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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 [...]
O-fucosylated CID spectra
O-linked fucose is easily lost in CID. A recent paper by Swearingen et al. in the Journal of Proteome Research discusses this in the context of identifying O-fucosylated thrombospondin type 1 repeats (TSRs) in Plasmodium parasites using database searching. The main problem is, the O-glycosidic bond is weaker than the peptide backbone. Collision energies typical for peptide fragmentation cause it [...]
Don’t wait: use spectral libraries now
The Human Proteome Project 2018 special issue in the Journal of Proteome Research contains a report from the 2017 Dagstuhl Seminar on Computational Proteomics. The paper by Deutsch et al. is titled Expanding the Use of Spectral Libraries in Proteomics, and the authors identify several challenges that slow down spectral library adoption. I’d like to address their main points. Adoption [...]
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 3: Quantitation statistics
Mascot Server and Distiller support a number of different quantitation methods. These methods are carried out at the peptide level, the peptides are then grouped into protein families, and the peptide quantitation results used to calculate protein ratio values. Mascot and Distiller perform a number of statistical procedures and tests to give you an indication of the quality and reliability [...]
Mascot Server cluster mode
Most modern Intel processors have at least 4 cores and some models have 12 cores or more. Mascot Server is licenced by the CPU, where each CPU corresponds to 4 physical cores, so a single PC is perfectly sufficient for licences of 1 or 2 CPU. If you have a larger licence, there comes a point where it is not [...]
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