Dataset Information

Simple, scalable and sensitive tip-based identification of protease substrates

ABSTRACT: Large-scale N-terminomics using COFRADIC and TAILS has revolutionized protease research, but is disseminated only in few labs. We present an alternative and simple protocol for N-terminal peptide enrichment, based on charge-based fractional diagonal chromatography (ChaFRADIC) and requiring only well-established protein chemistry and a pipette tip. We achieve unprecedented sensitivity quantifying >2000 unique N-terminal peptides from 10 µg per sample, allowing the identification of proteolytic targets and consensus motifs.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Laxmikanth Kollipara

LAB HEAD: Dr. René Peiman Zahedi

PROVIDER: PXD005954 | Pride | 2018-01-24

REPOSITORIES: Pride

ACCESS DATA

Dataset's files

Source:

Items per page:

1 - 5 of 21

Publications

Simple, scalable, and ultrasensitive tip-based identification of protease substrates.

Shema Gerta G Nguyen Minh T N MTN Solari Fiorella A FA Loroch Stefan S Venne A Saskia AS Kollipara Laxmikanth L Sickmann Albert A Verhelst Steven H L SHL Zahedi René P RP

Molecular & cellular proteomics : MCP 20180122 4

Proteases are in the center of many diseases, and consequently, proteases and their substrates are important drug targets as represented by an estimated 5-10% of all drugs under development. Mass spectrometry has been an indispensable tool for the discovery of novel protease substrates, particularly through the proteome-scale enrichment of so-called N-terminal peptides representing endogenous protein N termini. Methods such as combined fractional diagonal chromatography (COFRADIC)<sup>1</sup> an ...[more]

PMID: 29358340

Similar Datasets

Project description:We sought to determine the effect of TbPARN-1 overexpression on global mRNA steady-state levels in T. brucei. The mRNA expression profile of tet-induced TbPARN-1 OvEx procyclic cells was compared to the mRNA profile of control cells (expressing tet-induced TAP tag alone) by microarray analysis. Since overexpression of TbPARN-1 showed increased deadenylation activity in cytoplasmic extracts, overexpressing PARN-1 in vivo should result in more rapid deadenylation and decay of mRNAs targeted by PARN-1. Thus, mRNAs with lower steady state levels in PARN-1 OvEx cells can be considered likely targets for PARN-1-dependent deadenylation. Three different clones of the PARN-1 OvEx and Control cell lines were used for analysis. Each experiment was run in duplicate, with the dye-labeling reversed between duplicates. The T. brucei version 3 microarray chip (PFGRC) was used for this experiment. Each gene on the microarray was represented in duplicate, so for each experiment, mRNA levels were obtained from both points and averaged. As a control between arrays, we used a control probe set obtained from PFGRC. "The probe set consists of Cy3 and Cy5 end labeled 40-mer probes that are complementary to a set of 500 Arabidopsis thaliana 70-mer targets on PFGRC DNA microarrays. The UMSS is supplied as a ready to use mix of the Cy3 and Cy5 labeled oligonucleotides. A small aliquot is spiked into Cy-dye labeled nucleic acid samples just prior to hybridization on PFGRC microarrays. The UMSS produces a set of reference fluorescent signal intensities and ratios that are generated independent of the user-specific experimental samples. Since PFGRC intentionally targeted a heavy representation of Cy3 and Cy5 signals in the mid-range of detection, the Cy3 and Cy5 signal intensities and ratios generated by the UMSS should be highly reproducible. These signals serve as a reference point to judge the success of microarray experiments, and troubleshoot potential problems."

			Action	DRS
	QExactiveHF02_04436.raw	Raw
	QExactiveHF02_04437.raw	Raw
	QExactiveHF02_04438.raw	Raw
	QExactiveHF02_04439.raw	Raw
	QExactiveHF02_04440.raw	Raw