Glycosite Mapping and in situ Mass Spectrometry Imaging of MUC2 Glycopeptides via On-slide Mucinase Digestion
Ontology highlight
ABSTRACT:
INSTRUMENT(S):
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Colon, Salivary Gland, Esophagus
DISEASE(S): Salivary Gland Carcinoma,Esophageal Carcinoma,Mucinous Adenocarcinoma,Colon Mucinous Adenocarcinoma
SUBMITTER: 
Sarah Lowery
LAB HEAD: Stacy Malaker
PROVIDER: PXD055865 | Pride | 2026-04-06
REPOSITORIES: Pride
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| 20201218_Colon1b.sbd | Other | |||
| 20210806_Colon1a.sbd | Other | |||
| 20230508_Esophagus.sbd | Other | |||
| 20230919_SalivaryGland.sbd | Other | |||
| 20240723_Colon1a_R1-2_PHEA_Fraction1.xlsx | Xlsx |
Items per page: 5 1 - 5 of 93 |
Similar Datasets
Project description:Mucin domains are densely O-glycosylated modular protein domains found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are key players in a host of human diseases, especially cancer, but the scope of the mucinome remains poorly defined. Recently, we characterized a bacterial mucinase, StcE, and demonstrated that an inactive point mutant retains binding selectivity for mucins. In this work, we leveraged inactive StcE to selectively enrich and identify mucins from complex samples like cell lysate and crude ovarian cancer patient ascites fluid. Our enrichment strategy was further aided by an algorithm to assign confidence to mucin-domain glycoprotein identifications. This mucinomics platform facilitated detection of hundreds of glycopeptides from mucin domains and highly overlapping populations of mucin-domain glycoproteins from ovarian cancer patients. Ultimately, we demonstrate our mucinomics approach can reveal key molecular signatures of cancer from in vitro and ex vivo sources.
2022-06-29 | PXD024995 | Pride
Project description:Here we present an approach to identify N-linked glycoproteins and deduce their spatial localization using a combination of MALDI N-glycan MSI and spatially-resolved glycoproteomics. We subjected glioma biopsies to on-tissue PNGaseF digestion and MALDI-MSI and found that the glycan HexNAc4-Hex5-NeuAc2 was predominantly expressed in necrotic regions of high-grade canine gliomas. To determine the underlying sialo-glycoprotein, various regions in adjacent tissue sections were subjected to microdigestion and manual glycoproteomic analysis. Results identified haptoglobin as the protein associated with HexNAc4-Hex5-NeuAc2, making our study the first report that directly links glycan imaging with intact glycopeptide identification. In total, our spatially-resolved glycoproteomics technique identified over 400 N-, O-, and S- glycopeptides from over 30 proteins, demonstrating the diverse array of glycosylation present on the tissue slides and the sensitivity of our technique. Ultimately, this proof-of-principle work demonstrates that spatially-resolved glycoproteomics greatly complement MALDI-MSI in understanding dysregulated glycosylation.
2022-05-04 | PXD025537 | Pride
Project description:Protein glycosylation is a complex post-translational modification that is generally classified as N- or O-linked. Site-specific analysis of glycopeptides is accomplished with a variety of fragmentation methods, depending on the type of glycosylation being investigated and the instrumentation available. For instance, collisional dissociation methods are frequently used for N-glycoproteomic analysis with the assumption that one N-sequon exists per tryptic peptide. Alternatively, electron-based methods are indispensable for O-glycosite localization. However, the presence of simultaneously N- and O-glycosylated peptides could suggest the necessity of electron-based fragmentation methods for N-glycoproteomics, which is not commonly performed. Thus, we quantified the prevalence of N- and O-glycopeptides in mucins and other glycoproteins. A much higher frequency of co-occupancy within mucins was detected whereas only a negligible occurrence occurred within non-mucin glycoproteins. This was demonstrated from analyses of recombinant and/or purified proteins, as well as more complex samples. Where co-occupancy occurred, O-glycosites were frequently localized to the Ser/Thr within the N-sequon. Additionally, we found that O-glycans in close proximity to the occupied Asn were predominantly unelaborated core 1 structures, while those further away were more extended. Overall, we demonstrate electron-based methods are required for robust site-specific analysis of mucins, wherein co-occupancy is more prevalent. Conversely, collisional methods are generally sufficient for analyses of other types of glycoproteins.
2025-04-08 | PXD062628 | Pride
Project description:Protein glycosylation is a complex post-translational modification that is generally classified as N- or O-linked. Site-specific analysis of glycopeptides is accomplished with a variety of fragmentation methods, depending on the type of glycosylation being investigated and the instrumentation available. For instance, collisional dissociation methods are frequently used for N-glycoproteomic analysis with the assumption that one N-sequon exists per tryptic peptide. Alternatively, electron-based methods are indispensable for O-glycosite localization. However, the presence of simultaneously N- and O-glycosylated peptides could suggest the necessity of electron-based fragmentation methods for N-glycoproteomics, which is not commonly performed. Thus, we quantified the prevalence of N- and O-glycopeptides in mucins and other glycoproteins. A much higher frequency of co-occupancy within mucins was detected whereas only a negligible occurrence occurred within non-mucin glycoproteins. This was demonstrated from analyses of recombinant and/or purified proteins, as well as more complex samples. Where co-occupancy occurred, O-glycosites were frequently localized to the Ser/Thr within the N-sequon. Additionally, we found that O-glycans in close proximity to the occupied Asn were predominantly unelaborated core 1 structures, while those further away were more extended. Overall, we demonstrate electron-based methods are required for robust site-specific analysis of mucins, wherein co-occupancy is more prevalent. Conversely, collisional methods are generally sufficient for analyses of other types of glycoproteins.
2024-10-22 | PXD053713 | Pride
Project description:Glycosylation is an important post-translational modification characterized by extensive heterogeneity. While mass spectrometry (MS) is the premier technique to characterize glycoproteins, the study of intact glycopeptides presents challenges often not observed in the study of unmodified species. High-field asymmetric waveform ion mobility spectrometry (FAIMS) involves in-line gas-phase separation and offers the potential to analyze glycopeptides without prior enrichment. While FAIMS has been assessed for its use in glycoproteomics, most of these studies focus heavily or exclusively on N-glycoproteomics. Therefore, we evaluated FAIMS for O-glycoprotein and mucin analysis. We generated three samples: the mucin-domain glycoprotein podocalyxin, a recombinant glycoprotein mixture, and a WGA enriched human platelet sheddome. Although FAIMS allowed for the identification of new podocalyxin O-glycosites, it yielded less glycopeptide identifications and glycoforms. FAIMS seemed to be more useful in the increasingly complex samples, since we observed a higher number of identified glycosylated species. Because of the labile nature of glycosidic bonds, and presence of a buffer gas in FAIMS separation, we investigated the possibility of increased in-source fragmentation during FAIMS analysis. We compared total ion chromatograms of identifications eluting within a minute of identifications bearing larger glycan structures, albeit with the same peptide backbone. FAIMS experiments showed a 2-5-fold increase in spectral matches from in-FAIMS fragmentation compared to control experiments. These results were also replicated in other previously published data, indicating that this is a systematic behavior. In summary, our study highlights that, although there are potential benefits gained when using FAIMS separation, caution must be exercised when using FAIMS due to in-FAIMS fragmentation, which limits its applicability in the field of O-glycoproteomics.
2023-09-11 | PXD041217 | Pride
Project description:O-glycoproteases are an emerging class of enzymes that selectively digest glycoproteins at positions decorated with specific O-linked glycans. O-glycoprotease substrates range from any O-glycoprotein (albeit with specific O-glycan modifications) to only glycoproteins harboring specific O-glycosylated sequence motifs, such as those found in mucin domains. Their utility for multiple glycoproteomic applications is driving the search to both discover new O-glycoproteases and to understand how structural features of characterized O-glycoproteases influence their substrate specificities. One challenge of characterizing O-glycoprotease specificity restraints is the need to characterize O-glycopeptides with site-specific analysis of O-glycosites. Here, we demonstrate how O-Pair Search, a recently developed O-glycopeptide-centric identification platform that enables rapid searches and confident O-glycosite localization, can be used to determine substrate specificities of various O-glycoproteases de novo from LC-MS/MS data of O-glycopeptides. Using secreted protease of C1 esterase inhibitor (StcE) from enterohemorrhagic Escherichia coli and O-endoprotease OgpA from Akkermansia mucinophila, we explore numerous settings that effect O-glycopeptide identification and show how non-specific and semi-tryptic searches of O-glycopeptide data can produce candidate cleavage motifs that can be used to define new protease cleavage settings that lower search times and improve O-glycopeptide identifications. We use this platform generate the first consensus motif for the recently characterized immunomodulating metalloprotease (IMPa) from Pseudomonas aeruginosa and show that IMPa is a favorable O-glycoprotease for characterizing densely O-glycosylated mucin-domain glycoproteins.
2023-02-15 | PXD035775 | Pride
Project description:Synergism of Iraqi Sand/Cigarette Smoke Co-Exposure in Rats. 24 samples are used. A total of 102 rats will be separated into six exposure groups: each group consisting of 17 male CD Sprague-Dawley rats, 10 to 15 weeks old. The six exposure groups were each exposed via two routes: 1. nose-only inhalation exposures --- air or cigarette smoke; 2. whole-body inhalation exposures -- air or manufactured silica sand or Iraqi sand. The nose only exposures were conducted 3 hours per day, 5 days per week for 6 weeks. During the last two weeks of the nose - only exposures, whole-body exposure were also conducted 18-19 hours per day, 7 days per week.
2012-01-01 | E-GEOD-28804 | biostudies-arrayexpress
Project description:Formaldehyde (FA), an endogenous cellular aldehyde, is a rat nasal carcinogen. In this study, concentration- and exposure-duration transitions in FA mode of action (MOA) were examined with pharmacokinetic (PK) modeling for tissue formaldehyde acetal (FAcetal) and glutathione (GSH) and with histopathology and gene expression studies for tissue responses in nasal epithelium from rats exposed to 0, 0.7, 2, 6, 10 or 15 ppm FA 6 hr/day for 1, 4 or 13 weeks. The study had two goals. The first goal was to develop a basic PK model to estimate various forms of tissue formaldehyde and tissue glutathione (GSH). The second goal was to compare histopathology and gene expression changes in nasal tissues caused by inhalation of FA with changes in tissue FAcetal and free GSH calculated from the PK model. Patterns of gene expression varied with concentration and with duration. At 0.7 and 2 ppm, sensitive response genes (SRGs) - associated with cellular stress, thiol transport/reduction, inflammation, and cell proliferation - were similarly upregulated at all exposure durations. At 6 ppm and greater, gene expression changes showed enrichment of pathways involved in cell cycle, DNA repair, and apoptosis processes. ERBB, EGFR, WNT, TGF-β, Hedgehog, and Notch signaling were also enriched in differentially expressed genes. Benchmark doses (BMDs) for genes in significantly enriched pathways were lower at 13 weeks than at 1 or 4-week. The transcriptional and histological changes corresponded to PK model-predicted changes in free GSH at 0.7 and 2 ppm and in FAcetal at 6 ppm. DNA-replication stress, enhanced proliferation, metaplasia, and stem cell-niche activation appear to be associated with FA carcinogenesis at 6 ppm and above. Dose dependencies in MOA, the presence of high physiological FAcetal, and non-linear FAcetal/GSH tissue kinetics indicate that FA concentrations below 150 ppb (and probably any concentrations below irritant levels, i.e., ~ 1 ppm) would not increase cancer risks of inhaled FA in the nose or any other tissue. Closer examination of dose response relationships for endogenous compound toxicity could help guide biologically relevant approaches for chemical risk assessment. Eight week old male F344/CrlBR rats were exposed to formaldehyde through whole body inhalation. Whole-body exposures were performed at doses of 0, 0.7, 2, 6, 10, and 15 ppm (6 hours per day, 5 days per week). Inhalation animals were sacrified at 1, 4, and 13 weeks following initiation of exposure. Following sacrifice, tissue from the Level II region of the nose was dissected and digested with a mixture of proteases to remove the epithelial cells. The epithelial cells acquired from this section of the nose consisted primarily of transitional epithelium with some respiratory epithelium. Gene expression microarray analysis was performed on the epithelial cells.
2010-07-28 | E-GEOD-23179 | biostudies-arrayexpress
Project description:ß-chloroprene (2-chloro-1,3-butadiene), a monomer used in the production of neoprene elastomers, is of regulatory interest due to the production of multi-organ tumors in mouse and rat cancer bioassays. A significant increase in female mouse lung tumors was observed at the lowest exposure concentration of 12.8 ppm while a small, but not statistically significant, increase was observed in female rats only at the highest exposure concentration of 80 ppm. The metabolism of chloroprene results in the generation of reactive epoxides and the rate of overall chloroprene metabolism is highly species dependent. To identify potential key events in the mode-of-action of chloroprene lung tumorigenesis, dose response and time course gene expression microarray measurements were made in the lungs of female mice and female rats. The gene expression changes were analyzed using both a traditional analysis of variance approach followed by pathway enrichment analysis and a pathway-based benchmark dose (BMD) analysis approach. Pathways related to glutathione biosynthesis and metabolism were the primary pathways consistent with cross-species differences in tumor incidence and transcriptional BMD values for the pathway were more similar to differences in tumor response than were estimated target tissue dose surrogates based on the total amount of chloroprene metabolized per unit mass of lung tissue per day. The closer correspondence of the transcriptional changes with the tumor response are likely due to their reflection of the overall balance between metabolic activation and detoxication reactions whereas the current tissue dose surrogate reflects only oxidative metabolism. Female mice (B6C3F1/Crl) were exposed to chloroprene (CAS 126-99-8) by whole-body inhalation exposure at 0, 0.3, 2, 13, or 90 ppm. Exposures were performed for 6 hr/day, 5 days per week. The animals were sacrified at 5 days or 19 days (15 exposure days) post initiation of exposure. The right lung was excised and gene expression microarray analysis was performed using Affymetrix Mouse 430_2 arrays. Female rats (F344/NCrl) were exposed to chloroprene (CAS 126-99-8) by whole-body inhalation exposure at 0, 5, 30, 90, or 200 ppm. Exposures were performed for 6 hr/day, 5 days per week. The animals were sacrified at 5 days or 19 days (15 exposure days) post initiation of exposure. The right lung was excised and gene expression microarray analysis was performed using Affymetrix Rat 230_2 arrays.
2012-10-12 | E-GEOD-40795 | biostudies-arrayexpress
Project description:The number of legacy chemicals without toxicity reference values combined with the rate of new chemical development are overwhelming the capacity of the traditional risk assessment paradigm. More efficient approaches are needed to quantitatively estimate chemical risks. In this study, rats were dosed orally with multiple doses of six chemicals for 5 days, 2, 4, and 13 weeks. Target organs were analyzed for traditional histological and organ weight changes and transcriptional changes using microarrays. Histological and organ weight changes in this study and the tumor incidences in the original cancer bioassays were analyzed using benchmark dose (BMD) methods to identify noncancer and cancer points-of-departure. The dose-response changes in gene expression were also analyzed using BMD methods and the responses grouped based on signaling pathways. A comparison of transcriptional BMD values for the most sensitive pathway with BMD values for the noncancer and cancer apical endpoints showed a high degree of correlation at all time points. When the analysis included data from an earlier study with 8 additional chemicals, transcriptional BMD values for the most sensitive pathway were significantly correlated with noncancer (r = 0.827, p = 0.0031) and cancer-related (r = 0.940, p = 0.0002) BMD values at 13 weeks. The average ratio of apical-to-transcriptional BMD values was less than two suggesting that for the current chemicals, transcriptional perturbation did not occur at significantly lower doses than apical responses. Based on our results, we propose a practical framework for application of transcriptomic data to chemical risk assessment. Four to five week-old rats were exposed in dose-response and for multiple durations to 6 chemicals. The chemicals were 1,2,4-tribromobenzene (CAS No. 615-54-3), 2,3,4,6-tetrachlorophenol (CAS No. 58-90-2), bromobenzene (CAS No. 108-86-1), 4,4'-methylenebis(N,N-dimethyl)benzenamine (CAS No. 101-61-1), hydrazobenzene (CAS No. 122-66-7), N-nitrosodiphenylamine (CAS No. 86-30-6). For 1,2,4-tribromobenzene and 2,3,4,6-tetrachlorophenol, male Sprague Dawley (Rat/Crl:CD(SD)) rats were used. For bromobenzene and hydroazobenzene, male F344/DuCrl rats were used. For 4,4'-methylenebis(N,N-dimethyl)benzenamine and N-nitrosodiphenylamine, female F344/DuCrl rats were used. The exposure durations were 5 days, 2 weeks, 4 weeks, and 13 weeks. The dose and route of exposure are provided with the individual sample annotations. With each chemical treatment, a matched vehicle control group was run concurrently with the exposure. Gavage exposures were administered 7 days per week and feed exposures were provided 7 days per week. At the appropriate time point, animals were euthanized with a lethal intraperitoneal injection of sodium pentobarbital. For the liver, slices from each of the lobes were mixed together and used for microarray analysis. For the thyroid, the gland was transversely sectioned and the bottom portion was used for microarray analysis. For the bladder, the organ was longitudinally bisected, a slice was removed for histology and the remainder of the organ was minced and used for microarray analysis. Microarrays were run on five rats per dose per time point.
2013-04-22 | E-GEOD-45892 | biostudies-arrayexpress