Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Revealing Corynebacterium glutamicum proteoforms through top-down proteomics

ABSTRACT: Corynebacterium glutamicum is a bacterium widely employed in the industrial production of amino acids as well as a broad range of other biotechnological products. The present study describes the characterization of C. glutamicum proteoforms, and their post-translational modifications (PTMs) employing top-down proteomics. Despite previous evidence of PTMs having roles in the regulation of C. glutamicum metabolism, this is the first top-down proteome analysis of this organism. We identified 1125 proteoforms from 273 proteins, with 60 % of proteins presenting at least one mass shift, suggesting the presence of PTMs, including several acetylated, oxidized and formylated proteoforms. Furthermore, proteins relevant to amino acid production, protein secretion, and oxidative stress were identified with mass shifts suggesting the presence of uncharacterized PTMs and proteoforms that may affect biotechnologically relevant processes in this industrial workhorse. For instance, the membrane proteins mepB and SecG were identified as a cleaved and a formylated proteoform, respectively. While in the central metabolism, OdhI was identified as two proteoforms with potential biological relevance: a cleaved proteoform and a proteoform with PTMs corresponding to a 70 Da mass shift.

INSTRUMENT(S): LTQ Orbitrap Elite

ORGANISM(S): Corynebacterium Glutamicum Atcc 13032

SUBMITTER: Reynaldo Magalhaes Melo

LAB HEAD: Marcelo Valle de Sousa

PROVIDER: PXD038038 | Pride | 2022-12-10

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	F_RC_36H_US_1A18_00_HH_18.raw	Raw
	F_RC_36H_US_1A18_00_HH_18_181027185709.raw	Raw
	F_RC_36H_US_1A18_00_HH_18_181027185709_ms2_toppic_prsm.csv	Csv
	F_RC_36H_US_1A18_00_HH_18_181027204011.raw	Raw
	F_RC_36H_US_1A18_00_HH_18_181027204011_ms2_toppic_prsm.csv	Csv

Items per page:

1 - 5 of 53

Similar Datasets

Capillary zone electrophoresis-tandem mass spectrometry as an alternative to liquid chromatography-tandem mass spectrometry for top-down proteomics of histones with the identification of over 1000 histone proteoforms

Project description:Top-down proteomics (TDP) is an ideal approach for deciphering the histone code and it routinely employs reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS). Here we present capillary zone electrophoresis (CZE)-MS/MS via the electro-kinetically pumped sheath-flow CE-MS interface for large-scale top-down delineation of histone proteoforms. CZE-MS/MS identified 80% more proteoforms than RPLC-MS/MS (589 vs. 322) from a calf histone sample with more than 30-fold less sample consumption (75-ng vs. 3 µg), indicating its substantially higher sensitivity. The electrophoretic mobility (µef) of unmodified histone proteoforms can be predicted accurately (R2=0.98) with an optimized semi-empirical model based on our recent work, and we revealed that N-terminal acetylation had minimal effect on histone proteoforms’ µef. We identified 1108 histone proteoforms from the calf histone sample using two-dimensional size-exclusion chromatography (SEC)-CZE-MS/MS with less than 300-ng proteins consumed. We achieved delineation of histone proteoforms regarding post-translational modifications (PTMs) and their combinations, relative abundance among different proteoforms of the same protein, as well as the frequency of PTM occurrence on histone proteoforms. We identified histone proteoforms carrying various PTMs, e.g., acetylation, methylation (mono-, di-, and tri-), phosphorylation, and succinylation. We revealed that different histone proteins had drastically different patterns of PTMs. The results render CZE-MS/MS as a useful tool for deciphering the histone code in a proteoform-specific manner and on a global scale.

2022-04-04 | PXD021889 | Pride

Top Down Histone H4 - SUM159 Butyrate

Project description:Proteins physiologically exist and function as ‘proteoforms’ that arise from one gene but acquire additional function by further variation such as post-translational modification (PTM). When multiple PTMs coexist on single protein molecules top down proteomics becomes the only feasible method; however, most top down methods have limited quantitative capacity and insufficient throughput to truly address proteoform biology. Here we demonstrate that, with meticulous design and diligent consideration of statistics, top down proteomics is quantitative, reproducible, sensitive, and high throughput. The proteoforms of histone H4 are well-studied both as a challenging proteoform identification problem and due to their essential role in the regulation of all eukaryotic DNA templated processes, including gene expression. Much of histone H4’s function is obfuscated from prevailing methods due to combinatorial mechanisms. Starting from cells or tissues, after an optimized protein purification process the H4 proteoforms are physically separated by online C-3 chromatography, narrowly isolated in MS1 and sequenced with ETD fragmentation. With this method we achieve more than 30 replicates from a single 35mm tissue culture dish by loading 55ng of H4 on column. Parallelization and automation yield a sustained throughput of 12 replicates per day, operating continuously for weeks. We achieve reproducible quantitation (average biological Pearson correlations of 0.89) of hundreds of proteoforms (about 200-300) over almost six orders of magnitude and an estimated LLoQ of 0.001% abundance. We demonstrate the capacity of the method to precisely measure well-established changes with sodium butyrate treatment of SUM159 cells.

2019-11-22 | PXD013766 | Pride

Capillary Zone Electrophoresis-High Field Asymmetric Ion Mobility Spectrometry-Tandem Mass Spectrometry for Top-down Characterization of Histone Proteoforms

Project description:Characterization of histone proteoforms with various post-translational modifications (PTMs) is critical for a better understanding of functions of histone proteoforms in epigenetic control of gene expression. Mass spectrometry (MS)-based top-down proteomics (TDP) is a valuable approach for delineating histone proteoforms because it can provide us with a bird's-eye view of histone proteoforms carrying diverse combinations of PTMs. Here, we present the first example of coupling capillary zone electrophoresis (CZE), ion mobility spectrometry (IMS), and MS for online multi-dimensional separations of histone proteoforms. Our CZE-high-field asymmetric waveform IMS (FAIMS)-MS/MS platform identified 366 histone proteoforms from a commercial calf histone sample using a low microgram amount of histone sample as the starting material. CZE-FAIMS-MS/MS improved the number of histone proteoform identifications by about 3 folds compared to CZE-MS/MS alone (without FAIMS). The results indicate that CZE-FAIMS-MS/MS could be a useful tool for comprehensive characterization of histone proteoforms with high sensitivity.

2023-12-07 | PXD041357 | 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

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

Positional proteomics reveals differences in N-terminal proteoform stability

2015-10-23 | GSE74279 | GEO

Large-scale Top-down Proteomics of the Arabidopsis thaliana Leaf and Chloroplast Proteomes

Project description:We present a large-scale top-down proteomics study of plant leaf and chloroplast proteins, achieving the identification of over 4700 unique proteoforms. Using capillary zone electrophoresis coupled with tandem mass spectrometry analysis of offline size-exclusion chromatography fractions, we identify 3198 proteoforms for total leaf and 1836 proteoforms for chloroplast, with 1024 and 363 proteoforms having post-translational modifications (PTMs), respectively. The electrophoretic mobility prediction of CZE allowed us to validate PTMs that impact the charge state such as acetylation and phosphorylation. Identified PTMs included Trp (di)oxidation events on six chloroplast proteins that may represent novel targets of singlet oxygen sensing. Furthermore, our top-down proteomics data provides direct experimental evidence of the N- and C-terminal residues of numerous mature proteoforms from chloroplast, mitochondria, endoplasmic reticulum, and other sub-cellular localizations. With this information, we propose transit peptide cleavage sites and correct sub-cellular localization signal predictions. This large-scale analysis illustrates the power of top-down proteoform identification of PTMs and intact sequences that can benefit our understanding of both the structure and function of hundreds of plant proteins.

2023-01-05 | PXD034368 | Pride

Automated capillary isoelectric focusing-tandem mass spectrometry for top-down proteomics

Project description:Mass spectrometry (MS)-based top-down proteomics (TDP) delineates proteoforms in cells and requires high-resolution proteoform separation. Herein, for the first time, we employed an automated capillary isoelectric focusing-tandem MS (cIEF-MS/MS) system for large-scale TDP of complex proteomes. Single-shot cIEF-MS/MS identified 771 proteoforms from an Escherichia coli (E. coli) proteome consuming only nanograms of proteins. Coupling two-dimensional size exclusion chromatography (SEC)-cIEF, named “gel-free 2D-PAGE”, to ESI-MS/MS enabled the identification of nearly 2000 proteoforms from the E. coli proteome. Label-free quantitative TDP of zebrafish male and female brains using the SEC-cIEF-MS/MS quantified thousands of proteoforms and revealed sex-dependent proteoform profiles in brains. We discovered several proteolytic proteoforms of pro-opiomelanocortin and prodynorphin with significantly higher abundance in male brains as potential endogenous hormone proteoforms. Multi-level quantitative proteomic analyses (TDP and bottom-up proteomics (BUP)) of the brains revealed that majority of proteoforms having statistically significant difference in abundance between genders (TDP) showed no abundance difference at the corresponding protein group level (BUP), which highlights the importance of delineating proteins in a proteoform-specific manner for accurately understanding protein function.

2020-12-08 | PXD020342 | Pride

Combination of digital microfluidics and magnetic bead-based intact proteoform elution for top-down nanoproteomics of single C. elegans

Project description:While most nanoproteomics approaches for the analysis of low-input samples are based on bottom-up LC-MS workflows, top-down approaches enabling proteoform characterization are still underrepresented. Using mammalian cell proteomes, we here established a facile one-pot sample preparation protocol based on protein aggregation on magnetic beads and intact proteoform elution using 40% v/v formic acid. Performed on a digital microfluidics device, the workflow enabled sensitive analysis of single Caenorhabditis elegans nematodes, increasing the number of proteoform identifications compared to in-tube sample preparation by 46%. Label-free quantification of single nematodes grown under different conditions allowed to identify changes in abundance of proteoforms not distinguishable by bottom-up proteomics. The workflow presented here will help to elucidate cellular changes on the proteoform level in other samples of limited availability.

2023-06-19 | PXD039883 | Pride

Denaturing and native top-down venomics: Mapping proteoforms and protein complexes from king cobra venom

Project description:Characterizing whole proteins by top-down proteomics avoids a step of inference encountered in the dominant bottom-up methodology when peptides are assembled computationally into proteins for identification. The direct interrogation of whole proteins and protein complexes from the venom of Ophiophagus hannah (king cobra) provides a sharply clarified view of toxin sequence variation, transit peptide cleavage sites and post-translational modifications (PTMs) likely critical for venom lethality. A tube-gel format for electrophoresis (called GELFrEE) and solution isoelectric focusing were used for protein fractionation prior to LC-MS/MS analysis resulting in 131 protein identifications (18 more than bottom-up) and a total of 184 proteoforms characterized from 14 protein toxin families. Operating both GELFrEE and mass spectrometry to preserve non-covalent interactions generated detailed information about two of the largest venom glycoprotein complexes: the homodimeric L-amino acid oxidase (LAAO, ~130 kDa) and the multi-chain toxin cobra venom factor (~147 kDa). The LAAO complex exhibited two clusters of multi-proteoform complexes corresponding to the presence of 5 or 6 N-glycans moieties, each consistent with a distribution of N-acetyl hexosamines. Employing top-down proteomics in both native and denaturing modes provides unprecedented characterization of venom proteoforms and their complexes. A precise molecular inventory of venom proteins will propel the study of snake toxin variation and the targeted development of new anti-venoms or other biotherapeutics.

2016-05-18 | PXD003403 | Pride