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Posted by John Cottrell (January 12, 2018)

Results round-up for the ‘dark matter’ challenge

In June, we tried to harness the power of crowd-sourcing to explain some of the unidentified modifications found in open database searches. We selected 20 abundant and unassigned mass deltas from Supplementary Table 3 of the recent MSFragger paper from Alexey Nesvizhskii’s group at U. Michigan and offered prizes for the first credible explanations. There were 35 unannotated deltas in [...]

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Posted by John Cottrell (September 12, 2017)

How to create a spectral library for contaminants

An earlier article highlighted how modified and non-specific peptides from contaminants can be matched using a spectral library without increasing the search space for the target proteins. This is particularly useful for sequencing grade trypsin, which is modified by methylation or acetylation of the lysines, creating a large number of modified non-specific peptides that are missed by typical search strategies. [...]

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Posted by John Cottrell (June 26, 2017)

Trying to illuminate proteomics ‘dark matter’

The May 2017 issue of Nature Methods has a paper from Alexey Nesvizhskii’s group at U. Michigan describing a new open database search program called MSFragger. Strikingly, they also observed the two highly abundant but unidentified mass deltas reported in Steven Gygi’s 2015 mass tolerant paper: 301.9864 and 249.9803. The challenges of open searching were discussed in an earlier blog [...]

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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 (February 16, 2016)

Selenocysteine

David Fenyö and Ron Beavis have a short paper in J. Proteome Research that draws attention to a potential problem with peptides containing selenocysteine (1-letter code U, 3-letter code Sec). Samples are frequently alkylated, yet modified U is unlikely to be considered in the search. This need not be an issue for Mascot searches, but you may have no modifications [...]

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Posted by John Cottrell (September 22, 2015)

Mass-tolerant vs Error tolerant

"A mass-tolerant database search identifies a large proportion of unassigned spectra in shotgun proteomics as modified peptides" in Nature Biotechnology is from Steven Gygi’s lab at Harvard Medical School. It describes the use of a very wide precursor mass tolerance, +/- 500 Da, to identify modified peptides in a Sequest search. How does this approach, which the authors call an [...]

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Posted by John Cottrell (October 14, 2013)

Modifications round-up, part 2

This is the second of two articles dealing with topics relating to modifications. The first can be found here. Note that Site analysis was covered in an earlier article. Why aren’t amino acid substitutions listed in the search form? Amino acid substitutions are rare and there are lots of them, so the only practical way to use them is in [...]

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Posted by John Cottrell (September 19, 2013)

Modifications round-up, part 1

Much of the complexity in Mascot is associated with modifications. It can be hard to find information about some of aspects of handling modifications unless you already know what you are looking for. In this blog article, the first of two, I’ll collect together some of the topics that come up frequently in support emails. Note that Site analysis was [...]

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