Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

AP-MS-based interactome analysis of three pairs of N-terminal proteoforms and their canonical protein

ABSTRACT: N-terminal proteoforms stem from the same gene but differ at their N-terminus, and most of these are found to be truncated, though some are N-terminally extended caused by ribosomes starting translation from codons in the annotated 5’UTR, and/or carry modified N-termini different from those of the canonical protein. Biological functions of N-terminal proteoforms are emerging, however, it remains unknown to what extend N-terminal proteoforms further expand the functional complexity. To address this in a more global manner, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. For this, we first generated an in-depth catalogue of N-terminal proteoforms in the cytosol of HEK293T cells. From which 20 pairs of canonical protein and N-terminal proteoform(s) were selected. Protein-protein interaction profiling was done with Virotrap. Virotrap was selected as this methods avoids lysis before the isolation of the complexes and allows the detection of weak and cytosolic proteins as well. Our analysis of these pairs revealed that the overlap of the interactomes for both proteoforms is in general high, showing their functional relation. However, for all pairs tested we do report differences as well. We show that N-terminal proteoforms can be engaged in new/different interactions and as well can lose several interactions compared to the canonical protein. We used AP-MS to validate differences in the interaction networks reported for three of these 20 pairs of N-terminal proteoform and canonical counterpart.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Annelies Bogaert

LAB HEAD: Kris Gevaert

PROVIDER: PXD039085 | Pride | 2023-06-08

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Virotrap-based interactome analysis of 20 pairs of N-terminal proteoforms and their canonical protein

Project description:N-terminal proteoforms stem from the same gene but differ at their N-terminus, and most of these are found to be truncated, though some are N-terminally extended caused by ribosomes starting translation from codons in the annotated 5’UTR, and/or carry modified N-termini different from those of the canonical protein. Biological functions of N-terminal proteoforms are emerging, however, it remains unknown to what extend N-terminal proteoforms further expand the functional complexity. To address this in a more global manner, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. For this, we first generated an in-depth catalogue of N-terminal proteoforms in the cytosol of HEK293T cells. From which 20 pairs of canonical protein and N-terminal proteoform(s) were selected. Protein-protein interaction profiling was done with Virotrap. Virotrap was selected as this methods avoids lysis before the isolation of the complexes and allows the detection of weak and cytosolic proteins as well. Our analysis of these pairs revealed that the overlap of the interactomes for both proteoforms is in general high, showing their functional relation. However, for all pairs tested we do report differences as well. We show that N-terminal proteoforms can be engaged in new/different interactions and as well can lose several interactions compared to the canonical protein.

2023-06-08 | PXD039171 | Pride

Positional proteomics reveals differences in N-terminal proteoform stability

Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first study on protein turnover using positional proteomics and ribosome profiling to distinguish between N-terminal proteoforms of individual genes. Overall, we monitored the stability of 1,941 human N-terminal proteoforms, including 147 N-terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. Ribosome profiling of lactimidomycin and cycloheximide treated human Jurkat T-lymphocytes

2015-10-23 | E-GEOD-74279 | biostudies-arrayexpress

Positional proteomics reveals differences in N-terminal proteoform stability

2015-10-23 | GSE74279 | GEO

Mapping the cytosolic proteins of HEK293T cells

2023-06-08 | PXD039127 | Pride

N-terminal COFRADIC on cytosolic proteins of HEK293T cells - UniProt search

Project description:N-terminal proteoforms stem from the same gene but differ at their N-terminus, and most of these are found to be truncated, though some are N-terminally extended caused by ribosomes starting translation from codons in the annotated 5’UTR, and/or carry modified N-termini different from those of the canonical protein. Biological functions of N-terminal proteoforms are emerging, however, it remains unknown to what extend N-terminal proteoforms further expand the functional complexity. To address this in a more global manner, we mapped the interactomes of several pairs of N-terminal proteoforms and their canonical counterparts. For this, we first generated an in-depth catalogue of N-terminal proteoforms in the cytosol of HEK293T cells. As the N-terminal region is the part that differs between the proteoforms, we performed N-terminal enrichment via COFRADIC on the cytosol of HEK293T cells. We combined three digestion enzymes to increase the depth of analysis. Data was searched twise: once with a regular UniProt database and a second time with a custom database (combining the sequences of UniProt proteins, UniProt isoforms and publicly available Ribo-seq data). Data was filtered and this resulted in a catalogue of 3,306 N-termini from which 20 pairs of canonical protein and N-terminal proteoform(s) were selected for interactome analysis. Our analysis of these pairs revealed that the overlap of the interactomes for both proteoforms is in general high, showing their functional relation. However, for all pairs tested we do report differences as well. We show that N-terminal proteoforms can be engaged in new/different interactions and as well can lose several interactions compared to the canonical protein.

2023-06-08 | PXD039392 | Pride

Positional proteomics reveals differences in N-terminal proteoform stability.

Project description:To understand the impact of alternative translation initiation on a proteome, we performed the first large-scale study of protein turnover rates in which we distinguish between N-terminal proteoforms pointing to translation initiation events. Using pulsed SILAC combined with N-terminal COFRADIC we monitored the stability of 1,941human N-terminal proteoforms, including 147 proteoform pairs with heterogeneous N-termini originating from the same gene that result from alternative translation initiation and incomplete processing of the initiator methionine. N-terminally truncated proteoforms were on average less abundant than canonical proteoforms, many had different stabilities and exhibited both faster and slower turnover rates compared to their canonical counterparts. These differences in stability did not depend on the length of truncation but on individual protein characteristics. In silico simulation of N-terminal proteoforms in macromolecular complexes revealed possible consequences for complex integrity such as replacement of unstable canonical subunits. The extent of intrinsic disorder in N-terminal protein structures correlated with turnover times, indicating that a change in the structural flexibility of protein N-termini in truncated proteoforms might impact proteoform stability. Interestingly, removal of the initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms while susceptibility for N-terminal acetylation, another common co-translational modification, did not seem to impact on turnover rates. Taken together, our findings reveal differences in protein stability between N-terminal proteoforms and point to a role for alternative translation initiation and co-translational initiator methionine removal in the overall regulation of proteome homeostasis.

2013-03-14 | PXD002091 | Pride

Positional proteomics reveals differences in N-terminal proteoform stability.

2019-03-13 | MSV000083565 | MassIVE

Virotrap-based interactome analysis of a NTR protein

Project description:Ribosome profiling has revealed translation outside of canonical coding sequences (CDSs) including translation of short upstream ORFs, long non-coding RNAs, overlapping ORFs, ORFs in UTRs or ORFs in alternative reading frames. Studies combining mass spectrometry, ribosome profiling and CRISPR-based screens showed that hundreds of ORFs derived from non-coding transcripts produce (micro)proteins and might be functional, while other studies failed to find evidence for such types of non-canonical translation events. In this study we tried to detect (and characterize) proteins originating from these non-translated regions (NTRs). We attempted to discover translation products from non-coding regions at large scale by reducing the overall sample complexity (by enriching cytosolic N-terminal peptides) and combined it with an extend search space (combining UniProt proteins, UniProt isoforms and publicly available Ribo-seq data). Reasoning that this strategy would increase the likelihood of identifying proteins from NTRs. Further, we introduced rigorous data analysis and results curation workflows. This stringent filtering approach was found essential to retain confident translational evidence at the peptide level for NTRs. We show that, theoretically, our strategy facilitates the detection of translation events of transcripts from NTRs, but experimentally we find that less than 1% of all identified peptides might originate from such translation events. However, one NTR protein was further characterized by Virotrap based interaction analysis. This resulted in several potential interaction partners associated with membranes and vesicle transport. Showing that the non-translated regions that do result in proteins might be functional.

2022-07-12 | PXD030216 | Pride

Capillary zone electrophoresis-tandem mass spectrometry with activated ion electron transfer dissociation for large-scale top-down proteomics

Project description:Capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) has been recognized as an efficient approach for top-down proteomics recently for its high-capacity separation and highly sensitive detection of proteoforms. However, the commonly used collision-based dissociation methods often cannot provide extensive fragmentation of proteoforms for thorough characterization. Activated ion electron transfer dissociation (AI-ETD), that combines infrared photoactivation concurrent with ETD, has shown better performance for proteoform fragmentation than higher energy-collisional dissociation (HCD) and standard ETD. Here, we present the first application of CZE-AI-ETD on an Orbitrap Fusion Lumos mass spectrometer for large-scale top-down proteomics of Escherichia coli (E. coli) cells. CZE-AI-ETD outperformed CZE-ETD regarding proteoform and protein identifications (IDs). CZE-AI-ETD reached comparable proteoform and protein IDs with CZE-HCD. CZE-AI-ETD tended to generate better expectation values (E-values) of proteoforms than CZE-HCD and CZE-ETD, indicating higher quality of MS/MS spectra from AI-ETD respecting the number of sequence-informative fragment ions generated. CZE-AI-ETD showed great reproducibility regarding the proteoform and protein IDs with relative standard deviations less than 4% and 2% (n=3). Coupling size exclusion chromatography (SEC) to CZE-AI-ETD identified 3028 proteoforms and 387 proteins from E. coli cells with 1% spectrum-level and 5% proteoform-level false discovery rates. The data represents the largest top-down proteomics dataset using the AI-ETD method so far. Single-shot CZE-AI-ETD of one SEC fraction identified 957 proteoforms and 253 proteins. N-terminal truncations, signal peptide cleavage, N-terminal methionine removal and various post-translational modifications including protein N-terminal acetylation, methylation, S-thiolation, disulfide bonds, and lysine succinylation were detected.

2019-06-11 | PXD012247 | Pride