Spectrum Mill Frequently Asked Questions

Table of Contents


These are answers to questions we have received from Spectrum Mill users.

Can I search my own databases with Spectrum Mill?

Yes, Spectrum Mill allows you to create and search your own FASTA-format databases.

  1. To create a user database, click the Protein Databases from the Utilities section of the Spectrum Mill home page.
  2. Select  Create or append user database.
  3. Follow procedures in the online help. It is best if you read the entire document. As a minimum, read the following two sections:

How can I export Spectrum Mill results into HTML files?

  1. Generate a report in Protein/Peptide Summary.
  2. Right-click anywhere in the report and select View Source.
  3. Check that the report appears in Notepad.
  4. Select File > Save as... .
  5. Rename the file and give it an extension of .htm.
  6. Save the file. 
  7. Double-click the .htm file to view it.
  8. You may email the file as an attachment, or post it to a Web site.

Note: If you queue the summary request, it generates an HTML file automatically.


How can I export Spectrum Mill results into PowerPoint files?

If you have a full-featured program to edit graphics, do the following:

  1. Generate a report in Protein/Peptide Summary.
  2. Use your program's screen capture feature to capture the active Spectrum Mill window.
  3. Use your program's crop feature to crop the image to show the desired area.
  4. Copy the cropped image.
  5. Paste the image into Microsoft® PowerPoint.

If you do not have a full-featured program to edit graphics, do the following:

  1. Generate a report in Protein/Peptide Summary.
  2. To capture the active Spectrum Mill window, press ALT + PRINT SCREEN. (The PRINT SCREEN key is near the INSERT key.) The window is now copied to the clipboard.
  3. Paste the image into Microsoft Paint.
  4. Crop the image to show the desired area.
  5. Copy the cropped image.
  6. Paste the image into Microsoft PowerPoint.

Note: You may alternatively crop the image within PowerPoint.

How can I export Spectrum Mill results into Excel or upload to LIMS?

See Exporting to Excel or Uploading to LIMS.


Can I search for more than one cysteine modification?

Yes, you can. For example, if you use alkylate cysteines with two different reagents, you can search for both alkylations.

  1. Go to the MS/MS Search form.
  2. Click Choose... to open the Choose Modifications dialog.
  3. Select one of the cysteine modifications as a fixed modification.
  4. Select the other cysteine modification as a variable modification.
  5. Click OK.
  6. Set the Search mode to Variable modifications.
  7. Set other parameters.
  8. Start the search.

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.

How can I ensure that my species are represented in a species subset database?

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.

Why do I get more peptide spectrum matches (PSMs) with another database search engine than with Spectrum Mill?

During data extraction, Spectrum Mill 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 Spectrum Mill yields fewer false positives than another database search engines. Thus, while another search engine may tend to produce increased sequence coverage, the additional database matches will tend to be of poorer quality and may lead to increased false discovery rates.

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. 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.

How do I compare results of individual sample analyses from separate directories?

For example, suppose you have:

Control directory Disease directory
Control_repeat1.D Disease_repeat1.D
Control_repeat2.D Disease_repeat2.D
Control_repeat3.D Disease_repeat3.D

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:

Why do I see poor FWD-REV scores (low number) in validated results?

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.

How can I reduce the amount of disk space required for my Spectrum Mill data?

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