Calculations in Scaffold PTM

The following article contains a list of frequently asked question relating to calculations employed in Scaffold PTM. For specific questions not covered in our documentation we are available by telephone Monday through Friday from 8 AM to 5 PM PST. Our toll free number is 1-800-944-6027. Additionally support can be contacted via email at support@proteomesoftware.com.

Calculations in Scaffold PTM

How does Scaffold PTM quantify PTMs?
The PTM Spectrum Counts Tab offers semi-quantitative estimates of PTM abundances. It displays the label-free unweighted spectral count data that have a PTM at a specific site for each MS Sample or summarized for each Biosample or Category.

Scaffold Q+ and Scaffold Q+S calculate the reference value using quantitation options selected by the user before exporting SQML data. The PTM abundance is calculated by comparing the median log2 of the fold change measurements for each PTM site in every sample to the reference value. The reference value is the average (median or mean) peptide abundance measurement in the reference sample.

What is Ascore? How does it help me?
Ascore measures the probability of correct phosphorylation site localization based on the presence and intensity of site-determining ions in MS/MS spectra. The algorithm targets high-throughput phosphorylation analysis and site localization. Working in the Scaffold PTM environment, Ascore becomes an effective tool for automating large-scale, post-translational studies. 

Using Ascore, Scaffold PTM re-analyzes existing search engine results of previous searches done with Sequest, Mascot or other Scaffold compatible search engines. The new analysis attempts to best match the observed spectrum for PTM site. It graphically displays a new list of reported PTM sites and the evidence supporting the assignment of the PTM to the site. By comparing site determining ions, Scaffold PTM produces Ascore results assigning an ambiguity score to each reported PTM site.

The number of PTMs in each peptide can be determined by the precursor ion mass of the peptide’s spectra. Scaffold PTM adds this knowledge to the Ascore to derive a site location probability. Scaffold PTM then combines the site location probability estimates from all spectra matching peptides containing the site to obtain the best estimate of the probability that the PTM is at that site.

What is considered a "good" Ascore?
While this subjective measure will vary from data set to data set, an Ascore of >20 is generally a "good" Ascore.

How do I learn more about Ascore?
A probability-based approach for high-throughput protein phosphorylation analysis and site localization
Sean A Beausoleil, Judit Villén, Scott A Gerber, John Rush & Steven P Gygi. Nature Biotechnology 24, 1285 - 1292 (2006)

Phosphoproteome analysis of Drosophila melanogaster embryos.
Zhai B, Villén J, Beausoleil SA, Mintseris J, Gygi SP. J Proteome Res. 2008 Apr;7(4):1675-82.  

Phosphorylation Analysis of Primary Human T Lymphocytes Using Sequential IMAC and Titanium Oxide Enrichment
Carrascal M. et al. J Proteome Res 2008 Dec;7(12) 5167-76.

What does the Motif Score tell me?
The Motif Score is a comparison between the probability that a motif exists in the loaded data set and the probability that a specific sequence, regardless of modifications, exists in a background data set. For more information on Motif Scores and Motifs, see Schwartz, D and Gygi, SP. Nat Biotechnol. 2005 Nov;23(11):1391-8.

What is the difference between the Data set Percentage and the Background Percentage?
To measure the probability that a specific amino acid appears with a motif, Scaffold PTM uses a background, which can take the form of all proteins loaded into the program or an external FASTA database, to calculate a Background Percentage. This measurement is displayed in the Motif pane. To define the Motifs background Go to Edit>Preferences>Motif Background tab. Scaffold PTM also determines a Data set Percentage by calculating the probability that a modification occurs in a given motif throughout all identified peptides. This measurement is displayed in the Motif Pane.

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