Blog

Articles tagged: error tolerant

Return to blog archive

Posted by John Cottrell (November 15, 2018)

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 [...]

Full article and comments form

Posted by Richard Jacob (July 16, 2018)

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 [...]

Full article and comments form

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 [...]

Full article and comments form

Posted by John Cottrell (October 16, 2017)

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 [...]

Full article and comments form

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. [...]

Full article and comments form

Posted by John Cottrell (July 12, 2017)

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 [...]

Full article and comments form

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 [...]

Full article and comments form

Posted by John Cottrell (November 14, 2015)

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 [...]

Full article and comments form

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 [...]

Full article and comments form

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 [...]

Full article and comments form