therapeutics. [ABSTRACT FROM AUTHOR]

N -glycosylation is a common protein post translation modification, which has tremendous structure diversity and wide yet delicate regulation of protein structures and functions. Mass spectrometry-based N -glycoproteomics has become a state-of-the-art pipeline for both qualitative and quantitative characterization of N -glycosylation at the intact N -glycopeptide level, providing comprehensive information of peptide backbones, N -glycosites, monosaccharide compositions, sequence and linkage structures. For high-throughput analysis of large-cohort clinic samples, fast and high-performance separation is indispensable. Here we report our development of 1-h liquid chromatography gradient N -glycoproteomics method and accordingly optimized MS parameters. In the benchmark analysis of cancer and paracancerous tissue of hepatocellular carcinoma, 5,218 intact N -glycopeptides were identified, where 422 site- and structure-specific differential N -glycosylation on 145 N -glycoproteins was observed. The method, representing substantial increase of throughput, can be adopted for fast and efficient analysis of N -glycoproteomes at large scale. [Display omitted] • One-hour N -glycoproteomics with 1-h gradient is developed for fast analysis. • 85 % more IDs was achieved per elution time than previous 4-h gradient. • Fast N -glycoproteomics may find wide application of large-cohort study. [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]

Protein sialylation participates many biological processes in a linkage-specific manner, and aberrant sialylation has been associated with many malignant diseases. Mass spectrometry-based quantitative N-glycoproteomics has been widely adopted for quantitative analysis of aberrant sialylation, yet multiplexing method at intact N-glycopeptides level is still lacking. Here we report our study of sialic acid linkage-specific quantitative N-glycoproteomics using selective alkylamidation and multiplex tandem mass tags (TMT)-labeling. With lung cancer as a model system, differential sialylation in cancer tissues relative to adjacent non-tumor tissues was characterized at the intact N-glycopeptide level with N-glycosite information. TMT-labeled intact N-glycopeptides with and without sialic acid alkylamidation were subject to reversed-phase liquid chromatography-nano-electron spray ionization-tandem mass spectrometry (RPLC-nanoESI-MS/MS) analysis to provide comprehensive characterization of N-glycosylation with and without sialic acid at the intact N-glycopeptide level with structure and N-glycosite. In this study, 6384 intact N-glycopeptides without sialylation were identified and 521 differentially expressed intact N-glycopeptides from 254 intact N-glycoproteins were quantified. Eight intact N-glycoproteins responsible for N-glycan biosynthesis were identified as glycosyltransferases. In total, 307 sialylated intact N-glycopeptides with linkage-specific sialic acid residues were identified together with 29 N-glycans with α2,6-linked sialic acids and 55 N-glycans with α2,3-linked sialic acids. Intact N-glycoproteins with α2,6-sialylation were associated with coronavirus disease-(COVID)-19. Additionally, many types of N-glycosylation including terminal N-galactosylation, core and/or branch fucosylation, α2,6-sialylation and terminal bisecting N-acetylglucosamine were identified and quantified in intact N-glycoproteins from immunoglobulin family. [Display omitted] • Sialic acid linkage-specific quantitative N-glycoproteomics with selective alkylamidation and multiplex TMT-labeling. • Eight N-glycoproteins about N-glycan biosynthesis were identified as glycosyltransferases. • 307 sialylated intact N-glycopeptides identified with 29 α2,6-sialylated N-glycans and 55 α2,3-sialylated N-glycans. • Intact N-glycoproteins with α2,6-sialylation were associated with coronavirus disease-COVID-19. • Many N-glycosylation especially α2,6-sialylation quantified in intact N-glycopeptides from immunoglobulin family. [ABSTRACT FROM AUTHOR]

• HA degradation promotes odontoblastic differentiation and cell migration of mouse dental papilla cells. • During odontoblastic differentiation, HYAL2 is highly localized in odontoblasts in vivo. • Hyal2 knockdown leads to the accumulation of HA, which impedes dentinogenesis and cell migration. • Hyal2 silencing significantly affects the cytoskeletal networks and focal adhesion, and inhibits the activation of PI3K/Akt signaling pathway. The coordination between odontoblastic differentiation and directed cell migration of mesenchymal progenitors is necessary for regular dentin formation. The synthesis and degradation of hyaluronan (HA) in the extracellular matrix create a permissive niche that directly regulates cell behaviors. However, the role and mechanisms of HA degradation in dentin formation remain unknown. In this work, we present that HA digestion promotes odontoblastic differentiation and cell migration of mouse dental papilla cells (mDPCs). Hyaluronidase 2 (HYAL2) is responsible for promoting odontoblastic differentiation through degrading HA, while hyaluronidase 1 (HYAL1) exhibits negligible effect. Silencing Hyal2 generates an extracellular environment rich in HA, which attenuates F-actin and filopodium formation and in turn inhibits cell migration of mDPCs. In addition, activating PI3K/Akt signaling significantly rescues the effects of HA accumulation on cytodifferentiation. Taken together, the results confirm the contribution of HYAL2 to HA degradation in dentinogenesis and uncover the mechanism of the HYAL2-mediated HA degradation in regulating the odontoblastic differentiation and migration of mDPCs. [ABSTRACT FROM AUTHOR]

Non-imaging radar can achieve the detection and localization of moving targets, but it faces difficulties in the classification of targets. Aiming for the application of moving target classification by low/medium resolution non-imaging radar, this paper proposes a novel feature extraction method, based on the statistical characteristics of echo amplitude with the L-band fully polarimetric radar. A feature plane, composed of the 3rd-order central moment (skewness) and 4th-order central moment (kurtosis) as the statistical characteristics of the echo envelope, is established. In addition, two types of moving targets, pedestrians and non-motorized vehicles, are able to be effectively distinguished according to whether the echo signals of different polarizations have overlapping areas in the feature plane. The L-band fully polarimetric radar has been developed and field experiments have been conducted. The experimental results verify that the kurtosis and skewness of the fully polarimetric echo amplitude of the pedestrians are very close, and there is an overlapping area in the feature plane, while the statistical characteristics of the fully polarimetric echo signal of non-motor vehicles are different, and there is no overlapping area in the feature plane. This proposed feature extraction method has the advantage of being simple and robust, compared to the traditional imaging approach. [ABSTRACT FROM AUTHOR]

N-glycosylation is a common post-translational modification (PTM) in plants that possesses important biological functions including protein folding, protein quality control, protein stability and protein-protein interactions. Nowadays, the interest in analysis of glycoproteins using mass spectrometry is increasing because of advances in instrumentation and automatic data analysis tools. Here, we describe the progresses in mass spectrometry-based N-glycomics and N-glycoproteomics in plants. We also provide a comprehensive summary of N-glycan structures in plants according to the biosynthetic rules and up-to-date reports, which serves as a theoretical database for all N-glycan-related search engines. [Display omitted] • Methods and application progresses in mass spectrometry-based N-glycomics was comprehensively reviewed. • Methods and application progresses in mass spectrometry-based N-glycoproteomics was comprehensively reviewed. • Structural characteristics and core structures of plant N-glycans were summarized as a theoretical reference database. [ABSTRACT FROM AUTHOR]

• Site- and structure-specific identification of N-glycosylation of Arabidopsis at the intact N-glycopeptide level. • With spectrum-level FDR ≤ 1%, 5,687 intact N-glycopeptides coming from 3,713 N-glycosites of 3,140 N-glycoproteins were identified. • With structure-diagnostic fragment ions as experimental evidence, monosaccharide sequence structures of N-glycans on 630 intact N-glycopeptides were unambiguously distinguished from the other sequence isomers with the same monosaccharide compositions. The N-glycosylation is an important bioprocess in plant. Monosaccharide composition-level characterization at the intact N-glycopeptides has been extensively reported, yet structure-specific study to resolve multiple sequence structures of a single composition is still lacking. Here, we present a comprehensive structure-specific identification of intact N-glycopeptides of Arabidopsis with both HILIC and RAX enrichment, as well as GPSeeker and pGlyco database search. With target-decoy searches and spectrum-level FDR ≤ 1%, 5,687 N-glycopeptides from 3,713 N-glycosites of 3,140 N-glycoproteins were identified, which represents the currently most comprohensive profilling to our best knowledge. Wtih the experimental evidence support of structure-diagnostic fragment ions, 81 glycan structures from 54 glcan compostions were unambiguouly distinguished. The comprehensive experimental site- and structure-specific N-glycosylation data reported in this study will serve as a fundamental valuable reference for the coming functional studies of this widely adopted model organism of plant. [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]