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We have some tips and observations concerning the Mascot Server node in Proteome Discoverer.

This month's highlighted publication illustrates how peak intensities can give more accurate quantitation results than peak areas. If you have a recent publication that you would like us to consider for an upcoming Newsletter, please send us a PDF or a URL.

Mascot tip of the month describes the different types of neutral loss that can be associated with modifications.

Please have a read and feel free to contact us if you have any comments or questions.

Mascot Server workflows in Proteome Discoverer

For those of you that use Thermo's Proteome Discoverer (PD) as your primary user interface for database searching, we have a few tips and observations:

• Peak list size
  PD splits large peak lists into separate searches and transparently merges the results back together. If you want to view the search results using Mascot reports, and perform repeat searches, you can still do this by creating a combined result report, as described in our May 2017 newsletter.
   
• Missing functionality
  There are a few features in Mascot Server 2.6 that are not supported by the PD Mascot node, such as multiple precursor masses for chimeric spectra, error tolerant searches, and searching combinations of Fasta files and spectral libraries. To access these features for data processed through PD, repeat the search via the Mascot Server search log.
   
• Target Decoy PSM Validator
  For large search spaces, such as no enzyme searches, searches with many variable modifications, or very large databases, the Target Decoy PSM Validator can be wildly over conservative. For better sensitivity, use the Percolator node instead.

Go here to discover more tips and observations concerning the Mascot Server node in Proteome Discoverer.

Featured publication using Mascot

Here we highlight a recent interesting and important publication that employs Mascot for protein identification, quantitation, or characterization. If you would like one of your papers highlighted here please send us a PDF or a URL.

Sum of peak intensities outperforms peak area integration in iTRAQ protein expression measurement by LC-MS/MS using a TripleTOF 5600+ platform

Bastien Burat, Julien Gonzalez, Francois-Ludovic Sauvage, Hassan Aouad, Helene Arnion, Emilie Pinault, Pierre Marquet and Marie Essig

Bioscience Reports, 39(6) BSR20190904 (2019)

The authors investigated the quantitative accuracy of using peak areas versus peak intensities for 4-plex iTRAQ experiments of calcineurin inhibition on renal proximal tubular cells. They built an automated data processing algorithm called Customizable iTRAQ Ratio Calculator (CiR-C) to refine critical parameters related to peptide confidence and selection.

The data workflow encompassed Mascot Server for peptide and protein identification, jTRAQx software for computation of summed peak intensities at the peptide level, and the CiR-C algorithm for data integration and final protein quantitation. In comparison with the peak area workflow, the summed peak intensity approach had superior linear correlation with Western blot quantitation.

Mascot Tip

The neutral loss behaviour of a particular modification in Mascot is defined by its Unimod entry. As a rule, you should always choose 'scoring' for the type of neutral loss when creating or editing an entry in Unimod. Here are brief descriptions of all four types of neutral loss:

• Scoring: A neutral loss from the MS/MS fragments. The resultant fragments are considered for scoring, e.g. y-98 or b-98 for phosphopeptides. There can be up to 10 scoring neutral losses. During a search, Mascot iterates through the scoring neutral losses. The one that gives the highest score is chosen, and all the other neutral losses are treated as Satellite.
   
  The occurence of a particular neutral loss may depend on the ionisation method. Very gentle ionisation leaves the modification intact while higher energy fragmentation causes partial or complete loss of the modification. This is handled by including a neutral loss of zero (leave composition empty).
   
• Satellite: A neutral loss specified as satellite is never considered for scoring. If a Satellite neutral loss gives a match to a peak, that peak is removed from the list of noise peaks, which improves the score. None of the standard modifications in Unimod currently have satellite neutral losses.
   
• Peptide: A neutral loss from the intact peptide precursor. This peak is matched and so not treated as a noise peak for scoring purposes.
   
• Required Peptide: A required peptide neutral loss must be present in the spectrum. This carries some risk, because a perfectly good match could be rejected if this peak was missing.

About Matrix Science

Matrix Science is a provider of bioinformatics tools to proteomics researchers and scientists, enabling the rapid, confident identification and quantitation of proteins. Mascot software products fully support data from mass spectrometry instruments made by Agilent, Bruker, Sciex, Shimadzu, Thermo Scientific, and Waters.

Please contact us or one of our marketing partners for more information on how you can power your proteomics with Mascot.

Matrix Science Ltd, 64 Baker Street, London W1U 7GB, UK
T +44 (0)20 7486 1050  F +44 (0)20 7224 1344  E info@matrixscience.com