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Title: The problem with peptide presumption and low mascot scoring

item Cooper, Bret

Submitted to: Journal of Proteome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/10/2011
Publication Date: 1/13/2011
Citation: Cooper, B. 2011. The problem with peptide presumption and low mascot scoring. Journal of Proteome Research. 10:1432-1435.

Interpretive Summary: Mass spectrometry is used to identify proteins in biological samples. Given a mixture of peptides derived from the proteins, the mass spectrometer produces a spectrum which can be interpreted by software. The software assigns a score that says if the spectrum is a particular peptide versus some other peptide or some other substance like a sugar, fatty acid or oil. It is necessary to apply a scientific-based threshold to determine the lower end of acceptability for scores. However, some scientists are now accepting lower than normal scores. This study shows how the acceptance of low scores leads to questionable scientific data. This study also shows that the reasoning used to justify low scores is mathematically erroneous and has led to faulty judgments regarding protein identification. The purpose of this research is to encourage the development of standards that lead to better scientific data quality. These findings are important to scientists at universities, institutes, government agencies and companies who want to assign better confidence to the peptides and proteins they discover by mass spectrometry.

Technical Abstract: Mascot, a database-search algorithm, is used to deduce an amino acid sequence from a peptide tandem mass spectrum. The magnitude of the Ions score associated with each peptide mostly reflects the extent of b-y ion matching in a collision-induced dissociation spectrum. Recently, several studies report peptides identified with abnormally low Ions scores. While a majority of the spectra in these studies may be correctly assigned, the low scoring spectra could lack discernable b-y ion fragments that clearly delineate a peptide sequence. Instead, it appears that identification may be predicated primarily on judgmental parent ion mass accuracy and that justification to include such low-scoring peptides may be based on inaccurate false discovery rate modeling. It is likely that additional scientific experimentation is needed or appropriate methodologies adopted before substandard fragment ion matching can be considered proof of peptide identification.