Project description:This is the data acquired during the analysis of high pH reverse phase fractions from a glycopeptide-enriched sample from Arabidopsis, purified by lectin weak affinity chromatography using wheat germ agglutinin.
Project description:In this work, by using the lysates of PANC-1 (a pancreatic cancer cell line), we provided a head-to-head comparison of three affinity enrichment methods/materials (i.e., antibody, lectin AANL6, and an OGA mutant) for O-GlcNAc proteomics. The enrichment was performed before or after high-pH HPLC fractionation. The enriched peptides were analyzed by EThcD or HCD-product dependent-EThcD (i.e., HCD-pd-EThcD) mass spectrometry. The resulting data files were processed by using three different data analysis packages (i.e. Sequest HT, Byonic, and FragPipe).
Project description:Lectin is widely used to enrich glycoconjugates carrying glycans and to identify the abnormal glycosylated proteins in tumor with mass spectrometry, which is a useful tool for cancer diagnosis and prognosis monitoring. In our previously reported, we identified a novel lectin AANL from Agrocybe aegerita that has a higher affinity than other lectins when binding with GlcNAc, and the lectin AANL has been used for early diagnosis of non-small cell lung cancer. Subsequently, AANL6, a mutant of AANL, is cloned and purified, and has higher affinity to terminal GlcNAc than AANL. Therefore, in this study, we used the lectin AANL6 for enrichment of glycosylated peoteins from MM, and identified with quantitative proteomics, in order to find a potential sensitive biomarker for MM diagnosis.
Project description:We have compared three different glycopeptide enrichment techniques (SAX-ERLIC, HILIC, and lectin affinity) and a C18 control to a tryptic digest of proteins from depleted human plasma.
Project description:We have compared three different glycopeptide enrichment techniques (SAX-ERLIC, HILIC, and lectin affinity) and a C18 control to a tryptic digest of proteins from depleted human plasma.
Project description:O-GlcNAc is thought to regulate proteins in a mannaer analogous to other PTMs, modulating cellular functions including the cellular stress response. The aim of this study was to identify specific cellular networks and protein complexes that are differentially O-GlcNAcylated and/or expressed upon acute oxidative stress (H2O2 for 1 and 2 h). We achieved this by employing SILAC and a novel anti-O-GlcNAc G5 lectibody IP strategy that combind O-GlcNAc specific antibodies and the lectin WGA. We identified 990 proteins among all three treatments including 202 non-redundant O-GlcNAc modified peptides. Differentially expressed proteins clustered into canonical 14-3-3/PI3K signaling pathways including the 14-3-3 complex, chaperonins, RNA Pol II Mediator and nuclear pore complexes.
Project description:We applied the chemical reporter-based metabolic labeling method to acquire O-GlcNAc modified proteins chromatin loci. Human breast cancer cell line MCF-7, as well as the genotoxic stress (Adriamycin) adapted cells MCF-7/ADR, were fed with 1 mM GalNAz. Metabolic labeled O-GlcNAz chromatin were crosslinked, sonicated and enriched by bioorthogonal chemistry. Then, the genomic DNA fragments bounded by O-GlcNAc mark were de-crosslinked, and constructed into libraries following by next-generation sequencing (Chemoselective O-GlcNAc chromatin sequencing, COGC-seq). To verify the robustness of this chemical reporter-based metabolic labeling method, we compared the results in MCF-7 and MCF-7/ADR cells with classical lectin succinylated wheat germ agglutinin (sWGA) ChIP-seq strategy. We also analyzed gene expression MCF-7 and MCF-7/ADR cells by RNA-seq.
Project description:Efficient nucleic acid enrichment is pivotal for deciphering epigenetic modifications and disease biomarkers, yet current methods are constrained by insufficient specificity, poor versatility, and high costs. We developed a universal strategy named ‘Click-IP-Seq’ by leveraging the high-affinity binding between Protein G and Fc region of DBCO-modified IgG. This enabled the directional conjugation of DBCO-IgG with azide-modified nucleic acids via copper-free strain-promoted azide-alkyne cycloaddition (SPAAC) click chemistry, achieving specific capture and enrichment of modified nucleic acids. Firstly, this method efficiently enriched two major DNA modifications, 8-oxo-7,8-dihydroguanine (8-oxo-dG) and 5-hydroxymethylcytosine (5hmC) in model DNA systems. Then, genome-wide distribution of 8-oxodG from human cells and tissues align with previously reports. Finally, employing ‘Click-IP-Seq’, we performed the first comprehensive analysis of 8-oxo-dG spatial distribution and associated biological functions in human colorectal carcinoma tissues. This technology provides a high-specificity and versatile enrichment platform for nucleic acid modifications, which is expected to promote the application of cancer molecular diagnosis.
Project description:Protein post-translational modification (PTM) increases the functional diversity of the proteome and regulates numerous biological processes in eukaryotes. Two types of PTMs, O-linked-acetyl glucosamine modification (O-GlcNAc) and phosphorylation have been identified on the same amino acid, are considered as Yin-Yang modification for their antagonistic function recently. Vernalization, a prolonged cold exposure promoted flowering, is important for grain yield in temperate cereals, such as winter wheat. O-GlcNAcylation on TaGRP2 and phosphorylation on VER2 are involved in regulation of vernalization response (VRN) genes. However, less is known about how plant senses vernalization with general Yin-Yang modifications. Here we report that altering O-GlcNAc signaling by chemical inhibitors could change the vernalization response and affect flowering transition. Furthermore, we enriched O-GlcNAcylated and phosphorylated peptides from winter wheat plumules at different processing time points during vernalization by Lectin weak affinity chromatography (LWAC) and iTRAQ-TiO2, respectively. In total, about 200 O-GlcNAcylated proteins and 124 differential expressed phosphorylated proteins were identified by Mass Spectrum (MS). Based on GO enrichment, the identified O-GlcNAcylated proteins are mainly involved in response to abiotic stimulus and hormone, metabolic processing and gene expression. While dynamic phosphorylated proteins during vernalization participate in translation, transcription and metabolic processing. Of note, 31 proteins with both phosphorylation and O-GlcNAcylation modification were identified. Among them, TaGRP2 was further confirmed to participate in regulation of vernalization promoted flowering. The global modification profiles and genetic data at specific regulator suggested that the dynamic network of O-GlcNAcylation and phosphorylation on the key nodes regulate vernalization response and mediate flowering in wheat.
Project description:Protein post-translational modification (PTM) increases the functional diversity of the proteome and regulates numerous biological processes in eukaryotes. Two types of PTMs, O-linked-acetyl glucosamine modification (O-GlcNAc) and phosphorylation have been identified on the same amino acid, are considered as Yin-Yang modification for their antagonistic function recently. Vernalization, a prolonged cold exposure promoted flowering, is important for grain yield in temperate cereals, such as winter wheat. O-GlcNAcylation on TaGRP2 and phosphorylation on VER2 are involved in regulation of vernalization response (VRN) genes. However, less is known about how plant senses vernalization with general Yin-Yang modifications. Here we report that altering O-GlcNAc signaling by chemical inhibitors could change the vernalization response and affect flowering transition. Furthermore, we enriched O-GlcNAcylated and phosphorylated peptides from winter wheat plumules at different processing time points during vernalization by Lectin weak affinity chromatography (LWAC) and iTRAQ-TiO2, respectively. In total, about 200 O-GlcNAcylated proteins and 124 differential expressed phosphorylated proteins were identified by Mass Spectrum (MS). Based on GO enrichment, the identified O-GlcNAcylated proteins are mainly involved in response to abiotic stimulus and hormone, metabolic processing and gene expression. While dynamic phosphorylated proteins during vernalization participate in translation, transcription and metabolic processing. Of note, 31 proteins with both phosphorylation and O-GlcNAcylation modification were identified. Among them, TaGRP2 was further confirmed to participate in regulation of vernalization promoted flowering. The global modification profiles and genetic data at specific regulator suggested that the dynamic network of O-GlcNAcylation and phosphorylation on the key nodes regulate vernalization response and mediate flowering in wheat.