Loading and Organizing Quantitative Data in Scaffold Q+/Q+S

The following article contains a list of frequently asked question relating to loading and organizing data in Scaffold Q+. 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.

Loading Quantitative Data in Scaffold Q+

What types of data can Scaffold Q+ load? What about Q+S?
Please see the Scaffold file compatibility matrix for the most current information about loading data into Scaffold Q+/Q+S. Please note that not all search engines produce quantitative results that are compatible with Scaffold.

How do I load additional data into Scaffold Q+ or Q+S after I open it?
To load additional data files for analysis in Q+ or Q+S, you will need to return to Scaffold and load the files in Scaffold's data loading wizard. For more information on how this works, see these FAQs on loading data in Scaffold. 

For more information on organizing samples once loaded into Q+/Q+S, see the FAQ, Can I re-organize my samples after opening Q+ module? below.

What filters in Scaffold carry over into Q+?
Any protein or peptide that does not meet the probability thresholds set in Scaffold will not appear in Q+.

Certain filters do not carry over completely, however. Advanced filters set in Scaffold do not apply in Scaffold Q+ (such as GO term or taxonomy filters). The Req Mods filter applies on the protein level, but not on the peptide level.

Organizing Data in Scaffold Q+

What Quantitative Settings should I use?
Many of the settings you choose depend on your experimental design, which the documents below can advise. However, some suggestions are as follows:

Normalization Tab

Calculation Type: As stated in the FAQ, Should I use the median or the mean when quantifying my data set? the Median is the suggested setting UNLESS you have VERY well-behaved data.

Reference Type: See the FAQ, "Which reference type should I use?" below.

Normalization Type: Toggle this drop-down depending on whether you want Scaffold Q+ to normalize your data across biosamples or multiplexes.

Minimum Dynamic Range Tab

Because reporter ion peak heights below 1-5% of the highest peak are usually unreliable for quantification, we provide a graph with a slider to help you choose an appropriate level that trades off accuracy (not quantifying based on low confidence values with higher thresholds) versus breadth (including more values to improve statistics with lower thresholds). Higher dynamic range across the experiment (histogram leans to the right) is indicative of better quality data sets.

Other Settings Tab

Check the Use Non-Exclusive Peptides box to consider shared peptides during quantification. This might be useful if, for instance, there are groups of proteins which share a large number of peptides between them and have few unique peptides. Using non unique peptides in quantification would enable a user to quantify the protein group whereas before, little data might have been available.

Can I re-organize my samples after opening the Q+ module?
Yes, the Quant>Update Experimental Design... option allows you to re-organize the loaded quantitative samples. Here, you can add and remove samples from various categories, rename the existing categories, or add or remove various categories.

What is a reference sample?
This is the Quantitative Sample to which all others will be compared. Select which reporter ions or SILAC label from your experiment should be used as the reference or control group and click Add to assign them to the Reference Quantitative Sample.

Which reference type should I use?
Your experimental design will likely dictate which reference type you should use. 

For time series experiments where each sample represents a specific time, use the Individual Spectrum Reference type. More info on organizing these experiments below.

For a pooled reference sample, the Average Protein Reference is the appropriate type to choose.

How do I compare each sample to its own reference?
To compare each sample to its own reference, select and use the Individual Spectrum Reference Type in the Quantitative Settings menu option. If you are loading multiplexed data, you will have to load the data twice in Scaffold and separate out the Quant samples appropriate for your experimental design with Scaffold Q+.

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