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Posted by John Cottrell (June 18, 2016)

High FDRs for methylated peptides III

The MCP paper "Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates" raises some important questions concerning the accuracy and interpretation of database search results. In this third article, we look at the difference between using counts of matches (PSMs) and counts of distinct sequences to calculate the false discovery rate (FDR). filestarget [...]

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Posted by John Cottrell (May 18, 2016)

High FDRs for methylated peptides II

In a previous article, we discussed how the false discovery rate (FDR) for modified peptides would be higher than the global FDR for all PSMs if the proportion of modified peptides in the search space for false matches was higher than for true matches. This is only one factor in the very high FDRs for methylated peptides reported in the [...]

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Posted by John Cottrell (April 21, 2016)

High FDRs for methylated peptides

"Large Scale Mass Spectrometry-based Identifications of Enzyme-mediated Protein Methylation Are Subject to High False Discovery Rates" in the March edition of Molecular & Cellular Proteomics represents a very substantial and systematic piece of work from the University of New South Wales. Three types of sample preparation (coomassie gel, unstained gel, HILIC) were combined with three different ionisation methods (CID, ETD, [...]

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Posted by John Cottrell (July 15, 2015)

Search only for peptides we care about?

The July issue of Nature Methods contains a commentary from William Noble with the slightly provocative title "Mass spectrometrists should search only for peptides they care about". At first sight, this seems to contradict our general advice to include contaminant sequences when searching a single organism database or when using a taxonomy filter. One of Noble’s examples is a study [...]

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Posted by John Cottrell (January 19, 2015)

Creating a list of confidently identified proteins

This can be done very easily using Report Builder: Select the Decoy checkbox when submitting the search Open the result report as a Protein Family Summary Switch to the Report Builder tab Expand the decoy search section and set the peptide FDR to 1% Expand the filters section and set ‘Num of significant unique sequences’ > 1 Optionally, expand the [...]

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Posted by John Cottrell (November 22, 2013)

Does protein FDR have any meaning?

Its easy to grasp the concept of using a target/decoy search to estimate peptide false discovery rate. You search against a decoy database where there are no true matches available, so the number of observed matches provides a good estimate of the number of false matches in the results from the target. People debate implementation details, such as whether the [...]

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Posted by John Cottrell (December 1, 2012)

High mass accuracy: precursors

High performance instruments can deliver low ppm accuracy for both precursors and fragments. How does this impact database search? In this post, the first of two, we’ll look at the implications of very accurate precursor m/z. High mass accuracy and high resolution have transformed mass spectrometry over the last few years. Apart from increasing file sizes, and maybe the time [...]

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