Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Basic Strong Cation Exchange Chromatography, BaSCX, a Highly Efficient Approach for C-Terminomic Studies Using LysargiNase Digestion

ABSTRACT: Decoding protein C-termini is a challenging task in protein chemistry using conventional chemical/enzymatic approaches. With the rapid development in modern mass spectrometer, many advanced mass spectrometry (MS) based protein C-termini analysis approaches have been established. Although great progresses have been continually achieved, it is still nec-essary to develop more efficient approaches in order to discover a proteome-scale protein C-termini (C-terminome), and consequently to help understand their biological functions. In this report, we describe a simple method, termed BaSCX, for basic strong cation exchange chromatography, to study C-terminome.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human) Caenorhabditis Elegans Mus Musculus (mouse)

TISSUE(S): Cell Culture, Whole Body

SUBMITTER: Qingqing Li

LAB HEAD: Xumin Zhang

PROVIDER: PXD016317 | Pride | 2020-02-28

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	BaSCX-293T-1-acion.msf	Msf
	BaSCX-293T-1.raw	Raw
	BaSCX-293T-2-acion.msf	Msf
	BaSCX-293T-2.raw	Raw
	BaSCX-293T-3-acion.msf	Msf

Items per page:

1 - 5 of 63

Publications

Basic Strong Cation Exchange Chromatography, BaSCX, a Highly Efficient Approach for C-Terminomic Studies Using LysargiNase Digestion.

Li Qingqing Q Zhang Yang Y Huang Jingnan J Wu Zhen Z Tang Langlang L Huang Lin L Zhang Xumin X

Analytical chemistry 20200309 7

Decoding protein C-termini is a challenging task in protein chemistry using conventional chemical and enzymatic approaches. With the rapid development in modern mass spectrometer, many advanced mass spectrometry (MS)-based protein C-termini analysis approaches have been established. Although great progress is being continually achieved, it is still necessary to develop more efficient approaches in order to discover a proteome-scale protein C-termini (C-terminome) and consequently to help underst ...[more]

PMID: 32097557

Similar Datasets

Combination of multi-enzyme digestions and C-TAILS approach for deep screening of C-terminome

Project description:Protein C-termini study is still a challenging task and far behind its counterpart N-termini study. The identification of C-termini is often hampered by low ionization response in MS and fewer efficient enrichment methods existing. We previously optimized the C-TAILS (C-terminal amine-based isotope labeling of substrates) method to achieve 75% more identification of protein C-terminal peptides. Although with the significant improvement, the performance of method is not comparable with a regular proteome study since the identification number is very limited. Herein we reported an optimized method by introduction of multiple-enzyme digestions in order to increase the capacity and application of C-TAILS in biological and clinical study. To allow the application of other enzymes than Arg-C in C-TAILS method, we evaluated the effect of the omission of prior amine protection at protein level and revealed the negligible effect caused by. Subsequently we applied five different enzymes in the C-TAILS and evaluated the different performances on the basis of identification and complementariness. As a result, 722 protein C-termini of E. coli were identified in a triplicate experiments using 50 μg each, and the favored enzyme and enzyme combination were discovered and discussed.

2017-11-24 | PXD004275 | Pride

Systematic optimization of C-TAILS approach for deep screening of C-terminomics

Project description:It is well known that protein C-termini play important roles in various biological processes, and thus the precise characterization of C-termini is essential for fully elucidating protein structure and understanding protein functions. Although many efforts have been made in the fields during the latest two decades, the progress is still far behind its counterpart, N-termini, and it necessitates more novel or optimized methods. Herein, we reported an optimized C-termini identification approach based on the C-terminal amine-based isotope labeling of substrates (C-TAILS) method. We optimized the amidation reaction conditions to achieve higher yield of fully amidated product. We evaluated different carboxyl and amine blocking reagents and found the superior performance of Ac-NHS and ethanolamine. Replacement of dimethylation with acetylation for Lys blocking resulted in the identification of 232 C-terminal peptides in E. Coli sample, about 42% higher than the conventional C-TAILS. A systematic data analysis revealed that the optimized method is unbiased to amino acid composition, more reproducible and with higher MASCOT scores. Moreover, the introduction of SCII (Single-Charge Ion Inclusion) method to alleviate the charge deficiency of small peptides allowed an additional 26 % increase in identification number. With the optimized method, we identified 481 C-terminal peptides corresponding to 369 C-termini in E. Coli in a triplicate experiments using 80 μg each. Our optimized method would benefit the deep screening of C-terminomics and possibly help discover some novel C-terminal modifications.

2015-09-15 | PXD002409 | Pride

An Undesirable Side Effect of EDC-catalyzed Carboxyl Labeling Approach

Project description:Carbodiimide catalyzed carboxyl and amine conjugation (amidation) has been widely used to protect carboxyl groups. N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) is the most commonly used carbodiimide reagent in protein chemis-try due to its high efficiency and high solubility in aqueous media. It has also been applied in different proteomics studies including protein terminomics, protein glycosylation and crosslinking. Herein, we report that the EDC catalyzed amidation could cause a +155 Da side-modification at the tyrosine residue and severely hamper the identification of Tyr-containing peptides. We discovered the extremely low identification rate of Tyr-containing peptides in different published studies that employed EDC-catalyzed amidation. We carried out a systematic study and discovered a +155 Da side-modification occur-ring specifically on Tyr as the addition of EDC. Consideration of the side-modification in database search enabled the identification of 13 times more Tyr-containing peptides. Furthermore, we demonstrated that the treatment with carbonate-containing buffer could effectively reduce the side-product. Our study also implies that chemical reactions in proteomic studies should be carefully evaluated prior to their wide applications.

2020-12-10 | PXD020042 | Pride

Carboxypeptidase B-assisted charge-based fractional diagonal chromatography for deep screening of C-terminome

Project description:The determination of protein C-termini is of great significance for protein function annotation and proteolysis re-search. However, the progress of C-terminomics is still far behind its counterpart, N-terminomics, because of the low reactivity of the carboxyl group. Herein, we developed a negative selection strategy, termed carboxypeptidase B-assisted charge-based fractional diagonal chromatography (CPB-ChaFRADIC), to achieve a global C-terminome analysis. The highly reactive carboxypeptidase B cleavage was utilized to reduce the charge state of non-C-terminal peptides. Together with high-performance charge-based frac-tional diagonal chromatography, the C-terminal peptides could be isolated. Such a strategy was also applied for profiling C-termini from Escherichia coli cell lysates, and 441 canonical C-termini and 510 neo-C-termini originating from proteolytic processing were identified. These findings represent 2-fold and 5.8-fold that of identified C-termini via direct analysis, respectively. Using parallel digestion with trypsin and LysC, such a strategy enabled the identification of 604 canonical C-termini and 818 neo-C-termini, rep-resenting the largest C-terminome dataset of E. coli, and no deficiency in His/Lys/Arg-containing C-terminal peptides was observed. The presented CPB-ChaFRADIC strategy is therefore a highly efficient and unbiased strategy for large-scale C-terminome analysis. Furthermore, using the CPB-ChaFRADIC strategy, we identified 87 cleavage sites and 82 substrates of caspase-3 in Jurkat cells, demonstrating that the CPB-ChaFRADIC strategy shows great promise in promoting proteolysis research.

2020-05-25 | PXD018520 | Pride

Transcription profiling of mouse embryonic fibroblasts expressing wild type and heterozygous Plk4

Project description:A microarray comparison was performed to identify genes over expressed in both wild type and heterozygous Plk4 MEFs

2009-09-03 | E-MEXP-2161 | biostudies-arrayexpress

Transcription profiling of Saccharomyces cerevisiae pGAL1-MKS1, pGAL1-NNK1, and pGAL1-FMP48 overexpression

Project description:Analysis of the transcriptional effects of pGAL1-MKS1, pGAL1-NNK1, and pGAL1-FMP48 overexpression. BY4700 gal1::NAT ura3delta0 S. cerevisiae cells carrying a [pGAL1-MKS1-HA, URA3, CEN], [pGAL1-NNK1-HA, URA3, CEN], or [pGAL1-FMP48-HA, URA3, CEN] plasmid, or empty vector BG1805, were grown to early log phase, induced with 0.2% galactose, and harvested after 1.5 h and 6 h. RNA from a strain overexpressing MKS1, NNK1, or FMP48 was competitively hybridized with RNA from an identically treated strain carrying empty vector.<br><br>FMP48 = YGR052W. MKS1 = YNL076W. NNK1 = YKL171W.

2010-05-25 | E-MEXP-2171 | biostudies-arrayexpress

Posttranslation modification of NF-kB inducing kinase

Project description:2 This project is to identify the potential neddylation sites in human NF-kB inducing kinase (A.k.a. Map3K14). NIK-HA or NIK-HA in combination with FLAG-NEDD8 were co-expressed in HEK293T cells. NIK-HA were pulled down via denaturing immunoprecipitation protocol, subject to SDS-PAGE, in-gel trypsin digestion, and Nano LC-MS/MS Analysis. The potential peptides containing K-GG sites were identified.

2023-03-10 | PXD038653 | Pride

PKM2 functions as a histidine kinase to phosphorylate PGAM1 to increase glycolysis shunts in cancer

Project description:Phosphoglycerate mutase 1 (PGAM1) is a key-node enzyme that diverts the metabolic intermediates from glycolysis into its shunts to support macromolecule biosynthesis for rapid and sustainable cell proliferation. It is prevalent that PGAM1 activity is upregulated in various tumors; however, the underlying mechanism remains unclear. Here, we unveil that pyruvate kinase M2 (PKM2) moonlights as a histidine kinase in a phosphoenolpyruvate (PEP)-dependent manner to catalyze PGAM1 H11 phosphorylation, that is essential for PGAM1 activity. Moreover, the dimeric or monomeric PKM2 in tumor cells phosphorylates PGAM1 more efficiently than the tetrameric one. In response to epidermal growth factor (EGF), Src signaling triggered PGAM1 Y119 phosphorylation is a prerequisite for PKM2 binding and the subsequent H11 phosphorylation of PGAM1, which constitutes the discrepancy between tumor cells and normal ones. A PGAM1-derived pY119-containing cell-permeable peptide or Y119 mutation disrupts the interaction of PGAM1 with PKM2 and its H11 phosphorylation, and eventually dampens the glycolysis shunts and tumor growth. We not only identifes a histidine kinase function of PKM2, but also illustrates an enzymes-cross-talk regulatory mode during metabolic reprogramming.

2024-03-21 | PXD049235 | Pride

EGF promotes PKM2 O-GlcNAcylation by stimulating O-GlcNAc transferase phosphorylation at Y976 and their subsequent association

Project description:EGF is one of the most well-characterized growth factors and plays a crucial role in cell proliferation and differentiation. EGFR has been extensively explored as a therapeutic target against multiple types of cancers, such as lung cancer and glioblastoma. Recent studies have established a connection between deregulated EGF signaling and metabolic reprogramming, especially rewiring in aerobic glycolysis, which is also known as the Warburg effect and recognized as a hallmark in cancer. Pyruvate kinase M2 (PKM2) is a rate-limiting enzyme controlling the final step of glycolysis and serves as a major regulator of the Warburg effect. We previously showed that PKM2 T405/S406 O-GlcNAcylation, a critical mark important for PKM2 detetramerization and activity, was markedly upregulated by EGF. However, the mechanism by which EGF regulates PKM2 O-GlcNAcylation still remains uncharacterized. Here we demonstrated that EGF promoted O-GlcNAc transferase (OGT) binding to PKM2 by stimulating OGT Y976 phosphorylation. As a consequence, PKM2 O-GlcNAcylation and detetramerization were upregulated, leading to a significant decrease in PKM2 activity. Moreover, other than PKM2, the association of additional phosphotyrosine binding proteins, including STAT1, STAT3, STAT5, PKCδ and p85, which are reported factors bearing O-GlcNAcylation, with OGT was also enhanced when Y976 was phosphorylated. Together, EGF-dependent Y976 phosphorylation is critical for OGT-PKM2 interaction and we propose that this post-translational modification might be important for the substrate selection by OGT.

2022-08-22 | PXD031593 | Pride

Proteomic analysis reveals critical immunomolecular mechanisms in macrophage anti-tuberculosis processes

Project description:Tuberculosis infection activates the autoimmune system. However, the role of autoimmunity after Mycobacterium tuberculosis infection is unclear. In this study, liquid chromatography-tandem mass spectrometry (LC-MS/MS) combined with tandem mass spectrometry (TMT) was used to perform proteomic analysis of five spinal tuberculosis and five protruding disc tissue samples. The identification of key molecular mechanisms provides a molecular target for future treatment of spinal tuberculosis, which may significantly improve patients' quality of life and prognosis.

2021-01-04 | PXD022541 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

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