Positional proteomics reveals differences in N-terminal proteoform stability
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE74279 | GEO | 2015/10/23
REPOSITORIES: GEO
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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
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
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.
2019-03-13 | MSV000083565 | MassIVE
Project description:While previous studies suggest that many mRNAs containing more than one translation initiation site (TIS), the biological significance of most alternative TISs and their protein isoform (proteoform) products remains undetermined. Here we show that alternative translation initiation at a CUG and an AUG TIS in neuronal pentraxin receptor (NPR) mRNA produces two proteoforms, and their relative abundance is regulated by both neuronal activity as well as an adjacent RNA secondary structure. Downstream AUG initiation transformed the N-terminal transmembrane domain into a signal peptide, thereby converting NPR to a secreted factor sufficient to promote synaptic clustering of AMPA-type glutamate receptors. Changing the relative proteoform ratio, but not the overall NPR abundance reduces AMPA receptor in parvalbumin (PV)-positive interneurons and induces changes in learning behaviors in mice. In addition to NPR, N-terminal extensions of C1q-like synaptic organizers, mediated by upstream AUU start codons, anchor these otherwise secreted factors to the membrane. Thus, our results uncovered the plasticity of N-terminal signal sequences regulated by alternative TIS usage as a widespread mechanism to diversify protein localization and functions.
2024-09-23 | GSE255191 | GEO
Project description:Proteogenomics is an emerging research field yet lacking a standard method of analysis. In this article, we demonstrate the strength of proteogenomic analysis specific for N-terminal data that aims at the discovery of novel translational start sites. In summary, unidentified spectra were matched to a specific N-terminal peptide library encompassing all theoretical protein N-termini encoded in the genome. Gene prediction suggested 81 protein-coding models, of which several alternative proteoforms with unannotated protein starts. Next to the proteomic data, complementary ribosome footprinting data was generated from Arabidopsis thaliana cell cultures. Translation initiation site mapping by the ribosome footprinting data provided orthogonal evidence for 14 novel peptides identified by our proteogenomics pipeline.
2016-11-25 | GSE88790 | GEO
Project description:Here we report that the murine Tox gene encodes two proteins from a single mRNA and investigate the mechanism of production and function of these proteoforms. The annotated TOX coding sequence is predicted to produce a 526 amino acid protein (TOXFL). However, western blots reveal two bands. We found that the lower band consists of an N-terminally truncated variant of TOX (TOXDN), while the slower migrating band is TOXFL. The TOXDN proteoform is alternatively translated via leaky ribosomal scanning from an evolutionarily conserved translation initiation site downstream of the annotated translation initiation site. When expressed exogenously from a cDNA in murine CD8 T cells or HEK cells, or endogenously from the murine Tox locus, both forms are translated, although the ratio of TOXFL: TOXDN significantly varies with cellular context. This includes regulation of proteoform production during development of murine CD4 T cells in the thymus, where the positive selection of CD4+8+ cells and subsequent differentiation to CD4+8lo transitional and CD4SP cell subsets is associated with both an increase in total TOX protein and increased TOXDN production relative to TOXFL. Finally, we found that sole expression of TOXFL had a greater effect on gene regulation during chronic stimulation of murine CD8 T cells in culture mimicking exhaustion, than did TOXDN, including uniquely regulated cell cycle and other genes.
2023-07-10 | GSE236561 | GEO
Project description:Various forms of the protein, called proteoform, are generated by co- or post-translational modification, alternative splicing and alternative translation initiation site, resulting in interactions between ribosomes, mRNAs, tRNAs, and various enzymes. Here, we introduce a N-terminal peptide enrichment method (Nrich) using a negative selection on filter for the N-terminal peptides with two chemical reagents for labeling free amine and two endoproteases. We identified 6,525 acetylated (or partially acetylated) and 6,570 free protein N-termini from 5,727 proteins. The protein N-termini can be classified into nine groups, such as initial methionine removed or retained, non-terminal residue, signal/transit/pro-peptide removal, putative alternative translational initiation site (methionine removed or retained) and unknown processing, suggesting various proteoform in vivo. In addition, novel protein N-termini was identified in 5`-UTR sequence with pseudo start codon using customized database. Nrich can obtain information about protein stability, localization and function through the observation of finished N-terminal sequence of mature protein.
2017-08-17 | PXD005583 | Pride
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. 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.
2023-06-08 | PXD039085 | Pride
Project description:Large-scale top-down proteomics characterizes proteoforms in cells globally with high confidence and high throughput using reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) or capillary zone electrophoresis (CZE)-MS/MS. The false discovery rate (FDR) from the target-decoy database search is typically deployed to filter identified proteoforms to ensure high-confidence identifications (IDs). It has been demonstrated that the FDRs in top-down proteomics can be drastically underestimated. An alternative approach to the FDR can be useful for further evaluating the confidence of proteoform IDs after database search. We argue that predicting retention/migration time of proteoforms from the RPLC/CZE separation accurately and comparing their predicted and experimental separation time could be a useful and practical approach. Based on our knowledge, there is still no report in the literature about predicting separation time of proteoforms using large top-down proteomics datasets. In this pilot study, for the first time, we evaluated various semi-empirical models for predicting proteoforms’ electrophoretic mobility (µef) using large-scale top-down proteomics datasets from CZE-MS/MS. We achieved a linear correlation between experimental and predicted µef of E. coli proteoforms (R2=0.98) with a simple semi-empirical model, which utilizes the number of charges and molecular mass of each proteoform as the parameters. Our modeling data suggest that the complete unfolding of proteoforms during CZE separation benefits the prediction of their µef. Our results also indicate that N-terminal acetylation and phosphorylation both decrease proteoforms’ charge by roughly one charge unit.
2020-02-17 | PXD017265 | Pride
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