Linked Life Data

16335976

Source:http://linkedlifedata.com/resource/pubmed/id/16335976

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
6
pubmed:dateCreated
2005-12-12
pubmed:abstractText
A very popular approach in proteomics is the so-called "shotgun LC-MS/MS" strategy. In its mostly used form, a total protein digest is separated by ion exchange fractionation in the first dimension followed by off- or on-line RP LC-MS/MS. We replaced the first dimension by isoelectric focusing in the liquid phase using the Off-Gel device producing 15 fractions. As peptides are separated by their isoelectric point in the first dimension and hydrophobicity in the second, those experimentally derived parameters (pI and R(T)) can be used for the validation of potentially identified peptides. We applied this strategy to a cellular extract of Drosophila Kc167 cells and identified peptides with two different database search engines, namely PHENYX and SEQUEST, with PeptideProphet validation of the SEQUEST results. PHENYX returned 7582 potential peptide identifications and SEQUEST 7629. The SEQUEST results were reduced to 2006 identifications by validation with PeptideProphet. Validation of the PeptideProphet, SEQUEST and PHENYX results by pI and R(T) parameters confirmed 1837 PeptideProphet identifications while in the remainder of the SEQUEST results another 1130 peptides were found to be likely hits. The validation on PHENYX resulted in the fixation of a solid p-value threshold of <1 x 10(-04) that sets by itself the correct identification confidence to >95%, and a final count of 2034 highly confident peptide identifications was achieved after pI and R(T) validation. Although the PeptideProphet and PHENYX datasets have a very high confidence the overlap of common identifications was only at 79.4%, to be explained by the fact that data interpretation was done searching different protein databases with two search engines of different algorithms. The approach used in this study allowed for an automated and improved data validation process for shotgun proteomics projects producing MS/MS peptide identification results of very high confidence.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:issn
1535-3893
pubmed:author
pubmed:issnType
Print
pubmed:volume
4
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2273-82
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:articleTitle
Added value for tandem mass spectrometry shotgun proteomics data validation through isoelectric focusing of peptides.
pubmed:affiliation
DiagnoSwiss SA, Monthey, Switzerland.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't