Comprehensive characterization of differential glycation in hepatocellular carcinoma using tissue proteomics with stable isotopic labeling.

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.

Analysis of mutation-originated gain-of-glycosylation using mass spectrometry-based N-glycoproteomics.

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.

Hyaluronan degradation by HYAL2 is essential for odontoblastic differentiation and migration of mouse dental papilla cells.

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.

High-throughput N-glycoproteomics with fast liquid chromatographic separation.

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.

Investigation of the Catalytic Mechanism of a Soluble N-glycosyltransferase Allows Synthesis of N-glycans at Noncanonical Sequons.

The soluble N-glycosyltransferase from Actinobacillus pleuropneumoniae (ApNGT) can establish an N-glycosidic bond at the asparagine residue in the Asn-Xaa-Ser/Thr consensus sequon and is one of the most promising tools for N-glycoprotein production. Here, by integrating computational and experimental strategies, we revealed the molecular mechanism of the substrate recognition and following catalysis of ApNGT. These findings allowed us to pinpoint a key structural motif (215DVYM218) in ApNGT responsible for the peptide substrate recognition. Moreover, Y222 and H371 of ApNGT were found to participate in activating the acceptor Asn. The constructed models were supported by further crystallographic studies and the functional roles of the identified residues were validated by measuring the glycosylation activity of various mutants against a library of synthetic peptides. Intriguingly, with particular mutants, site-selective N-glycosylation of canonical or noncanonical sequons within natural polypeptides from the SARS-CoV-2 spike protein could be achieved, which were used to investigate the biological roles of the N-glycosylation in membrane fusion during virus entry. Our study thus provides in-depth molecular mechanisms underlying the substrate recognition and catalysis for ApNGT, leading to the synthesis of previously unknown chemically defined N-glycoproteins for exploring the biological importance of the N-glycosylation at a specific site.

Structural N-glycoproteomics characterization of cell-surface N-glycosylation of MCF-7/ADR cancer stem cells.

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 ß1,6-branching N-glycan from integrin beta-5 were found.

Gain-of-glycosylation in breast multi-drug-resistant MCF-7 adenocarcinoma cells and cancer stem cells characterized by site- and structure-specific N-glycoproteomics.

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.

The role of O-GlcNAcylation in innate immunity and inflammation.

O-linked ß-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.

Aberrant N-glycolylneuraminic acid in breast MCF-7 cancer cells and cancer stem cells.

N-Glycolylneuraminic acid (Neu5Gc) is not normally detected in humans because humans lack the hydroxylase enzyme that converts cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) to CMP-Neu5Gc; thus, any Neu5Gc appearing in the human body is aberrant. Neu5Gc has been observed in human cancer cells and tissues. Moreover, antibodies against Neu5Gc have been detected in healthy humans, which are obstacles to clinical xenotransplantation and stem cell therapies. Thus, the study of Neu5Gc in humans has important pathological and clinical relevance. Here, we report the N-glycoproteomics characterization of aberrant Neu5Gc in breast MCF-7 cancer cells and cancer stem cells (CSCs) at the molecular level of intact N-glycopeptides, including comprehensive information (peptide backbones, N-glycosites, N-glycan monosaccharide compositions, and linkage structures) based on a target-decoy theoretical database search strategy and a spectrum-level false discovery rate (FDR) control ≤1%. The existence of Neu5Gc on N-glycan moieties was further confirmed according to its characteristic oxonium fragment ions in the MS/MS spectra of either m/z 308.09816 (Neu5Gc) or 290.08759 (Neu5Gc-H2O). The results are an important addition to previously reported Neu5Ac data and can be further validated with targeted MS methods such as multiple and parallel reaction monitoring and biochemical methods such as immunoassays. This MS-based N-glycoproteomics method can be extended to the discovery and characterization of putative aberrant Neu5Gc in other biological and clinical systems.

Sialic acid linkage-specific quantitative N-glycoproteomics using selective alkylamidation and multiplex TMT-labeling.

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.

Proteoform Identification and Quantification Using Intact Protein Database Search Engine ProteinGoggle.

Proteomics studies the proteome of organisms, especially proteins that are differentially expressed under certain physiological or pathological conditions; qualitative identification of protein sequences and posttranslational modifications (PTMs) and their positions can help us systematically understand the structure and function of proteoforms. With the development and relative popularity of soft ionization technology (such as electrospray ionization technology) and high mass measurement accuracy and high-resolution mass spectrometers (such as orbitrap), the mass spectrometry (MS) characterization of complete proteins (the so-called top-down proteomics) has become possible and has gradually become popular. Corresponding database search engines and protein identification bioinformatics tools have also been greatly developed. This chapter provides a brief overview of intact protein database search algorithm "isotopic mass-to-charge ratio and envelope fingerprinting" and search engine ProteinGoggle.

Exploration of quantitative site-specific serum O-glycoproteomics with isobaric labeling for the discovery of putative O-glycoprotein biomarkers.

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.

Comprehensive site- and structure-specific characterization of N-glycosylation in model plant Arabidopsis using mass-spectrometry-based N-glycoproteomics.

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.

Structure-Specific N-Glycoproteomics Characterization of NIST Monoclonal Antibody Reference Material 8671.

The characteristics of monoclonal antibodies (mAbs) cohering various function effectors show great expectation in therapy. Glycosylation, one of the common post-translational modifications, deeply influences cohesion. It is necessary to grasp monosaccharide composition/sequence and glycan structures in mAbs. There has been comprehensive mass spectrometry characterization of N-glycosylation of mAbs, and monosaccharide compositions are deduced according to known biosynthetic rules. Our recently developed intact N-glycopeptide search engine GPSeeker has made structure-specific characterization of N-glycosylation possible with structure-diagnostic fragment ions from selective fragmentation of N-glycan moieties. Here, we report our structure-specific N-glycoproteomics characterization of NIST monoclonal antibody reference material 8671 using GPSeeker, and 59 N-glycan structures (including 16 pairs of isomers) are characterized.

Progress in quantification of nicotine content and form distribution in electronic cigarette liquids and aerosols.

Each electronic cigarette (e-cigarette) is a battery-powered system which converts electronic cigarette liquids (e-liquids) into the inhalable phase by heating the solution when it is in use. After four generations of development, e-cigarettes tend to be more customized and user-operable. The main components in the e-liquid and the aerosol are vegetable glycerin, propylene glycol, nicotine, organic acid and some flavor ingredients. Among them, nicotine is closely associated with the irritation and physiological satisfaction caused by tobacco products, and it is the core functional substance of e-cigarettes. For this reason, the quantification of nicotine content and nicotine form distribution mainly focuses on the components of the e-liquid and the released aerosol. Up to now, various technologies and methods have been applied in the analysis and research of nicotine content and nicotine form distribution in the e-liquid and its aerosol. GC-MS is often used as the most viable tool for the analysis of volatile organic compounds and can be widely applied in the measurement of nicotine related chemicals; there are a number of quantitation strategies using LC-MS, LC-MS/MS or 1H NMR for the analysis of e-cigarette samples. We also reviewed the four main methods for determining the distribution of nicotine forms, which are pH value derivation, solvent extraction, SPME and NMR methods. These research methods are of great significance to the upgrading and development of e-cigarette products.

The glycosylation in SARS-CoV-2 and its receptor ACE2.

Coronavirus disease 2019 (COVID-19), a highly infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 235 million individuals and led to more than 4.8 million deaths worldwide as of October 5 2021. Cryo-electron microscopy and topology show that the SARS-CoV-2 genome encodes lots of highly glycosylated proteins, such as spike (S), envelope (E), membrane (M), and ORF3a proteins, which are responsible for host recognition, penetration, binding, recycling and pathogenesis. Here we reviewed the detections, substrates, biological functions of the glycosylation in SARS-CoV-2 proteins as well as the human receptor ACE2, and also summarized the approved and undergoing SARS-CoV-2 therapeutics associated with glycosylation. This review may not only broad the understanding of viral glycobiology, but also provide key clues for the development of new preventive and therapeutic methodologies against SARS-CoV-2 and its variants.

Genome­wide association study and genomic prediction for growth traits in yellow-plumage chicken using genotyping-by-sequencing.

BACKGROUND: Growth traits are of great importance for poultry breeding and production and have been the topic of extensive investigation, with many quantitative trait loci (QTL) detected. However, due to their complex genetic background, few causative genes have been confirmed and the underlying molecular mechanisms remain unclear, thus limiting our understanding of QTL and their potential use for the genetic improvement of poultry. Therefore, deciphering the genetic architecture is a promising avenue for optimising genomic prediction strategies and exploiting genomic information for commercial breeding. The objectives of this study were to: (1) conduct a genome-wide association study to identify key genetic factors and explore the polygenicity of chicken growth traits; (2) investigate the efficiency of genomic prediction in broilers; and (3) evaluate genomic predictions that harness genomic features. RESULTS: We identified five significant QTL, including one on chromosome 4 with major effects and four on chromosomes 1, 2, 17, and 27 with minor effects, accounting for 14.5 to 34.1% and 0.2 to 2.6% of the genomic additive genetic variance, respectively, and 23.3 to 46.7% and 0.6 to 4.5% of the observed predictive accuracy of breeding values, respectively. Further analysis showed that the QTL with minor effects collectively had a considerable influence, reflecting the polygenicity of the genetic background. The accuracy of genomic best linear unbiased predictions (BLUP) was improved by 22.0 to 70.3% compared to that of the conventional pedigree-based BLUP model. The genomic feature BLUP model further improved the observed prediction accuracy by 13.8 to 15.2% compared to the genomic BLUP model. CONCLUSIONS: A major QTL and four minor QTL were identified for growth traits; the remaining variance was due to QTL effects that were too small to be detected. The genomic BLUP and genomic feature BLUP models yielded considerably higher prediction accuracy compared to the pedigree-based BLUP model. This study revealed the polygenicity of growth traits in yellow-plumage chickens and demonstrated that the predictive ability can be greatly improved by using genomic information and related features.

Methylation of PhoP by CheR Regulates Salmonella Virulence.

The two-component system PhoP/PhoQ is essential for Salmonella enterica serovar Typhimurium virulence. Here, we report that PhoP is methylated extensively. Two consecutive glutamate (E) and aspartate (D)/E residues, i.e., E8/D9 and E107/E108, and arginine (R) 112 can be methylated. Individual mutation of these above-mentioned residues caused impaired phosphorylation and dimerization or DNA-binding ability of PhoP to a different extent and led to attenuated bacterial virulence. With the help of specific antibodies recognizing methylated E8 and monomethylated R112, we found that the methylation levels of E8 or R112 decreased dramatically when bacteria encountered low magnesium, acidic pH, or phagocytosis by macrophages, under which PhoP can be activated. Furthermore, CheR, a bacterial chemotaxis methyltransferase, was identified to methylate R112. Overexpression of cheR decreased PhoP activity but increased PhoP stability. Together, the current study reveals that methylation plays an important role in regulating PhoP activities in response to environmental cues and, consequently, modulates Salmonella virulence. IMPORTANCE Posttranslational modifications (PTMs) play an important role in regulating enzyme activities, protein-protein interactions, or DNA-protein recognition and, consequently, modulate many biological functions. We demonstrated that PhoP, the response regulator of PhoP/PhoQ two-component system, could be methylated on several evolutionally conserved amino acid residues. These amino acid residues were crucial for PhoP phosphorylation or dimerization, DNA-binding ability of PhoP, and Salmonella virulence. Interestingly, methylation negatively regulated the activity of PhoP. A bacterial chemotaxis methyltransferase CheR was involved in PhoP methylation. Methylation of PhoP could stabilize it in an inactive conformation. Our work provides a more informative depiction of PhoP PTM and markedly improves our understanding of the coordinate regulation of bacterial chemotaxis and virulence.

Promising Immunotherapies against COVID-19.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to a severe pandemic and deeply affected the livelihood of people worldwide. In response to the pandemic, researchers have been rapidly studying different aspects of COVID-19, such as virus detection, vaccinations, and epidemiological aspects of the disease. It has been reported that SARS-CoV-2 can induce uncontrolled inflammation and cause a lack of antiviral response, thereby aggravating the disease. Therefore, recovery of immune functions is key to COVID-19 treatment. Many clinical trials are exploring suitable therapies, and some progress has been made. Early administration of interferons may prevent COVID-19 exacerbation and/or promotes recovery from the diseases. Inhibitors of inflammation can prevent cytokine storms and multi-organ damage. Convalescent plasma containing neutralizing antibodies has played an important role in therapeutic options at the beginning of the pandemic owing to the lack of other effective methods. To aid the development of treatment options for COVID-19, this review focuses on immunotherapies, including treatment with interferons, inhibition of pro-inflammatory mechanisms, and the use of convalescent plasma.

Putative N-glycoprotein markers of MCF-7/ADR cancer stem cells from N-glycoproteomics characterization of the whole cell lysate.

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.