Upload for CID/HCD/ETD MetaSPS paper (JPR, 2013)
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ABSTRACT:
PROVIDER: MSV000078436 | MassIVE | Wed May 29 00:00:00 BST 2013
REPOSITORIES: MassIVE
Similar Datasets
Project description:3 hour nLC-MS/MS runs used to compare/combine CID/ETD/HCD spectra for peptides from four separate protease digesions.
2013-12-23 | MSV000078517 | MassIVE
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
Project description:UnlabelledCore-fucosylation (CF) plays important roles in regulating biological processes in eukaryotes. Alterations of CF-glycosites or CF-glycans in bodily fluids correlate with cancer development. Therefore, global research of protein core-fucosylation with an emphasis on proteomics can explain pathogenic and metastasis mechanisms and aid in the discovery of new potential biomarkers for early clinical diagnosis. In this study, a precise and high throughput method was established to identify CF-glycosites from human plasma. We found that alternating HCD and ETD fragmentation (AHEF) can provide a complementary method to discover CF-glycosites. A total of 407 CF-glycosites among 267 CF-glycoproteins were identified in a mixed sample made from six normal human plasma samples. Among the 407 CF-glycosites, 10 are without the N-X-S/T/C consensus motif, representing 2.5% of the total number identified. All identified CF-glycopeptide results from HCD and ETD fragmentation were filtered with neutral loss peaks and characteristic ions of GlcNAc from HCD spectra, which assured the credibility of the results. This study provides an effective method for CF-glycosites identification and a valuable biomarker reference for clinical research.Biological significanceCF-glycosytion plays an important role in regulating biological processes in eukaryotes. Alterations of the glycosites and attached CF-glycans are frequently observed in various types of cancers. Thus, it is crucial to develop a strategy for mapping human CF-glycosylation. Here, we developed a complementary method via alternating HCD and ETD fragmentation (AHEF) to analyze CF-glycoproteins. This strategy reveals an excellent complementarity of HCD and ETD in the analysis of CF-glycoproteins, and provides a valuable biomarker reference for clinical research.
| S-EPMC5953178 | biostudies-literature
Project description:Comprehensive analysis of the ubiquitylome is a prerequisite to fully understand the regulatory role of ubiquitylation. However, the impact of key mass spectrometry parameters on ubiquitylome analyses has not been fully explored. In this study, we show that using electron transfer dissociation (ETD) fragmentation, either exclusively or as part of a decision tree method, leads to ca. 2-fold increase in ubiquitylation site identifications in K-ε-GG peptide-enriched samples over traditional collisional-induced dissociation (CID) or higher-energy collision dissociation (HCD) methods. Precursor ions were predominantly observed as 3+ charged species or higher and in a mass range 300-1200 m/z. N-ethylmaleimide was used as an alkylating agent to reduce false positive identifications resulting from overalkylation with halo-acetamides. These results demonstrate that the application of ETD fragmentation, in addition to narrowing the mass range and using N-ethylmaleimide yields more high-confidence ubiquitylation site identification than conventional CID and HCD analysis.
| S-EPMC4711353 | biostudies-literature
Project description:Protein posttranslational methylation and acetylations have been reported to occur in archaea, including members of the genus Sulfolobus, but have not been characterized on a proteome-wide scale. Sulfolobus chromatin proteins are known to be methylated and acetylated on lysine side chains, resembling eukaryotic histones in this respect. We utilized bottom-up and top-down proteomic approaches to perform a global and deep methylation study in the hyperthermoacidophylic archaeon S. islandicus LAL 14/1 with a particular focus on chromatin proteins. Without specific enrichment, 1931 methylation sites on 731 protein were found by bottom-up proteomic analysis. The dynamics of protein methylation has been investigated on 424-526 proteins throughout 3 cell culture growth stages. We also confirm the relaxed specificity and steady abundance of the previously described methyltransferase aKMT4 which is implicated in the massive proteome methylation. We also detected an abundant N-terminal acetylation of S. islandicus proteins, with more than one third of the detected N-terminal peptides being acetylated
2022-03-01 | PXD003102 | Pride
Project description:We developed TMMF strategy (Targeted MS strategy combined with Multi-Fragmentation) which provided plenty information of targeted glycopeptides based on complementary MS2 spectra from HCD, ETD and CID in a single MS run.
2021-03-12 | PXD024705 |
Project description:Full-length de novo sequencing of unknown proteins remains a challenging open problem. Traditional methods that sequence spectra individually are limited by short peptide length, incomplete peptide fragmentation, and ambiguous de novo interpretations. We address these issues by determining consensus sequences for assembled tandem mass (MS/MS) spectra from overlapping peptides (e.g., by using multiple enzymatic digests). We have combined electron-transfer dissociation (ETD) with collision-induced dissociation (CID) and higher-energy collision-induced dissociation (HCD) fragmentation methods to boost interpretation of long, highly charged peptides and take advantage of corroborating b/y/c/z ions in CID/HCD/ETD. Using these strategies, we show that triplet CID/HCD/ETD MS/MS spectra from overlapping peptides yield de novo sequences of average length 70 AA and as long as 200 AA at up to 99% sequencing accuracy.
| S-EPMC4591044 | biostudies-literature
Project description:Splenic B cell subsets (IgM+CD138+ ASC, FO B cells, MZ+B1 B cells) sorted from 8 month LDL-r KO mice with or without c-myb hypomorphism fed a 12 week HCD diet
2019-04-09 | GSE129450 | GEO
Project description:The vulnerability of bone marrow hematopoiesis to perturbations of cholesterol metabolism is well documented, while the underlying cellular and molecular mechanisms remain poorly understood. Here we reveal a distinct cholesterol metabolic signature of hematopoietic stem cells (HSCs) within the hematopoietic compartment.To identify the phenotype switching and function variation in BM LT-HSCs with HCD treatment, we performed RNA-seq of LT-HSCs from the bone marrow of mice with or without3-month HCD treatment.
2023-03-29 | GSE214035 | GEO
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