Scaffold extended output

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Contents 1 Scaffold - Phenyx integration 1.1 Introduction to the issue 1.2 The solution in Phenyx 1.3 What does it do, what does it change? 1.4 How to retrieve the scaffold-pidres.xml file

Scaffold - Phenyx integration

Introduction to the issue

• Scaffold is using a peptide prophet approach to validate an identification job. To do this, it assumes that the result contains two populations of peptide hits: a population of correct peptide hits and a population of incorrect peptide hits. It therefore models a bimodal distribution to define its probabilities. This model has been created to optimally work with SEQUEST on ion-trap data, which provides a result for each spectrum submitted to the search engine. In general, the number of "false hits" is larger than the number of "true hits". As Phenyx tends to report by default only peptide matches above a given confidence level, the population of "false hits" is too small to be modeled by Scaffold or peptide prophet. The solution to this is to ask Phenyx to generate a result (a peptidematch) for all spectra submitted, even if the scores are below the acceptance criteria. The result can then be exported to a file that Scaffold can read.

The solution in Phenyx

• A check box named 'Scaffold Extended Output' in the Submission page allows to prepare a Phenyx job ready to be imported into Scaffold

• It is not necessary to modify your submission parameters or profiles, just check this box, launch a job and collect the dedicated export file
• Once imported into Scaffold (2.0), Phenyx results will be read and statistically analyzed

What does it do, what does it change?

• At least one peptide match per spectrum (and possibly per calculation node used, depending on the configuration) will be reported, even if hits are below the selected score thresholds

• Most of these additional peptide matches are protein-orphan because of too low z-score value (it means that those peptide matches are not associated to a protein)

• A 'pidres-scaffold.xml' file is automatically created in addition to the regular files in the job directory

• The Proteins Overview page will display validated proteins and associated peptides as usual

• The Size of the Compounds Overview page will considerably increase due to report of all identified peptides independently of the z-score value (default "non scaffold extended report" setting: only peptides that reach the z-score threshold are reported)

• caution: the display of the Compounds Overview page may cause browser crash

• caution: if you resubmit the job, you have to reselect the box in the submission page

How to retrieve the scaffold-pidres.xml file

1. select the job in the Phenyx Desktop
2. select 'export text' from the left-hand list of links
3. in the appearing Console Management page, select 'browse'
4. click and download the file named 'scaffold-pidres.xml'

You are ready to import into Scaffold and to allow Prophet to work correctly on Phenyx results.