Dataset Information

Enhanced Peptide Identification by Electron Transfer Dissociation Using an Improved Mascot Percolator Algorithm

ABSTRACT: E.Coli Full Tryptic Digest CID Triplicate Analysis

INSTRUMENT(S): LTQ Orbitrap, instrument model

ORGANISM(S): Homo Sapiens (human) Escherichia Coli Escherichia Coli (strain K12)

SUBMITTER: James Wright

PROVIDER: PRD000544 | Pride | 2013-06-27

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	PRIDE_Exp_Complete_Ac_18989.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_18989.pride.mztab.gz	Mztab
	PRIDE_Exp_Complete_Ac_18989.xml.gz	Xml
	PRIDE_Exp_Complete_Ac_18990.pride.mgf.gz	Mgf
	PRIDE_Exp_Complete_Ac_18990.pride.mztab.gz	Mztab

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Publications

Enhanced peptide identification by electron transfer dissociation using an improved Mascot Percolator.

Wright James C JC Collins Mark O MO Yu Lu L Käll Lukas L Brosch Markus M Choudhary Jyoti S JS

Molecular & cellular proteomics : MCP 20120406 8

Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifica ...[more]

PMID: 22493177

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Project description:Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation. But, once the protein is already oxidized at least once, the spatial distribution of any consecutive oxidation no longer truly reflects initial protein solvent accessibility and residue reactivity, because the site preference of further oxidation may also be influenced by the changes caused by the prior oxidation. Thus, it would be interesting to obtain an assessment of the protein structure based on the FPOP data, by analyzing only singly oxidized protein populations. This requires utilization of more specific top-down mass spectrometry approaches. The key element of any top-down experiment is selection of a suitable method of ion isolation, excitation and fragmentation. Here we employ and compare collision-induced dissociation (CID), electron-transfer dissociation (ETD), and electron-capture dissociation (ECD) combined with multi continuous accumulation of selected ions (CASI). Singly oxidized subpopulation of FPOP labeled ubiquitin was used to optimize the method. The usefulness was then demonstrated further by using it to visualize structural changes induced by cofactor removal from holo/apo myoglobin system. The top-down data were compared with the literature and with the bottom-up data set obtained on the same samples. The top-down results were found to be in a good agreement, which indicates that monitoring singly FPOP oxidized ion population by top-down is a functional workflow for oxidative protein footprinting.