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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. [...]
Exporting spectral library search results
Mascot 2.6 integrates spectral library searching. Today we’ll describe how these searches can be exported. Please ensure you’ve installed the Mascot 2.6.1 patch, as support for exporting library search data was not complete in the initial Mascot 2.6.0 release. Library searches can be either library-only or integrated searches. Integrated means the search is against both a spectral library and a [...]
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 [...]
Ion series for EThcD
The ion series considered during a Mascot search are selected by choosing an instrument type. If you have Mascot Server in-house, you can edit existing instrument types and create new ones. There are 17 ion series available, and Mascot takes an iterative approach to scoring. If the number of matched peaks in a particular ion series is no better than [...]
Mascot Security: priority settings
An earlier article looked at privacy settings in Mascot security. This article looks at priority settings; the settings that allow one user group to run bigger or longer or more complex searches than another. Many aspects of searches can be limited by user group. The important ones are: SEARCH: Allow msms no enzyme searches SEARCH: Maximum number of concurrent searches [...]
Mascot Security: privacy settings
It is perfectly possible to share a single Mascot Server between individuals or groups who require privacy for their searches. Mascot Security provides the mechanism to limit access to result reports, databases, and even custom modifications. Mascot security is not designed to be highly secure – a determined hacker could probably find ways around it without too much difficulty – [...]
Exporting search results: tips and tricks
The Mascot Server export utility allows search results to be exported in a wide range of formats, together with the native result file and the MGF peak list. Exporting the result file can be useful if you need it for a third party application and don’t have file share access to the Mascot Server. Exporting the MGF might be useful [...]
How many of you are there in there? Processing and searching chimeric MS/MS spectra with Mascot Distiller and Mascot Server
In the typical shotgun proteomics experiment, the assumption is that each MS/MS spectrum is derived from a single precursor selected by the Mass Spectrometer for fragmentation. However, in practice, near isobaric precursors can co-elute and undergo co-fragmentation resulting in chimeric MS/MS spectra containing fragments from multiple different precursor peptides. With high resolution data, it is possible for these overlapping isotope [...]
Protein inference for spectral library searches
The major new feature of Mascot Server 2.6, now running on this web site, is that searches of spectral libraries have been fully integrated with ‘conventional’ Mascot searches of Fasta files. The search engine for spectral library searches is MSPepSearch from Steve Stein and colleagues at NIST. We didn’t have any revolutionary ideas for improving spectral library scoring so, rather [...]
Benchmarking your Mascot Server
In an earlier blog post we talked about choosing the right hardware for a Mascot Server and mentioned that the major performance bottle neck is the CPU performance. We also looked at the PassMark PerformanceTest CPUBenchmark as a way of predicting the performance of a Mascot Server. In this article we will look at how to measure Mascot Server performance [...]
Retention time
Although retention time is not part of the Mascot scoring algorithm, it can be used by Percolator to improve the re-scoring of search results and it is important or essential information for many types of quantitation. This article examines how retention time is represented in the peak list, the search results, and various export formats. Mascot Server works off a [...]
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