Dataset Information

Cleavable Hydrophobic Derivatization Strategy for Enrichment and Identification of Phosphorylated Lysine Peptides

ABSTRACT: Herein, a cleavable hydrophobic derivatization (CHD) strategy was established for the enrichment and identification of pLys peptides. By CHD, 2,5-dioxopyrrolidin-1-yl-3-(decyldisulfanyl)propanoate (Dec-disulf-NHS) was synthesized to react with lysine dephosphorylated peptides, and then the derived peptides were captured by a C18 column, followed by the cleavage of hydrophobic chain to leave the specific label on the target peptides.

ORGANISM(S): Escherichia Coli

SUBMITTER: Lihua Zhang

PROVIDER: PXD012682 | iProX | Tue Feb 12 00:00:00 GMT 2019

REPOSITORIES: iProX

ACCESS DATA

Publications

Cleavable hydrophobic derivatization strategy for enrichment and identification of phosphorylated lysine peptides.

Hu Yechen Y Li Yang Y Gao Hang H Jiang Bo B Zhang Xiaodan X Li Xiao X Wu Qiong Q Liang Zhen Z Zhang Lihua L Zhang Yukui Y

Analytical and bioanalytical chemistry 20190416 18

Because of structural flexibility and acid lability, the identification of phosphorylated lysine (pLys) peptides is a great challenge. We report here a cleavable hydrophobic derivatization (CHD) strategy for the enrichment and identification of pLys peptides. First, 2,5-dioxopyrrolidin-1-yl-3-(decyldisulfanyl)propanoate was synthesized to react with dephosphorylated lysine peptides, and then the derived peptides were captured by a C<sub>18</sub> column, followed by cleavage of the hydrophobic ch ...[more]

PMID: 30993368

Similar Datasets

Project description:Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 - TEV cleavage site - two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex. Identification of targets for individual exosome subunits in wild-type and mutant yeast cells.

Dataset Information

Cleavable Hydrophobic Derivatization Strategy for Enrichment and Identification of Phosphorylated Lysine Peptides

Publications

Cleavable hydrophobic derivatization strategy for enrichment and identification of phosphorylated lysine peptides.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets