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N-linked glycosylation is a common posttranslational modification of proteins that results in macroheterogeneity of the modification site. However, unlike simpler modifications, N-glycosylation introduces an additional layer of complexity with tens of thousands of possible structures arising from various dimensions, including different monosaccharide compositions, sequence structures, linking structures, isomerism, and three-dimensional conformations. This results in additional microheterogeneity of the modification site of N-glycosylation, i.e., the same N-glycosylation site can be modified with different glycans with a certain stoichiometric ratio. N-glycosylation regulates the structure and function of N-glycoproteins in a site- and structure-specific manner, and differential expression of N-glycosylation under disease conditions needs to be characterized through site- and structure-specific quantitative analysis. Numerous advanced methods ranging from sample preparation to mass spectrum analysis have been developed to distinguish N-glycan structures. Chemical derivatization of monosaccharides, online liquid chromatography separation and ion mobility spectrometry enable the physical differentiation of samples. Tandem mass spectrometry further analyzes the macro/microheterogeneity of intact N-glycopeptides through the analysis of fragment ions. Moreover, the development of search engines and AI-based software has enhanced our understanding of the dissociation patterns of intact N-glycopeptides and the clinical significance of differentially expressed intact N-glycopeptides. With the help of these modern methods, structure-specific N-glycoproteomics has become an important tool with extensive applications in the biomedical field.

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]

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]

Salt stress is one of the significant environmental stresses that severely affect plant growth and development. Here, we report quantitative N-glycoproteomics characterization of differential N-glycosylation in Sorghum bicolor under low, median and high salinity stress. 21,621 intact N-glycopeptides coming from the combination of 127 N-glycan structures on 6574 N-glycosites from 5321 proteins were identified; differential N-glycosylation was observed for 682 N-glycoproteins which are mainly involved in the pathways of biosynthesis of secondary metabolites, biosynthesis of amino acids and several metabolic pathways. 41 N-glycan structures modifying on 338 N-glycopeptides from 122 glycoproteins were co-quantified and deregulated under at least one salt stress, including enzymes of energy production and carbohydrate metabolisms, cell wall organization related proteins, glycosyltransferases and so on. Intriguingly, with increasing salt concentration, there was an increase in the percentage of complex N-glycans on the altered N-glycopeptides. Furthermore, the observation of glycoproteins with distinct salt sensitivity is noteworthy, particularly the upregulated hyposensitive glycoproteins that predominantly undergo complex N-glycan modification. This is the first N-glycoproteome description of salt stress response at the intact N-glycopeptide level in sorghum and a further validation of data reported here would likely provide deeper insights into the stress physiology of this important crop plant., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a highly dynamic and widespread post-translational modification (PTM) that regulates the activity, subcellular localization, and stability of target proteins. O-GlcNAcylation is a reversible PTM controlled by two cycling enzymes: O-linked N-acetylglucosamine transferase and O-GlcNAcase. Emerging evidence indicates that O-GlcNAcylation plays critical roles in innate immunity, inflammatory signaling, and cancer development. O-GlcNAcylation usually occurs on serine/threonine residues, where it interacts with other PTMs, such as phosphorylation. Thus, it likely has a broad regulatory scope. This review discusses the recent research advances regarding the regulatory roles of O-GlcNAcylation in innate immunity and inflammation. A more comprehensive understanding of O-GlcNAcylation could help to optimize therapeutic strategies regarding inflammatory diseases and cancer., (© The Author(s) (2022). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, CEMCS, CAS.)

Purpose: Exploration study of site-specific isobaric-TMT-labeling quantitative serum O-glycoproteomics for the discovery of putative O-glycoprotein cancer biomarkers., Experimental Design: Sera of 10 breast cancer patients was used as the exploration cohort. More abundant N-glycosylation was first removed with PNGase F. After tryptic digestion of de-N-glycosylated serum proteome, the TMT-labeled O-glycopeptides mixture was prepared and analyzed with RPLC-MS/MS. Site-specific qualitative and quantitative database search of O-glycopeptides was carried out with pGlyco 3.0. The same raw datasets were also searched with intact N-glycopeptide search engine GPSeeker to exclude possible interference of N-glycosylation. The final IDs were checked manually with GlcNAc-containing glycosite-determining fragment ions for confirmation., Results: With the control of spectrum-level FDR ≤ 1% and manual validation, 299 O-glycopeptides corresponding to 83 O-glycosites and 66 O-glycoproteins were identified, and 13 O-glycopeptides were found differentially expressed. Most interestingly, differential O-glycosylation was observed for IgG1 and IgG3, which is an interesting putative biomarker panel., Conclusion and Clinical Relevance: Isobaric-labeling site-specific quantitative O-glycoproteomics is currently a state-of-the-art instrumental platform for discovery of putative seral cancer biomarkers. Differential seral O-glycosylation was observed in the IgG1 and IgG3., (© 2022 Wiley-VCH GmbH.)