Scaffold is compatible with numerous search engines; however, they must be configured properly for Scaffold to read results. This document provides information on the configuration of Mascot to allow for the loading of quantitative data into Scaffold. SILAC and precursor intensity results must be processed using Mascot Distiller, a separate program that interfaces with the Mascot server, in order to be compatible with Scaffold Q+S. For data set specific inquiries or errors Proteome Software support is available from 8 AM to 5 PM PST Monday through Friday and can be contacted via firstname.lastname@example.org or by phone at 1-800-944-6027.
Please see the Scaffold File Compatibility Matrix for the most up to date information on search engines currently supported in Scaffold.
Viewing quantitative results requires the proper Scaffold quantitative module. Precursor intensity values can be viewed in Scaffold, Q+ and Q+S; SILAC data requires Scaffold Q+S. While Scaffold can view precursor intensities, the Q+ modules provide much more complex normalization and statistical analysis.
- Open Mascot Distiller and select the File menu. Here you have two options: New Project for processing a single RAW file or New Multi-File Project for processing multiple RAW files.
- Select the manufacturer of the instrument you have and select your RAW files
- Mascot Distiller starts the process by loading the file and displaying the Total Ion Current (TIC)
- To search the RAW data using your in house Mascot server select Processing > Process and Search... This will bring up the Mascot search dialog.
- Define the parameters that Mascot should use in the MS/MS search. These include the FASTA database, modifications, digestion enzyme and search tolerances. Note that SILAC modifications do not need to be selected as variable or fixed.
- Next the quantitation type must be selected. For precursor intensity choose Average [MD] from the dropdown. There are multiple options for quantitation via SILAC, make sure to choose the one that corresponds to the labels used in your experiment.
Figure 1. Average [MD] selected for precursor intensity
Figure 2. An example of SILAC quantitation
- You do not need to select a data file when running Mascot through Distiller
- Click start to return to Distiller, Mascot will process the data in the background.
- Once the search is finished the quantitation must be done. This can be done using the Analysis > Quantitate menu. Make sure to select the All Families radio button.
- Once the quantitative values are calculated save the quantitative XML report using the Analysis > Quantitation Reports... menu. Select the Save Complete XML option.
- Save the project using File > Save As, this will create a Mascot Distiller Project file (ROV). This ROV file is required for loading into Scaffold.
- The data is now ready to be loaded into Scaffold. Make sure the ROV and XML file are located in the same directory. If Scaffold cannot access the Mascot search DAT file from the Mascot server then make sure to add the DAT file to the same directory as the ROV and XML files.
- When loading the results into Scaffold make sure to select the proper quantitation method in the Load Data Wizard (Stable Isotope Labeling (Multiplex) for SILAC and Precursor Intensity (Standard) for precursor intensity).
Figure 3. Dimethylation [MD] selected for Dimethylation
A note about Dimethyl labeling. The process for processing Scaffold compatible data using Dimethyl labeling is almost identical to the procedure above with the one difference being the quantitation method. Select Dimethylation [MD] as the quantitation method. Scaffold Q+S is required to process this type of quantitative data.