Dataset Information

Peptide gene sequences of substrate phage for different proteases

ABSTRACT: We present a method that employs two genetically encoded substrate phage display libraries coupled with next generation sequencing (SPD-NGS) that allows up to 10,000-fold deeper sequence coverage of the typical 6 to 8 residue protease cleavage sites compared to state-of-the-art synthetic peptide libraries or proteomics. We applied SPD-NGS to two classes of proteases, the intracellular caspases 2, 3, 6, 7 and 8, and the ectodomains of the membrane sheddases, ADAMs 10 and 17. The first library (Lib 10AA) was used to determine substrate cleavage motifs. Lib 10AA contains a highly diverse randomized 10-mer substrate peptide sequences (10^9 unique members) that was displayed mono-valently on filamentous phage and bound to magnetic beads via an N-terminal biotin. The protease was allowed to cleave the SPD beads, and the released phage subjected to up to three total rounds of positive selection followed by next generation sequencing (NGS). This allowed us to identify from 10^4 to 10^5 unique cleavage sites over a broad dynamic range of NGS counts (ranging from 3-5000), and produced consensus and optimal cleavage motifs based positional sequencing scoring matrices and state-of the-art machine learning algorithm that closely matched synthetic peptide data. A second SPD-NGS library (Lib hP) was constructed that allowed us to identify candidate human proteome sequences. Lib hP displayed virtually the entire human proteome tiled in contiguous 49AA sequences with 25AA overlaps (nearly 1 million members). After three rounds of positive selection we identified up to 10^4 natural linear cut sites depending on the protease and captured most of the examples previously identified by proteomics (ranging from 30 to 1000) and predicted 10 to 100-fold more.

ORGANISM(S): synthetic construct

PROVIDER: GSE154923 | GEO | 2020/07/23

REPOSITORIES: GEO

ACCESS DATA

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Project description:Granzymes are a family of structurally related serine proteases involved in immunity. To date four out of five human granzymes have been assigned orthologues in mice; however for granzyme H, no clear murine orthologue has been suggested and its role in cytotoxicity remains controversial. In this study, we demonstrate that granzyme H is an inefficient cytotoxin. Besides analyzing the substrate specificity profile of granzyme H by means of substrate phage display, we assessed human granzyme H and mouse granzyme C cleavage susceptibility on a proteome-wide level. The extended specificity profiles of granzyme C and granzyme H (i.e. beyond P4-P4') match those previously observed for granzyme B. We demonstrate conservation of these extended specificity profiles among various granzymes since granzyme B cleavage susceptibility of an otherwise granzyme H/C specific cleavage site can be conferred simply by altering the P1-residue to aspartate, the preferred P1-residue of granzyme B. Our results thus indicate a conserved, but hitherto generally underappreciated specificity-determining role of extended protease-substrate contacts in steering cleavage susceptibility. Bio-IT: The spectra were searched with Mascot Daemon 2.2. The following search parameters were used. Peptide mass tolerance was set at 0.2 Da and peptide fragment mass tolerance at 0.1 Da; with the ESI-QUAD-TOF as selected instrument for peptide fragmentation rules for the Q-TOF Premier data. Endoproteinase semiArg-C/P (i.e., no restriction towards arginine-proline cleavage) was set as enzyme allowing one missed cleavage. Variable modifications were pyroglutamate formation of N-terminal glutamine, pyrocarbamidomethyl formation of N-terminal alkylated cysteine, deamidation of asparagine, acetylation or tri-deuteroacetylation of the alpha-N-terminus. Fixed modifications were methionine oxidation (sulfoxide), carbamidomethyl for cysteine, tri-deuteroacetylation of lysine and for identifying heavy labelled peptides, [13C6] or [13C615N4]-arginine were additionally set as fixed modifications. The spectra and identifications were stored in ms_lims

Project description:Shotgun and positional proteomics study of a mouse embryonic stem cell line. We devised a proteogenomic approach constructing a custom protein sequence search space, built from both SwissProt and RIBO-seq derived translation products, applicable for LC-MSMS spectrum identification. To record the impact of using the constructed deep proteome database we performed two alternative MS-based proteomic strategies: (I) a regular shotgun proteomic and (II) an N-terminal COFRADIC approach. The obtained fragmentation spectra were searched against the custom database (combination of UniProtKB-SwissProt and RIBO-seq derived translation sequences) using three different search engines: OMSSA (version 2.1.9), X!Tandem (TORNADO, version 2010.01.01.04) and Mascot (version 2.3). The first two were run from the SearchGUI graphical user interface (version 1.10.4). A combination of X!Tandem and Mascot was used for the N-terminal COFRADIC analysis, a combination of all three search engines for the shotgun proteome analysis. Note that OMMSA cannot cope with the protease setting semi-ArgC/P needed to analyze N-terminal COFRADIC data.For the shotgun proteome data, trypsin was set as cleavage enzyme allowing for one missed cleavage, and singly to triply charged precursors or singly to quadruple charged precursors were taken into account respectively for the Mascot or X!Tandem/OMSSA search engines, and the precursor and fragment mass tolerance were set to respectively 10 ppm and 0.5 Da. Methionine oxidation to methionine-sulfoxide, pyroglutamate formation of N-terminal glutamine and acetylation (protein N-terminus) were set as variable modifications. For the N-terminal COFRADIC analysis the protease setting semi-ArgC/P (Arg-C specificity with arginine-proline cleavage allowed) was used. No missed cleavages were allowed and the precursor and fragment mass tolerance were also set to respectively 10 ppm and 0.5 Da. Carbamidomethylation of cysteine and methionine oxidation to methionine-sulfoxide and 13C3D2-acetylation of lysines were set as fixed modifications. Peptide N-terminal acetylation or 13C3D2-acetylation and pyroglutamate formation of N-terminal glutamine were set as variable modifications and instrument setting was put on ESI-TRAP. Protein and peptide identification in addition to data interpretation was done using the PeptideShaker algorithm (http://code.google.com/p/peptide-shaker, version 0.18.3), setting the false discovery rate to 1% at all levels (protein, peptide, and peptide to spectrum matching). Aforementioned tools and algorithms (SearchGui, X!Tandem, OMSSA, and PeptideShaker) are freely available as open source.

Dataset Information

Peptide gene sequences of substrate phage for different proteases

Dataset's files

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets