These are answers to questions we have received from Spectrum Mill users.
Yes, the Spectrum Mill workbench allows you to create and search your own FASTA-format databases.
Note: If you queue the summary request, it generates an HTML file automatically.
If you have a full-featured program to edit graphics, do the following:
If you do not have a full-featured program to edit graphics, do the following:
Note: You may alternatively crop the image within PowerPoint.
See Exporting to Excel or Uploading to LIMS.
Yes, you can. For example, if you use alkylate cysteines with two different reagents, you can search for both alkylations.
The search will consider each cysteine as modified by either the fixed or variable modification. Thus, if more than one cysteine is present in a peptide, the software will consider the various combinations of modified forms. It will not consider unmodified forms.
If you need to consider unmodified forms and the same peptide can only contain all of one modification, or all of the other, or none, then ask your system administrator to modify smconfig.custom.xml to create a mix cycle. (In this case it would be a three-cycle search.) Then do an identity mode search.
In MS/MS Search, when you go to Search Parameters, and choose one of the predefined Species, the software gets the species definitions from the file: E:\SpectrumMill\msparams_mill\species.txt. To check whether your species are represented in a particular species definition, open the species.txt file and determine the included species under the species subset definition in question. For additional details, see Spectrum Mill Species Definitions.
If your species are not present, you can edit existing definitions or add new ones. See To Add/Change a Species Filter.
During data extraction, the Spectrum Mill workbench employs filtering that identifies and excludes noise spectra and poor quality spectra. This is in part why searches proceed so quickly, and is a major reason why the Spectrum Mill workbench yields fewer false positives than other database search engines. Thus, while other search engines tend to produce increased sequence coverage, the additional database matches are of poorer quality and may lead to an increase in the number of false positives.
We do not recommend that you turn off Spectrum Mill's spectral quality filtering. However, if you wish to turn it off temporarily so you can compare results directly with other database search engines, first configure the ability to disable quality filtering. Then mark the check boxes for Disable quality filtering in the Data Extractor and Disable match filtering in MS/MS Search. To perform the comparison, use a well-characterized sample (for example, BSA digest). You will likely find that while you do get more comprehensive search results (that is, increased sequence coverage), the quality of the results are poorer.
For more details on Spectrum Mill's intelligent spectral extraction, see Spectral Preprocessing for MS/MS Data.
For example, suppose you have:
|Control directory||Disease directory|
The filename wildcard (Search result files:, near the lower left corner of the Protein/Peptide Summary page) is a simple and powerful way to limit which files you compare. For example, in Protein - Protein Comparison Columns mode:
To compare Disease versus Control:
To compare Disease_repeat1.D versus Control_repeat1.D:
When you observe poor FWD-REV scores (low numbers) in validated results, that probably means that very few reversed hits were present in the dataset that enabled the target FDR to be reached with little or no filtering of hits. This could happen because:
Finally, you may see the effect only on charge states for which there are small numbers per run in the dataset. For that reason, on charges greater than 4, if you have multiple runs per directory, use Autovalidation by directory rather than by file.
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