Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

"How to deal with internal fragment ions ?" (synthetic unmodified histone H3 Tail)

ABSTRACT: Synthetic histone H3.1were analyzed via MSMS on and Orbitrap Fusion Lumos. Various fragmentation parameters (e.g Reaction time, supplementary activation) were used in order to study the fragmentation efficiency and formation of internal fragment ions.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Arthur Grimaud

LAB HEAD: Veit Schwammle

PROVIDER: PXD050421 | Pride | 2025-02-24

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	0unmod_Agc1e5Z10ETD20SA15Uscan3INJT150Res30000_H31tail.raw	Raw
	0unmod_Agc1e5Z10ETD20SA15Uscan3INJT150Res30000_centroided.mzML	Mzml
	0unmod_Agc1e5Z10ETD20SA15Uscan3INJT150Res30000_deconvoluted.mzIdentML	Mzid
	0unmod_Agc1e5Z10ETD20SA15Uscan3INJT150Res30000_deconvoluted.mzML	Mzml
	10unmod_Agc1e5Z10ETD30SA0Uscan3INJT150Res30000_H31tail.raw	Raw

Items per page:

1 - 5 of 171

Similar Datasets

"How to deal with internal fragment ions ?" (synthetic unmodified histone H3, H4)

Project description:Synthetic histones (H3.1, H4) were analyzed via MSMS on an Eclipse mass spectrometer. Various fragmentation parameter and fragmentation methods were used in order to study the fragmentation efficiency and formation of internal fragment ions.

2025-02-24 | PXD050615 | Pride

Benchmarking multiple fragmentation methods on an Orbitrap Fusion for top-down phospho-proteoform characterization

Project description:Top-down analysis of intact proteins by mass spectrometry provides an ideal platform for comprehensive proteoform characterization, in particular, for the identification and localization of post-translational modifications (PTM) co-occurring on a protein. One of the main bottlenecks in top-down proteomics is insufficient protein sequence coverage caused by incomplete protein fragmentation. Based on previous work on peptides, increasing sequence coverage and PTM localization by combining sequential ETD and HCD fragmentation in a single fragmentation event, we hypothesized that protein sequence coverage and phospho-proteoform characterization could be equally improved by this new dual fragmentation method termed EThcD, recently been made available on the Orbitrap Fusion. Here, we systematically benchmark the performance of several (hybrid) fragmentation methods for intact protein analysis on an Orbitrap Fusion, using as a model system a 17.5 kDa N-terminal fragment of the mitotic regulator Bora. During cell division Bora becomes multiply phosphorylated by a variety of cell cycle kinases, including Aurora A and Plk1, albeit at distinctive sites. Here, we monitor the phosphorylation of Bora by Aurora A and Plk1, analyzing the generated distinctive phospho-proteoforms by top-down fragmentation. We show that EThcD and ETciD on a Fusion are feasible and capable of providing richer fragmentation spectra compared to HCD or ETD alone, increasing protein sequence coverage, and thereby facilitating phosphosite localization and the determination of kinase specific phosphorylation sites in these phospho-proteoforms.

2015-07-16 | PXD001845 | 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

Comprehensive identification of crosslinked peptides from methyltransferase Hmt1 and its substrate Npl3: use of multiple crosslinkers, mass spectrometry approaches and software platforms

Project description:Chemical-Crosslinking Mass Spectrometry (XLMS) using MS-cleavable crosslinkers is fast becoming an established technique in the study of protein-protein interactions for both small and large scale samples. With the increased uptake of XLMS as a technique, different combinations of crosslinker types, fragmentation strategies and analysis programs are being applied to a diverse array of biological samples. This study is focused on understanding how the three variables of an XLMS experiment – crosslinker type, fragmentation and program – could generate differences in identifications, leading to increased biological coverage of a sample. Here we probe for the first time, the known enzyme:substrate interaction between yeast arginine methyltransferase Hmt1p and its substrate, heterologous nucleolar protein Npl3p. We use this known interaction as a platform to compare two analysis programs, MeroX and XlinkX2.0, using two crosslinker types, DSSO and DSBU, and two mass-spectrometry fragmentation strategies, CID/ETD and SteppedHCD. Through these we also compare different algorithm strategies, Precursor and Reporter-Ion, as well as assess the impact of restricting data searches to lysine only crosslinks versus the inclusion of serine, threonine and tyrosine as reactive residues. From this analysis we show direct evidence of Hmt1p in contact with its known methylation sites on Npl3p, the intrinsically disordered “SRGG” region. We also show through our multi-crosslinker, multi-fragmentation and multi-software approach that two approaches leads to greater understanding and depth of a crosslinked sample, and this is due to some combinations of experimental variables creating greater coverage of crosslinks than others.

2018-07-20 | PXD008348 | Pride

Finding and using Diagnostic Ions in Collisional Induced Crosslinked Peptide Fragmentation Spectra

Project description:Crosslinking mass spectrometry (XL-MS) has emerged as a powerful tool in its own right for the investigation of protein structures and interactions. Utilizing standard shotgun MS mass spectrometry equipment and specialized database search software, crosslinked peptide-pairs can be identified and directly translated into distance constraints for protein structure and protein-protein interaction investigations. Whereas the gas-phase dissociation behavior of linear peptides is well understood, less is however known about the gas-phase dissociation behavior of crosslinked peptides. In this work, we set out to expose the behavior of commonly used crosslinking reagents, both non- as well as gas-phase cleavable, using synthetic peptides to establish mechanistic insights. We describe that crosslinked peptide pairs generate specific fragmentation patterns under HCD and CID fragmentation conditions, distinct from mono-linked peptide and non-modified peptides. We discuss in detail the resulting diagnostic ions that can help distinguishing linear peptides from mono-linked and crosslinked peptide pairs and how that may be used to further increase the efficiency of XL-MS analysis.

2019-07-16 | PXD013083 | Pride

Ultraviolet Photodissociation of ESI and MALDI generated protein ions on a Q-Exactive mass spectrometer

Project description:UVPD was implemented on an Orbitrap Q-Exactive plus equipped with a ESI/EP-MALDI. UVPD of MALDI generated ions was benchmarked against MALDI-ISD, MALDI-HCD and ESI-UVPD. MALDI UVPD outperformed MALDI HCD and ISD efficiently sequencing small proteins ions.

2019-01-24 | PXD011526 | Pride

Electron Transfer/ Higher Energy Collisional Dissociation of Doubly Charged Peptide Ions: Identification of Labile Protein Phosphorylations

Project description:Fragmentation behaviour of labile phosphorylated peptides

2019-05-27 | PXD012989 | Pride

Quantifying Positional Isomers (QPI) by top-down mass spectrometry

Project description:Proteomics has exposed a plethora of post-translational modifications, but demonstrating functional relevance requires new approaches. Top-down proteomics can characterize co-occurring modifications in terms of localization, abundance and hierarchy. Here, we present a top-down MS analysis workflow for the discovery and quantification of proteoforms. Confident fragment assignment allows for localization of modification sites and quantification of all proteoforms, including positional isomers, as validated by investigating synthetic isoforms of ubiquitin and hyper-phosphorylated Bora.

2021-03-18 | PXD012084 | Pride

Mirroring the charged termini; ETD/HCD fragmentation characteristics of LysargiNase and tryptic peptides and their benefits for peptide sequencing in proteomics

Project description:A key step in proteomics is the digestion of proteins into peptides, so far largely done by using trypsin. Tryptic digestion leads to peptides that in ESI-MS attain predominantly two charges, via protonation at the free N-terminus and at the C-terminal basic residue Arginine or Lysine. These peptides can be readily sequenced and identified by collision-induced dissociation (CID) or higher-energy collisional dissociation (HCD), as the fragmentation rules are well understood. Here we explore the trypsin mirror protease, LysargiNase, which cleaves equally specifically at Arg and Lys, albeit at the N-terminal end. The resulting peptides are therefore practically tryptic-alike in length and sequence, except that the two charges are now both positioned at the N-terminus. We compare the chromatographic separation properties, gas phase fragmentation behavior and (phospho)proteome sequence coverage of tryptic and LysargiNase peptides using electron-transfer dissociation (ETD), and for comparison HCD. We find that tryptic and LysargiNase peptides fragment nearly as mirror images. For LysargiNase predominantly N-terminal peptide ions (c-ions/ETD, b-ions/HCD) are formed, whereas for trypsin C-terminal fragment ions dominate (z-ions/ETD, y-ions/HCD). Especially during ETD LysargiNase peptides fragment into low-complexity, but information rich sequence ladders. We observe that trypsin and LysargiNase chart distinct parts of the (phospho)proteome. Therefore, we conclude that the collective use LysargiNase and Trypsin will benefit a more in-depth and reliable analysis of (phospho)proteomes.

2016-12-02 | PXD004447 | Pride

Middle-Down Proteomic Analyses with Ion Mobility Separations of Endogenous Isomeric Proteoforms

Project description:We integrated FAIMS into the WCX-HILIC ETD MS/MS system to improve identification of histonetail proteforms

2020-04-02 | PXD016918 | Pride