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Cysteine capping with CAM reagent

ABSTRACT: CAM reagent was synthesized and used to uniquely convert cysteine residues, under mild conditions, into trypsin-recognsable sites. We apply this cysteine-capping methodology in the identification of missense variants in HCT-15 cell lines.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Colon

SUBMITTER: Samuel Ofori

LAB HEAD: Keriann Marie Backus

PROVIDER: PXD047844 | Pride | 2025-03-13

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	2023-04-10-KB-noFAIMS-SO-1A_HighpH_II_f1.pepXML	Pepxml
	2023-04-10-KB-noFAIMS-SO-1A_HighpH_II_f1.raw	Raw
	2023-04-10-KB-noFAIMS-SO-1A_HighpH_II_f2.pepXML	Pepxml
	2023-04-10-KB-noFAIMS-SO-1A_HighpH_II_f2.raw	Raw
	2023-04-10-KB-noFAIMS-SO-1A_HighpH_II_f3.pepXML	Pepxml

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Publications

Generating cysteine-trypsin cleavage sites with 2-chloroacetamidine capping.

Ofori Samuel S Desai Heta S HS Shikwana Flowreen F Boatner Lisa M LM Dominguez Iii Emil R ER Castellón José O JO Backus Keriann M KM

Chemical communications (Cambridge, England) 20240815 67

An electrophilic arginine mimetic, 2-chloroacetamidine (CAM), was deployed to enable trypsin-mediated proteolysis at cysteine residues and to enhance mass spectrometry-based proteomic detection of cysteine-containing peptides. Illustrating the value of the CAM-capping strategy, proteogenomic analysis using a two-stage false discovery rate (FDR) search revealed >50% enhanced coverage of missense variants, when compared to established workflows. ...[more]

PMID: 39081146

Similar Datasets

Cysteine capping with CAM reagent

Project description:CAM reagent was synthesized and used to uniquely convert cysteine residues, under mild conditions, into trypsin-recognsable sites. We apply this cysteine-capping methodology in the identification of missense variants in HCT-15 cell lines.2-bromoethylamine (BEA)was used as controls, for cysteine capping and subjected to similar trypsin treatment.

2025-03-13 | PXD052935 | Pride

Identification of re-capping substrates for the cytoplasmic capping enzyme complex

Project description:The analysis of capped RNAs by massively parallel sequencing has identified a large number of previously unknown transcripts, some of which are small RNAs and others are 5M-bM-^@M-^Y truncated forms of RefSeq genes. The latter may be generated by endonuclease cleavage or by stalling of Xrn1 at defined sites. With the exception of promoter-proximal transcripts the caps on all of these are added post-transcriptionally by a cytoplasmic capping enzyme complex that includes capping enzyme and a kinase that converts 5M-bM-^@M-^Y-monophosphate ends to a diphosphate capping substrate. We previously described a modified form of capping enzyme with dominant negative activity against cytoplasmic capping (DN-cCE). A tet-inducible form of this was used to identify substrates for cytoplasmic capping by treating cytoplasmic RNA from control and induced cells with and without Xrn1. Surviving RNA was analyzed on Affymetrix Human Exon 1.0 arrays and scored for changes in probe intensity as a function of its position on each RefSeq gene to derive a factor (alpha) that could be compared between sets. Notably, transcriptome-wide changes were not evident unless RNA was treated with Xrn1. This analysis identified 2,666 uncapped mRNAs in uninduced cells, 672 mRNAs that appeared in the uncapped pool in cells expressing DN-cCE, and 835 mRNAs that were in both populations. Changes in cap status of 10 re-capping targets and 5 controls were assessed by 3 independent measures; susceptibility to Xrn1, recovery with a biotin-tagged DNA primer after ligating a complementary RNA oligonucleotide to uncapped 5M-bM-^@M-^Y ends, and binding or exclusion from a high affinity cap-binding matrix comprised of immobilized eIF4E and the corresponding binding domain of eIF4G. 3 biological replicates of 4 different samples comparing XRN1 treatment/non-treatment and Dox induction/non-induction of K294A

2012-12-20 | E-GEOD-36729 | biostudies-arrayexpress

Identification of re-capping substrates for the cytoplasmic capping enzyme complex

Project description:The analysis of capped RNAs by massively parallel sequencing has identified a large number of previously unknown transcripts, some of which are small RNAs and others are 5’ truncated forms of RefSeq genes. The latter may be generated by endonuclease cleavage or by stalling of Xrn1 at defined sites. With the exception of promoter-proximal transcripts the caps on all of these are added post-transcriptionally by a cytoplasmic capping enzyme complex that includes capping enzyme and a kinase that converts 5’-monophosphate ends to a diphosphate capping substrate. We previously described a modified form of capping enzyme with dominant negative activity against cytoplasmic capping (DN-cCE). A tet-inducible form of this was used to identify substrates for cytoplasmic capping by treating cytoplasmic RNA from control and induced cells with and without Xrn1. Surviving RNA was analyzed on Affymetrix Human Exon 1.0 arrays and scored for changes in probe intensity as a function of its position on each RefSeq gene to derive a factor (alpha) that could be compared between sets. Notably, transcriptome-wide changes were not evident unless RNA was treated with Xrn1. This analysis identified 2,666 uncapped mRNAs in uninduced cells, 672 mRNAs that appeared in the uncapped pool in cells expressing DN-cCE, and 835 mRNAs that were in both populations. Changes in cap status of 10 re-capping targets and 5 controls were assessed by 3 independent measures; susceptibility to Xrn1, recovery with a biotin-tagged DNA primer after ligating a complementary RNA oligonucleotide to uncapped 5’ ends, and binding or exclusion from a high affinity cap-binding matrix comprised of immobilized eIF4E and the corresponding binding domain of eIF4G.

2012-12-20 | GSE36729 | GEO

Transcriptional profile of bovine chorioallantoic membrane explants in response to wild type, M-NM-^TvirB2 or M-NM-^TbtpB Brucella abortus

Project description:In this study the transcription profile of bovine trophoblastic cells infected with Brucella samples was evaluated. Chorioallantoic membrane (CAM) explants inoculated with wild type (strain 2308), and to compare with the transcription profile of trophoblastic cells infected with mutant strains lacking virB2 or btpB.M-NM-^TvirB2 or M-NM-^TbtpB by microarray analysis at 4 hours post infection. Genes with significant variation in levels of transcripts (fold change > 2 and P < 0.05) were functionally classified, and transcripts related to defense and inflammation were assessed by quantitative real time RT-PCR. Chorioallantoic membrane explant culture (CAM) were obtained from 7 intact pregnant bovine uteruses at the final third of gestation. Three-condition experiment, wild type B. abortus 2308, DvirB2 or DbtpB in 4 control replicates and Microarray analysis.

2014-09-04 | E-GEOD-58216 | biostudies-arrayexpress

U87 cells implanted on a chick CAM (Chorioallantoic Membrane)

Project description:Glioblastoma multiforme (GBM) brain tumours have one of the shortest mean survival times (<1 year). In vivo models of GBM in mice and rats have been developed to study aspects of glioma that cannot be observed in cell culture such as angiogenesis, invasion and metastasis. Gliomas can be induced by implantation of rodent glioma cell lines into the brain or flank of nude mice. The disadvantages of rodent models however include variable growth rate and poor penetrance, which leads to difficulties in collecting clearly graded samples (pre-vascular/vascular). Bikfalvi et al have previously established a human GBM model that addresses these issues based on the chicken egg chorio-allantoic membrane (CAM), a highly vascularised extra-embryonic tissue. We have used DNA microarrays and a CAM model of GBM to study gene expression during the recruitment and development of the tumour vasculature. Over a 5 day period samples were taken every 12 hours from the tumour implantation site consisting of tumour cells and stroma cells, and also from a site distant from the implantation site consisting of just CAM cells. This study will shed light on the dynamic transcriptional signature of pathological angiogenesis. On day 10 of embryonic development 3-5 million U87 cells were deposited onto the surface of the CAM after gentle laceration. The cells were contained within a plastic ring and each tumour was size matched based on its volume. Tumour/stroma and distant CAM samples were cut out at 12 hour intervals post implantation for 5 days, equalling 10 time points. Each time point consisted of three replicates. U87 cells in culture (pre-implantation) were also included in the study in triplicate.

2013-12-02 | E-GEOD-43674 | biostudies-arrayexpress

Mouse Stereocilia Capping Protein Immunoaffinity Purification

Project description:Capping protein controls stereocilia length and width during hair bundle development. To determine what other proteins are involved in capping protein regulation, we carried out immunoaffinity purifications targeted at either CAPZA or CAPZB2. The starting material for immunopurification was crude stereocilia membranes isolated from mouse inner ear.

2017-08-25 | PXD006077 | Pride

Genome-wide study of the MSL1 effect on gene expression and mRNA capping efficiency

Project description:Measurement of capping efficiency (by 5'CAP and 5'noCAP sequencing) in male (S2) and female (Kc) Drosophila melanogaster cells upon depletion of MSL1 by dsRNA compared to the eGFP RNAi control. The measurement of capping efficiency was combined with gene expression measurement by strand specific RNA-Seq in female (Kc) Drosophila melanogaster cells

2023-09-30 | E-MTAB-2235 | biostudies-arrayexpress

The eukaryotic translation initiation factor eIF4E elevates steady-state m7G capping of coding and noncoding transcripts

Project description:Methyl-7-guanosine (m7G) “capping” of coding and some noncoding RNAs is critical for their maturation and subsequent activity. Here, we discovered that eukaryotic translation initiation factor 4E(eIF4E), itself a cap-binding protein, drives the expression of the capping machinery and the increased capping efficiency of ∼100 coding and noncoding RNAs. This dataset collects transcriptomic data for quantitative cap immunoprecipitation (CapIP) assay in eIF4E-Flag or vector stable U2Os cells.

2020-09-30 | GSE158728 | GEO

Hepacivirus RNA capping study

Project description:Analysis of non-canonical RNA capping in the Hepacivirus genus

| PRJEB89091 | ENA

Chorioallantoic membrane (CAM) and embryonic blood serum proteomics

Project description:In chickens, embryonic development begins upon egg formation and lasts for 21 days of incubation until hatching. The CAM is an extraembryonic membrane that serves a critical role in acid-base balance, gaseous exchange, calcium solubilization, and antimicrobial protection. Comparative proteomic analyses of CAM at two developmental stages (ED12 and ED19), in comparison to the proteome of embryonic blood serum, revealed protein groups that are relatively or highly specific to the CAM. The specific CAM functions include gaseous exchange, Ca2+ transport, vasculature development, and protection against pathogen invasion. Overall, our results highlight the structure-function relationship of the CAM protein constituents that potentially could expand its biomedical applications.

2022-08-12 | PXD027129 | Pride

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