Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

ABSTRACT: Cross-linking/mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition proper-ties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

INSTRUMENT(S): Orbitrap Fusion Lumos, LTQ Orbitrap Elite

ORGANISM(S): Homo Sapiens (human) Schizosaccharomyces Pombe

SUBMITTER: Therese Dau

LAB HEAD: Juri Rappsilber

PROVIDER: PXD011459 | Pride | 2019-03-07

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	HSA_active.fasta	Fasta
	HSA_elastase-trypsin_crosslinks.csv	Csv
	HSA_elastase-trypsin_rep1.apl	Other
	HSA_elastase-trypsin_rep1.raw	Raw
	HSA_elastase-trypsin_rep2.apl	Other

Items per page:

1 - 5 of 119

Similar Datasets

Proteomics Using Proteases Alternative to Trypsin Benefits from Sequential Digestion with Trypsin

Project description:High specificity and ease of use make trypsin the most used enzyme in proteomics. Proteases with complementary cleavage specificity to trypsin have been applied to obtain additional data. However, use of proteases with broad specificity proved especially challenging. In this work, we analyzed the characteristics of five protease alternatives to trypsin for protein identification and sequence coverage when applied to S. pombe whole cell lysates. The specificity of the protease heavily impacted on the number of proteins identified. Proteases with higher specificity let to the identification of more proteins than proteases with lower specificity. However, AspN, GluC, chymotrypsin and proteinase K largely benefited from being paired with trypsin in sequential digestion, as had been shown by us for elastase before. In the most extreme case, the addition of trypsin to a proteinase K digest increased the number of identified proteins by 524 %. Also, AspN (82 %) and GluC (74 %) protein identifications largely improved following the additional digestion with trypsin. In general, protein identifications improved most over the use of the single protease when the enzymes followed on an initial digestion with trypsin. In the most extreme case, the sequential digest with trypsin and AspN yielded even higher number of protein identifications than digesting with trypsin alone.

2021-06-07 | PXD017321 | Pride

β-TrCP and Casein Kinase II mediated degradation of cyclin F controls timely mitotic progression

Project description:Orderly progressions of events in the cell division cycle are necessary to ensure the replication of DNA and cell division. Checkpoint systems allow the accurate execution of each cell cycle phase. The precise regulation of the levels of cyclin proteins is fundamental to coordinate cell division with checkpoints, avoiding genome instability. Cyclin F has important functions in regulating the cell cycle during the G2 checkpoint; however, the mechanisms underlying the regulation of cyclin F are poorly understood. Here, we observe that cyclin F is regulated by proteolysis through β-TrCP. β-TrCP recognizes cyclin F through a non-canonical degron site (TSGXXS) after its phosphorylation by Casein Kinase II. The degradation of cyclin F mediated by β-TrCP occurs at the G2/M transition and this event is required to promote mitotic progression and favours the activation of a transcriptional programme required for mitosis.

2018-10-02 | PXD010737 | Pride

Tissue Microfibril Comparison - Structural and compositional diversity of fibrillin microfibrils in human tissues

Project description:Fibrillin microfibrils are large supramolecular ECM assemblies which play different functional roles in different tissues. We aim to compare the ultrastructure and biological composition of fibrillin microfibrils purified from human eye, skin and cultured dermal fibroblasts. This will allow us to understand whether these their structure and compositions have evolved to suit the functions they play in different tissues.

2018-02-16 | PXD008450 | Pride

A truncated MRE11 form induced by SPRTN is deficient in DNA repair and radiosensitises cancer cells

Project description:he human MRE11/RAD50/NBS1 (MRN) complex plays a crucial role in sensing and repairing DNA DSB. MRE11 possesses 3’-5’ exonuclease and endonuclease activity and forms the core of the multifunctional MRN complex. We previously identified a C-terminally truncated form of MRE11 (TR-MRE11) associated with post-translational MRE11 degradation. Here we identified the approximate cleavage site of TR-MRE11 at 559-580 amino acids, its DNA damage repair function and the factors regulating TR-MRE11 accumulation. The nuclease enzymatic activity of TR-MRE11 was dramatically reduced, associated with a lack of DNA binding efficiency, whilst TR-MRE11 still interacted efficiently with RAD50 and NBS1. Lack of the MRE11 C-terminal resulted in deficient HR repair and increased cellular radiosensitivity. Knockdown of SprT-like N-terminal domain (SPRTN), an essential metalloprotease for DNA-protein crosslink repair, resulted in failure of MRE11 cleavage, with TR-MRE11 protein levels being positively correlated with SPRTN protein expression. The presence of this DNA repair-defective C-terminal truncation could explain the finding of high MRE11 expression, by immunohistochemistry using an antibody against MRE11 prior to the C-terminal, being associated with survival following radical radiotherapy in cancer patients. Ultimately, understanding the functional differences between intact and repair-defective MRE11 may lead to improvements in patient outcomes through a more informed choice of treatment.

2021-02-23 | PXD017964 | Pride

Sequence and structural Sequence and structural variations determining the recruitment of WNK kinases to the KLHL3 E3 ligase

Project description:The BTB-Kelch protein KLHL3 is a Cullin3-dependent E3 ligase that mediates the ubiquitin-dependent degradation of kinases WNK1-4 to control blood pressure and cell volume. A crystal structure of KLHL3 has defined its binding to an acidic degron motif containing a PXXP sequence that is strictly conserved in WNK1, WNK2 and WNK4. Mutations in the second proline abrograte the interaction causing the hypertension syndrome pseudohypoaldosteronism type II. WNK3 shows a diverged degron motif containing 4 amino acid substitutions that remove the PXXP motif raising questions as to the mechanism of its binding. To understand this atypical interaction, we determined the crystal structure of the KLHL3 Kelch domain in complex with a WNK3 peptide. The electron density enabled the complete 11-mer WNK-family degron motif to be traced for the first time revealing several conserved features not captured in previous work, including additional salt bridge and hydrogen bond interactions. Overall, the WNK3 peptide adopted a conserved binding pose except for a subtle shift to accommodate bulkier amino acid substitutions at the binding interface. At the centre, the second proline was substituted by WNK3 Thr541, providing a unique phosphorylatable residue among the WNK-family degrons. Fluorescence polarisation and structural modelling experiments revealed that its phosphorylation would abrogate the KLHL3 interaction similarly to hypertension-causing mutations. Together, these data reveal how the KLHL3 Kelch domain can accommodate the binding of multiple WNK isoforms and highlight a potential regulatory mechanism for the recruitment of WNK3.

2022-03-07 | PXD031606 | Pride

Improved analysis of crosslinking mass spectrometry data with Kojak 2.0, advanced by integration into the Trans-Proteomic Pipeline

Project description:Fragmentation ion spectral analysis of chemically crosslinked proteins is an established technology in the proteomics research repertoire for determining protein interactions, spatial orientation, and structure. Here we present Kojak version 2.0, a major update to the original Kojak algorithm, which was developed to identify crosslinked peptides from fragment ion spectra using a database search approach. A substantially improved algorithm with updated scoring metrics, support for cleavable crosslinkers, and identification of crosslinks between 15N-labeled homomultimers are among the newest features of Kojak 2.0 presented here. Kojak 2.0 is now integrated into the Trans-Proteomic Pipeline, enabling access to dozens of additional tools within that suite. In particular, the PeptideProphet and iProphet tools for validation of crosslinks improve the sensitivity and accuracy of correct crosslink identifications at user-defined thresholds. These new features improve the versatility of the algorithm, enabling its use in a wider range of experimental designs and analysis pipelines. Kojak 2.0 remains open-source and multi-platform.

2023-01-18 | PXD037492 | Pride

Cross-Linked/Mass-spectrometry analysis of RNA-Polimerase II

Project description:The data of a previous publication (Chen et.al. 2009 doi:10.1038/emboj.2009.401) where reprocesed and then used to validate false discovery rate calculations for unique residue pairs.

2017-03-09 | PXD004749 | Pride

Cebpa regulates gene transcription in basophils

Project description:To investigate the mechanisms by which C/EBPa drives basophil differentiation and maintains basophil identity, we examined whether or not C/EBPa promotes basophil molecular programming and simultaneously represses mast cell molecular programming. We performed genome-wide gene expression profiling on basophils and mast cells and found that 6798 genes were shared by mast cells and basophils; 2033 genes were expressed 2-10 (log2 1-3.3)-fold higher in basophils (differentially expressed in basophils); and 413 genes were expressed greater than 10 (log2 3.3)-fold in basophils (highly expressed in basophils). On the other hand, we found 569 genes were expressed 2-10 (log2 -1 to -3.3) fold higher in mast cells and 171 genes were highly expressed in mast cells [greater than 10 fold (log2 -3.3)]. We treated purified basophils prepared from Cebpaf/f RosaYFP/creER mice and Cebpa+/+ RosaYFP/creER control mice with or without 4HT treatment for five days. Gene expression in the treated basophils was analyzed using microarray analysis. Overall, deletion of C/EBPa in basophils resulted in a reduction of mRNA expression for 248 genes and led to an increase in mRNA expression for 255 genes. The majority of the C/EBPa-regulated genes were either differentially or highly expressed in basophils or mast cells. In this study, we compared gene expression in basophils and mast cell and identified genes which specifically expressed in basophils and mast cells. By using Cebpa conditional knock out mice, we identified Cebpa regulated genes in basophils.

2013-04-01 | E-GEOD-41596 | biostudies-arrayexpress

Cross-Linking/mass spectrometry (CLMS) analysis of RNA polymerase II

Project description:The mass spectrometry data of a previous publication (Chen et.al. 2009 doi:10.1038/emboj.2009.401) were reprocesed and is used for method developing, teaching and training purpose.

2018-07-20 | PXD010481 | Pride

The E. coli effector EspJ blocks Src kinase activity via amidation and ADP-ribosylation

Project description:In vitro ADP-ribosylated Src-K295M was separated by SDS-PAGE and the Coomassie stained protein band excised and digested with either trypsin, thermolysin or elastase. All digests were analysed by nanoLC-MS/MS using Higher-energy C-trap Dissociation (HCD) as well as Electron-Transfer Dissociation (ETD) fragmentation.

2014-12-19 | PXD001461 | Pride