Sequential Phosphoproteomic Enrichment through Complementary Metal-Directed Immobilized Metal Ion Affinity Chromatography
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo Sapiens (human)
SUBMITTER: Chia-Feng Tsai, Chuan-Chih Hsu and Yu-Ju Chen
PROVIDER: PXD004903 | JPOST Repository | Wed Sep 07 00:00:00 BST 2016
REPOSITORIES: jPOST
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| HCC_Fe_IMAC_20120229_01.wiff | Wiff | |||
| HCC_Fe_IMAC_20120229_02.wiff | Wiff | |||
| HCC_Fe_IMAC_20120229_03.wiff | Wiff | |||
| HCC_Fe_IMAC_20120229_04.wiff | Wiff | |||
| HCC_Fe_IMAC_20120229_05.wiff | Wiff |
Items per page: 5 1 - 5 of 20 |
Publications
Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography.
Tsai Chia-Feng CF Hsu Chuan-Chih CC Hung Jo-Nan JN Wang Yi-Ting YT Choong Wai-Kok WK Zeng Ming-Yao MY Lin Pei-Yi PY Hong Ruo-Wei RW Sung Ting-Yi TY Chen Yu-Ju YJ
Analytical chemistry 20131217 1
Methodologies to enrich heterogeneous types of phosphopeptides are critical for comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinct binding affinities of Ga(3+) and Fe(3+) for phosphopeptides, we designed a metal-directed immobilized metal ion affinity chromatography for the sequential enrichment of phosphopeptides. In Raji B cells, the sequential Ga(3+)-Fe(3+)-immobilized metal affinity chromatography (IMAC) strategy displayed a 1.5-3.5-fold superior ph ...[more]
Similar Datasets
Project description:Abstract: Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, methodologies to enrich the heterogeneous types of phosphopeptides are critical towards comprehensive mapping of the under-explored phosphoproteome. Taking advantage of the distinctive binding affinity of Ga3+ and Fe3+ towards phosphopeptides, we designed a tip-based metal-directed immobilized metal ion affinity (MD-IMAC) chromatography for sequential enrichment of phosphopeptides. On the analysis of Raji B cell, this sequential Ga3+-Fe3+-IMAC strategy demonstrated 1.5-3.5 fold superior phosphoproteomic coverage compared to the single IMAC (Fe3+, Ti4+, Ga3+ and Al3+). In addition, as high as 92% among the 6283 phosphopeptides was uniquely enriched by either 1st Ga3+-IMAC fraction (41%) or 2nd Fe3+-IMAC fraction (51%). The complementary property of Ga3+ and Fe3+ was further shown on the exclusively superior efficiency to enriched almost all the 1214 multiply phosphorylated peptides (99.4%) by 1st Ga3+-IMAC, while as low as 10% of 5069 monophosphorylated phosphopeptides was commonly enriched by both fractions. Application of our sequential Ga3+-Fe3+-IMAC approach to a human lung cancer tissue allowed the identification of 2560 unique phosphopeptides with only 8% overlapping. The fractionation ability was shown not only on the mono phosphopeptides and multiply phosphopeptides but also on the basic and acidic phosphopeptides; acidiphilic phosphorylation sites were predominately present in 1st Ga3+-IMAC (72%) and 85% Pro-directed and 79% basophilic phosphorylation sites were enriched by 2nd Fe3+-IMAC. Most interestingly, this strategy complementarily mapped different kinase substrate on the protein as well as site levels in multiple cellular pathways related to cancer invasion and metastasis of lung cancer ., Given the demonstrated fractionation ability, reproducibility, sensitivity and ease of tip preparation, we hope that this Ga3+-Fe3+-IMAC allow more comprehensive characterization of phosphoproteome in vitro and in vivo. Database search: The raw MS/MS data obtained by TripleTOF 5600 were processed using AB_SCIEX MS Data Converter with default parameters. All MS/MS files were analyzed using Mascot (Matrix Science, London, UK; version 2.3) against the SwissPort database (version 57.8) with the following constraints: an allowance for tryptic peptides of up to two missed cleavage sites, a fragment ion mass tolerance of 0.05 Da, and a parent ion tolerance of 10 ppm. Phosphorylation (S, T, Y) and oxidation (M) were selected as variable modifications. Searching on a randomized decoy database created by Mascot was required to evaluate the false discovery rate associated with protein identification. The false discovery rates with a Mascot score (p< 0.05) ranged between 0% and 1% in this study.
2014-01-29 | PXD000314 | Pride
Project description:At a pH > 5, phosphopeptides have two negative charges per residue and are well-retained in anion-exchange chromatography. However, the peptides with one or two phosphate groups are not separated from the peptides with multiple Asp or Glu residues, which interfere with the identification of phosphopeptides in tryptic digests. At a pH around 2, phosphate residues have just a single negative charge but Asp and Glu have none. This facilitates the separation of phosphopeptides from unmodified acidic peptides. The singly phosphorylated peptides are retained too weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode (electrostatic repulsion-hydrophilic interaction chromatography). Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, both with peptide standards and with a complex tryptic digest. The SAX column exhibited higher capacity at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified at pH 6 than via ERLIC at pH 2. In one run, 12,467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions the performances of the SAX and WAX materials were comparable.
2015-04-23 | PXD001333 | Pride
Project description:Previous experiments have demonstrated that c-Maf can undergo liquid-liquid phase separation (LLPS) in MM. In order to search for the downstream target genes of c-Maf based on its LLPS regulation, we successfully constructed three kinds of cells with EV, c-Maf and already c-Maf-IDR mutation, and simultaneously subjected the three kinds of cells to ChIP sequencing (selecting the FLAG-tagged antibody), and then analysed them for the differential genes, thus screening the downstream targets of c-Maf based on its LLPS regulation.
2024-08-31 | GSE264392 | GEO
Project description:At a pH > 5, phosphopeptides have two negative charges per residue and are well-retained in anion-exchange chromatography. However, the peptides with one or two phosphate groups are not separated from the peptides with multiple Asp or Glu residues, which interfere with the identification of phosphopeptides in tryptic digests. At a pH around 2, phosphate residues have just a single negative charge but Asp and Glu have none. This facilitates the separation of phosphopeptides from unmodified acidic peptides. The singly phosphorylated peptides are retained too weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode (electrostatic repulsion-hydrophilic interaction chromatography). Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, both with peptide standards and with a complex tryptic digest. The SAX column exhibited higher capacity at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified at pH 6 than via ERLIC at pH 2. In one run, 12,467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions the performances of the SAX and WAX materials were comparable.
2017-03-29 | MSV000080774 | MassIVE
Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.
2020-05-14 | GSE150460 | GEO
Project description:Phosphosites cluster, a set of co-regulation sites, extends a variety of protein function via the distinct combination of phosphorylation events. The coordination of proximal multisite phosphorylation is a universal mechanism to regulate protein function, activity, and interaction pattern. The common method for efficiently separating heterogeneous types of phosphopeptides is insufficient thereby hampering the complete mapping of phosphoproteome. By taking the advantages of distinct binding affinities of Ga2O3 and TiO2 toward phosphopeptides under the same buffer condition, we present a simple and rapid strategy, metal-directed metal oxide chromatography (MD-MOC), for sequential separation of singly and multiply phosphorylated peptides. Among the 6,585 phosphopeptides identified from PC9 cells, up to 92% was uniquely enriched in either the first Ga2O3 or subsequent TiO2 fractions and more than 90 % of phosphopeptides in Ga2O3 fraction were multiply phosphorylated peptide, demonstrating the high complementary properties of these two materials. The high separation efficiency (<10% overlapping of identified phosphopeptides) results in a 1.9-fold superior phosphoproteomic coverage compared to conventional TiO2-based MOC in PC9 cells. Finally, the MD-MOC approach was applied to the large-scale phosphoproteomic profiling of ten mouse organ tissues allowed the identification of 42,086 unique phosphopeptides from 6,669 phosphoproteins using single-shot LC/MS/MS analysis. To our knowledge, the identification of 14,854 multiply phosphorylated peptides presented the largest dataset for multiply phosphorylated peptide from rodent tissues to date. Also, around half identified novel phosphosites (n=2,752) were uniquely came from the multiply phosphorylated peptide dataset further emphasizing the need of improving the detection sensitivity of multiply phosphorylated peptides. Given the good separation ability, stable reproducibility, and ease of tip-based preparation, this MD-MOC presents a sensitive approach for deep phosphoproteome identification and quantification from limited sample for shedding light on in vivo phosphoregulations.
2018-01-11 | PXD005685 | JPOST Repository
Project description:Combustion ion chromatography (CIC) has found a role in environmental analytical chemistry for fluorine content analysis. It is used for extractable organofluorine (EOF) analysis to evaluate perfluoroalkyl and polyfluoroalkyl substances (PFASs) and other organofluorine burden. The prevailing assumption has been that all PFASs are incinerated in CIC and matrix components have no impact on this process, but this has not been experimentally evaluated. In this work, the combustion efficiencies of 13 different PFASs were determined (66-110%). A notable difference was observed between calibrating the CIC with inorganic fluorine or organofluorine. Potential interferences from cations and coextracted matrix components from whole blood and surface water samples were evaluated. These observations should be acknowledged when performing EOF analysis using CIC, overlooking either non-100% combustion efficiencies or the differences in calibrating the CIC with inorganic fluorine or organofluorine could lead to underestimating EOF content and through that to misguide policy decisions.
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Project description:Acute basophilic leukemia is a rare subtype of acute myeloblastic leukemia. We recently described a recurrent t(X;6)(p11;q23) translocation occurring in male infants with acute basophilic leukemia, generating a MYB-GATA1 fusion gene. To better understand the role of MYB-GATA1 in the leukemogenesis of acute basophilic leukemias, we expressed this chimeric transcription factor in the UT-7 cell line under normal or differentiating conditions. A gene expression analysis revealed 9 genes deregulated by both MYB-GATA1 and the basophilic differentiation. Three of these genes (CCL23, IL1RL1 and NTRK1) were also increased by MYB-GATA1 in CD34-positive cells, as indicated by quantitative RT-PCR.
2017-05-31 | GSE72913 | GEO
Project description:Phosphoproteomics requires better separation of phosphopeptides to boost the coverage of the phosphoproteome. We argue that an alternative separation method that produces orthogonal phosphopeptide separation to the widely used LC needs to be considered. Capillary zone electrophoresis (CZE) is one important alternative because CZE and LC are orthogonal for phosphopeptide separation and because the migration time of peptides in CZE can be accurately predicted. In this work, we coupled strong cation exchange (SCX)-reversed-phase LC (RPLC) to CZE-MS/MS for large-scale phosphoproteomics of the colon carcinoma HCT116 cell line. The CZE-MS/MS-based platform identified 11,555 phosphopeptides. The phosphopeptide dataset is at least 100% larger than that from previous CZE-MS/MS studies and will be a valuable resource for building a model for predicting the migration time of phosphopeptides in CZE. Preliminary investigations demonstrated that predicted and observed electrophoretic mobility of phosphopeptides containing one phosphoryl group had good linear correlations (R2≥0.94). Adding one phosphoryl group on a peptide decreased its electrophoretic mobility dramatically because the phosphoryl group reduced the peptide’s charge by roughly 1 based on our experimental data. Phosphopeptides tended to migrate significantly slower than unphosphopeptides in the CZE separation capillary and phosphorylated and unphosphorylated forms of peptides were separated well by CZE. The CZE-MS/MS and LC-MS/MS were complementary in large-scale phosphopeptide identifications and produced different phosphosite motifs from the HCT116 cell line. The data highlight the value of CZE-MS/MS for phosphoproteomics as a complementary separation approach for not only improving the phosphoproteome coverage but also providing more insight into the phosphosite motifs.
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Project description:The localization of condensin along chromosomes is crucial for their accurate segregation in anaphase. Condensin is enriched at telomeres but how and for what purpose had remained elusive. Here we show that fission yeast condensin accumulates at telomere repeats through the balancing acts of Taz1, a core component of the shelterin complex that ensures telomeric functions, and Mit1, a nucleosome-remodeler associated with shelterin. We further show that condensin takes part in sister-telomere separation in anaphase, and that this event can be uncoupled from the prior separation of chromosomes arms, implying a telomere-specific separation mechanism. Consistent with a cis-acting process, increasing or decreasing condensin occupancy specifically at telomeres modifies accordingly the efficiency of their separation in anaphase. Genetic evidence suggests that condensin promotes sister-telomere separation by counteracting cohesin. Thus, our results reveal a shelterin-based mechanism that enriches condensin at telomeres to drive in cis their separation during mitosis.
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