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Articles tagged: error tolerant
Error tolerant searches now show statistical significance
The latest release of Mascot Server introduces some important changes to error tolerant searches. Matches from the second pass search now have expect values attached, indicating confidence levels. These are either estimates based on counting trials or empirical values derived from searching a decoy database. If you are not familiar with the error tolerant search, now is the time to [...]
Variable Modifications in Mascot 2.7
Most protein samples will exhibit some degree of modification which needs to be considered when carrying out a database search In this article we’ll take a look at some important changes we introduced in Mascot 2.7 in how Mascot handles variable modifications. Variable modification permutation in Mascot 2.6 and earlier In Mascot 2.6 or earlier, variable modification permutation is handled [...]
The plus one dilemma
There are several common modifications that can add approximately 1 Da to a peptide mass. Even if you have high accuracy data, it can sometimes be difficult to figure out which one is correct. Delta Lys->Glu substitution 0.947630 Leu->Asn or Ile->Asn substitution 0.958863 Deamidation at N or Asn->Asp substitution 0.984016 Deamidation at Q or Gln->Glu substitution 0.984016 Citrullination at [...]
Back to Basics: Optimize your search parameters
Every now and then you need to determine good search parameters for a data set. They may be different from the normal ones you use due to a change in instrumentation, you may be analyzing data from a public resource like PRIDE/Proteome exchange or it could be data from a collaborator. Whatever the reason, here’s a quick overview on how [...]
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 [...]
Step away from the iodoacetamide
In our July newsletter, we featured a paper from Torsten Müller and Dominic Winter, University of Bonn, concerning alkylation artefacts. Some of their findings were quite shocking. For example, differences of more than 9 fold in numbers of identified methionine-containing peptides for in-gel digested samples between iodine- and non-iodine-containing alkylation reagents. This is important because a glance at the literature [...]
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. [...]
The most analysed protein is …
Trypsin, of course. The Journal of Proteome Research has a paper from the Medical University of Graz concerning the importance of correctly identifying spectra from contaminant proteins. In particular, trypsin autolysis peptides. The authors point out that sequencing grade trypsin is modified by methylation or acetylation of the lysines, to inhibit autolysis. Unless these variable modifications are selected in a [...]
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 [...]
Shooting fish in a barrel
We sometimes get asked about searching a sequence database with just one entry, or maybe a small database where the entries are variants of the same protein. Details tend to be sketchy or confidential, so we assume this is usually QA of a recombinant protein, rather than protein identification in the conventional sense. Maybe Mascot Server is just a convenient [...]