Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

2 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Tandem Enrichment and Simultaneous Profeling

ABSTRACT: Tandem Enrichment and Simultaneous Profeling of N-glycopeptides and phosphopeptides

ORGANISM(S): Homo Sapiens

SUBMITTER: Weijie Qin

PROVIDER: PXD033282 | iProX | Mon Apr 18 00:00:00 BST 2022

REPOSITORIES: iProX

ACCESS DATA

Json Xml

Publications

A New Strategy for High-Efficient Tandem Enrichment and Simultaneous Profiling of N-Glycopeptides and Phosphopeptides in Lung Cancer Tissue.

Du Zhuokun Z Yang Qianying Q Liu Yuanyuan Y Chen Sijie S Zhao Hongxian H Bai Haihong H Shao Wei W Zhang Yangjun Y Qin Weijie W

Frontiers in molecular biosciences 20220524

N-glycosylation and phosphorylation, two common posttranslational modifications, play important roles in various biological processes and are extensively studied for biomarker and drug target screening. Because of their low abundance, enrichment of N-glycopeptides and phosphopeptides prior to LC-MS/MS analysis is essential. However, simultaneous characterization of these two types of posttranslational modifications in complex biological samples is still challenging, especially for tiny amount of ...[more]

PMID: 35685241

Similar Datasets

TIHAMAC: Streamlined Tandem IMAC-HILIC Workflow for Simultaneous and High-Throughput Plant Phosphoproteomics and N-glycoproteomics Analysis

Project description:Protein post-translational modifications (PTMs) are pivotal molecular events in plant cells, encompassing processes such as protein folding and enzyme activation. Among these PTMs, N-glycosylation and phosphorylation hold particular importance, as they play essential roles in regulating plant growth and stress responses. The intricate PTM landscape is exemplified by the identification of multiple plant membrane-localized proteins subject to both phosphorylation and N-glycosylation, hinting at potential interplay influencing protein functionality. To investigate the crosstalk between phosphorylation and glycosylation in plant signaling, immobilized metal affinity chromatography (IMAC) and hydrophilic interaction chromatography (HILIC) have been primarily utilized for large-scale phosphopeptide and glycopeptide enrichment, respectively. In this work, we introduce a novel streamlined tandem S-Trap-IMAC-HILIC strategy, termed TIMAHAC, to enable serial enrichment of phosphopeptides and glycopeptides. This approach successfully integrates the two methods through tandem tip enrichment, eliminating the need for buffer exchange and sample transfer. As a result, it significantly minimizes sample loss during serial enrichment steps, and streamlines experimental procedures, ultimately saving time. We demonstrated the efficiency of the TIHAMAC strategy in the glycoproteomics and phosphoproteomics analyses using 200 μg of Arabidopsis digests. Up to 1,954 N-glycopeptides and 11,255 phosphopeptides were identified by timsTOF HT. Remarkably, 69% of N-glycopeptides were consistently quantified with a coefficient of variation less than 20%. Furthermore, more than one-third identified glycoproteins exhibited phosphorylation modifications. These results suggest that the applicability of the TIMAHAC method for studying the potential crosstalk of N-glycoproteome and phosphoproteome within complex plant signaling networks.

2024-04-03 | PXD047065 | Pride

ATP-Coated Dual-functionalized Titanium (IV) IMAC Material for Simultaneous Enrichment and Separation of Glycopeptides and Phosphopeptides

Project description:Protein glycosylation and phosphorylation are two of the most common post-translational modifications (PTMs), which plays an important role in many biological processes. However, low abundance and poor ionization efficiency of phosphopeptides and glycopeptides make direct MS analysis challenging. Previously, we explored the electrostatic and hydrophilic properties of commercial centrifuge-assisted-extraction Titanium (IV) IMAC (CAE-Ti-IMAC) material and its application in simultaneously enriching and separating common glycopeptides, phosphopeptides, and M6P glycopeptides in dual-mode. In this study, we developed a hydrophilicity enhanced dual-functional Ti-IMAC material with adenosine triphosphate as grafted group (denoted as: epoxy-ATP-Ti4+) to achieve better enrichment performance in dual-mode separation. The epoxy-ATP-Ti4+ IMAC material was prepared from commercially available epoxy functionalized silica particles in a facile way, which only required two steps of reaction. The ATP molecule not only provided superiorly strong and active metal phosphate sites to bind phosphopeptides, but also contributed significantly to the hydrophilicity to enrich glycopeptides. The epoxy-ATP-Ti4+ IMAC material showed great selectivity and sensitivity for phosphopeptide enrichment in conventional IMAC mode. With optimized buffer and fractionation, the material successfully separated glycopeptides and phosphopeptides with high specificity. Besides standard protein samples, the material was further applied to HeLa cells and mouse lung tissue samples. Our method allows simple and effective enrichment and separation of glycopeptides and phosphopeptides, which paves the way for studying the potential crosstalk between these two PTMs.

2023-07-20 | PXD029775 | Pride

A Spin-Tip Enrichment Strategy for the Simultaneous Analysis of N-Glycopeptides and Phosphopeptides from Human Pancreatic Tissues

Project description:Post-translational modifications (PTMs) change protein structures and functions. Methods for the simultaneous enrichment of multiple PTM types can maximize coverage in analyses. We present a protocol using dual-functional Ti(IV)-immobilized metal affinity chromatography followed by mass spectrometry for the simultaneous enrichment and analysis of protein N-glycosylation and phosphorylation in pancreas tissues.

2022-08-12 | PXD033065 | Pride

Simultaneous Detection of DNA Adenine and Cytosine Methylation using NT-seq [series1: no enrichment]

Project description:Simultaneous Detection of DNA Adenine and Cytosine Methylation using NT-seq [series1: no enrichment]

| PRJNA764234 | ENA

Probing the global kinome and phosphoproteome in Chlamydomonas reinhardtii via sequential enrichment and quantitative proteomics

Project description:Identifying dynamic protein phosphorylation events is critical for understanding kinase/phosphatase regulated signaling pathways. To date, protein phosphorylation and kinase expression have been examined independently in photosynthetic organisms. Presented here is a method to study the global kinome and phosphoproteome in tandem for the first time in a photosynthetic organism, the model alga Chlamydomonas reinhardtii (Chlamydomonas), using mass spectrometry-based label-free proteomics. A dual enrichment strategy first targets intact protein kinases via capture on immobilized multiplexed inhibitor beads (MIBs) with subsequent proteolytic digestion of unbound proteins and peptide-based phosphorylation enrichment. To increase depth of coverage, both data-dependent and data-independent (via SWATH) mass spectrometric acquisitions were performed to obtain a >50% increase in coverage of the enriched Chlamydomonas kinome over coverage found with no enrichment. The quantitative phosphoproteomic dataset yielded 2,304 phosphopeptides and 1,765 localized phosphosites with excellent reproducibility across biological replicates (90% of quantified sites with coefficient of variation below 11%). This approach enables simultaneous investigation of kinases and phosphorylation events at the global level to facilitate understanding of kinase networks and their influence in cell signaling events.

2016-10-05 | PXD004681 | Pride

Tandem repeat sequencing in XLID

Project description:Tandem repeat sequencing in XLID

| PRJEB21659 | ENA

Simultaneous Detection of DNA Adenine and Cytosine Methylation using NT-seq [series2: 6mA DIP enrichment]

Project description:Simultaneous Detection of DNA Adenine and Cytosine Methylation using NT-seq [series2: 6mA DIP enrichment]

| PRJNA764233 | ENA

Array CGH data of A. oryzae strains bearing targeted tandem chromosomal duplications

Project description:We here describe the first successful construction of a targeted tandem duplication of a large chromosomal segment in Aspergillus oryzae. The targeted tandem chromosomal duplication was achieved by using strains that had 5M-bM-^@M-^YM-NM-^TpyrG upstream of the region targeted for tandem chromosomal duplication and 3M-bM-^@M-^YM-NM-^TpyrG downstream of the target region. Consequently, strains bearing a 210-kb targeted tandem chromosomal duplication near the centromeric region of chromosome 8 and strains bearing a targeted tandem chromosomal duplication of a 700-kb region of chromosome 2 were successfully constructed. The strains bearing the tandem chromosomal duplication were efficiently obtained from the regenerated protoplast of the parental strains. However, the generation of the chromosomal duplication did not depend on the introduction of double-stranded breaks (DSBs) by I-SceI. The chromosomal duplications of these strains were stably maintained after five generations of culture under non-selective conditions. The strains bearing the tandem chromosomal duplication in the 700-kb region of chromosome 2 showed highly increased protease activity in solid-state culture, indicating that the duplication of large chromosomal segments could be a useful new breeding technology and gene analysis method. A. oryzae strain bearing a 210-kb targeted tandem chromosomal duplication, A. oryzae strain bearing a 700-kb targeted tandem chromosomal duplication, and A. oryzae RIB40 (wild type strain), were cultivated in Polypeptone-dextrin medium. After 3 days cultivation, genomic DNAs from the samples were extracted, and array CGH analysis was carried out to confirm the chromosomal duplications in the strains.

2014-06-30 | E-GEOD-57246 | biostudies-arrayexpress

An Internal Standard for Assessing Phosphopeptide Recovery from Metal Ion/Oxide Enrichment Strategies

Project description:Phosphorylation-mediated signaling pathways have major implications in cellular regulation and disease. Mass spectrometry-based proteomics is a well-established approach for quantifying thousands of phosphorylation events in a single experiment. However, proteins with roles in signaling pathways are frequently less abundant and phosphorylation is often sub-stoichiometric. As such, the efficient enrichment and subsequent recovery of phosphorylated peptides is vital. To understand better phosphopeptide recovery from enrichment strategies, we designed a peptide internal standard-based assay directed toward sample preparation strategies for mass spectrometry analysis. We coupled mass-differential tandem mass tag (mTMT) reagents (specifically, TMTzero and TMTsuper-heavy), nine mass spectrometry-amenable phosphopeptides (phos9), and peak area measurements from extracted ion chromatograms to determine phosphopeptide recovery. We showcase this mTMT/phos9 recovery assay by evaluating three commercial phosphopeptide enrichment workflows. Our assay provides data on the recovery of phosphopeptides, which complement other metrics, namely the number of identified phosphopeptides and enrichment specificity. Our mTMT/phos9 assay is applicable to any enrichment protocol in a typical experimental workflow irrespective of sample origin or labeling strategy.

2018-12-04 | PXD008966 | Pride

Finding the Sweet Spot in ERLIC Mobile Phase for Simultaneous Enrichment of N-Glyco and Phosphopeptides

Project description:Simultaneous enrichment of glyco- and phospho- peptides will benefit the studies of biological processes regulated by these post-translational modifications (PTMs). It will also reveal potential crosstalk between these two ubiquitous PTMs. Unlike custom designed multifunctional solid phase extraction (SPE) materials, operating strong-anion exchange (SAX) resin in electrostatic repulsion-hydrophilic interaction chromatography (ERLIC) mode provides a readily available strategy to analytical labs for enrichment of these PTMs for subsequent mass spectrometry (MS)-based characterization. However, the choice of mobile phase has largely relied on empirical rules from hydrophilic interaction chromatography (HILIC) or ion-exchange chromatography (IEX) without further optimization and adjustments. In this study, ten mobile phase compositions of ERLIC were systematically compared; the impact of multiple factors including organic phase proportion, ion pairing reagent, pH and salt on the retention of glycosylated and phosphorylated peptides were evaluated. This study demonstrated good enrichment of glyco- and phospho- peptides from the nonmodified peptides in a complex tryptic digest. Moreover, the enriched glyco- and phospho- peptides elute in different fractions by orthogonal retention mechanisms of hydrophilic interaction and electrostatic interaction in ERLIC, maximizing the LC-MS identification of each PTM. The optimized mobile phase can be adapted to the ERLIC HPLC system, where the high resolution in separating multiple PTMs will benefit large-scale MS-based PTM profiling and in-depth characterization.

2019-09-06 | PXD015179 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data