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The Third Column: MS/MS fragment ion decharging in Mascot Server 2.7
In the Mascot Generic (mgf) peak list format, MS/MS fragment ions are typically defined as pairs of values. The first value is the m/z of the fragment ion peak and the second the intensity of the peak. However, a third value can also be supplied – the charge state of the fragment ion. In Mascot 2.6 or earlier, the fragment [...]
Protein FDR in Mascot Server 2.7
One of the new features in Mascot Server 2.7, now running on this web site, is an estimate of protein FDR. This is displayed in the Protein Family Summary for Fasta searches whenever automatic decoy is selected. The basis is the number of proteins inferred in the target database compared with the number in the decoy database. Conceptually, this is [...]
Disaster recovery
There are a number of potential issues that can cause your Mascot Server or other computer infrastructure to stop working. In the worst case, you may need to do a full recovery from backups. This can be an involved procedure, but it is straightforward as long as you have backed up key configuration and data files. For disk or storage [...]
Is your database search reproducible?
A lot has been written about the reproducibility of shotgun proteomics workflows, as well as controlling measurement variability between instruments and laboratories. An equally important factor is data analysis transparency and reproducibility, which is gaining increasing visibility. This boils down to two essentials: recording all software parameters used, and ensuring the software produces the same output given the same input [...]
Common myths about protein scores
Mascot Server is used in many different application areas by both mass spectrometry experts and non-experts. Over the years, we’ve spotted a few recurring misconceptions about how protein scores are interpreted and used. All the examples come from recent peer-reviewed papers. Protein scores in PMF searches The very first thing to check is, what type of experiment is being reported. [...]
The three Ds: Diethylation, Dimethylation and the Deuterium effect
Dimethylation Mascot Server and Mascot Distiller support a number of precursor intensity based quantitation techniques using a number of different techniques and chemistries. The software also allows you to flexibly define your own methods, allowing you to implement quantitation protocols which we have not supplied ‘out of the box’. One isotope labelling method which Distiller supports ‘out of the box’ [...]
Identify proteins by more than ‘gut’ feeling
Last month, we discussed benchmarking protein inference and the role of shared peptide matches. Excluding shared matches may be beneficial to protein identification accuracy if the sequence database contains perfect representations of all proteins in the sample. Many real-life data sets don’t meet this condition. Metaproteomics and environmental samples, such as the various human body sites, peat bog and ocean [...]
What are you inferring?
Benchmarking protein inference is notoriously difficult. Artificial samples of known content tend to be too simple while real samples lack ground truth. An interesting approach was adopted for the ABRF iPRG 2016 study, and has been the subject of a publication from The et al. A collection of human Protein Epitope Signature Tags (PrESTs) were expressed in E. coli and [...]
Back to basics 5: Peptide-spectrum match statistics
Mascot can identify peptides in uninterpreted MS/MS data. Observed spectra are submitted to Mascot as search queries. A query specifies the precursor ion m/z and charge state as well as the MS/MS peak list. Mascot digests protein sequences from the chosen database and selects peptide sequences whose mass is within the specified tolerance of the query’s precursor mass. The software [...]
Mascot workflows in Proteome Discoverer
For many users of Thermo instruments, Proteome Discoverer (PD) is their primary user interface for database searching, and Mascot is represented by a node in the workflow. This article collects together a few tips and observations concerning Proteome Discoverer 2.3 and Mascot Server 2.6. Proteome Discoverer Configuration Under Administration; Mascot Server, the setting Max. MGF File Size [MB] has a [...]
Back to basics 4: Mascot Daemon
Mascot Daemon is a client to Mascot Server that can automate the processing of raw data to peak lists and submit multiple searches to a central Mascot Server. It is included with the Mascot Server licence and can be installed on as many computers in the lab as you like. Processing raw data files will use CPU resources, so you [...]
X!Hunter MGF libraries
Mascot 2.6 supports three file formats for spectral libraries: the NIST MSP format, the SpectraST sptxt format and the X!Hunter MGF format. The sptxt format is a variation on MSP and will be covered in a future blog post. The main functional difference between MSP and MGF is in the level of annotation. The X!Hunter MGF format is minimalistic and [...]
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