Agilent Spectrum Mill MS Proteomics Workbench
Tips and Tricks
Table of Contents
This collection of tips and troubleshooting information will help your Spectrum Mill workbench run smoothly.
To Configure Client PCs
Client PCs can run on any version of Windows that supports Internet Explorer
To view your operating system:
- Select Start > Run.
- In the Open box, type winver.
While the Spectrum Mill workbench requires Internet Explorer 11, you may
choose another browser as your default browser. However, you must access
Spectrum Mill from Internet Explorer. A desktop icon to start Spectrum Mill in
Internet Explorer is created on the Spectrum Mill server. See
To Create a Shortcut to the Spectrum Mill Workbench on
Spectrum Mill requires Internet Explorer 11. To check your browser version:
- Open Internet Explorer.
- Select Help > About Internet Explorer.
If you do not see the Internet Explorer menu, press the Alt key, or
right-click in the title bar and select the Menu bar option.
To avoid potential issues, it is best to add your Spectrum Mill server as a
Notes about Compatibility View
In prior versions of Spectrum Mill, it was necessary to configure the browser to use Compatibility View.
Now, the opposite is true. You must not enable
Compatibility View for the Spectrum Mill server.
If you get a warning to disable Compatibility View when you access the
Spectrum Mill server, do the following:
- In Internet Explorer, click Tools > Compatibility View Settings.
If the Spectrum Mill server is listed in the Compatible Sites,
- Clear the check box for Display intranet sites in Compatibility View.
Check that Internet Explorer on the client is configured for OracleTM JRE.
JRE 1.8 must be installed. Install the 32-bit version first, then
the 64-bit version.
A compatible version of Java is provided on the Spectrum Mill
It is best to uninstall any prior versions before you install an
If the Spectrum Mill Java controls do not display:
- Open the Configure Java program to display the Java Control Panel.
- In the Security tab, add your Spectrum Mill server to the Exception Site
If step 2 does not work. then add the Spectrum Mill server to Internet
Explorer's Trusted Sites list: :
- Select Tools > Internet Options... .
- Click the Security tab.
- Under Select a zone to view or change security setting, click
- Click the Sites button.
- For Add this website to the zone, type or copy in the
address of the Spectrum Mill home page.
- Clear the check box for Require server verification (https:) for
all sites in this one.
- Click Add.
Spectrum Mill works best with the highest screen resolution your monitor
If you do not see the most recent content or results from your Spectrum Mill
server, you may need to enable the browser to check for newer versions. Note
that the following steps were required in versions of the Spectrum Mill
workbench prior to B.05.00, but should not be necessary now. However, if you do
not see the most recent content:
- Select Tools > Internet Options... .
- Click the General tab.
- Under Browsing history, click Settings....
- Click Every time I visit the webpage.
Enable cookies so you can save form settings.
- Select Tools > Internet Options... .
- Click the Privacy tab and set to Low, or click the Advanced button and enable cookies.
- For more detailed instructions to enable cookies, search Internet Explorer help for cookies.
- If the drop-down menus in the Spectrum Mill workbench appear empty, you may need to add the Spectrum
Mill server as a trusted site. For instructions, search Internet Explorer help for trusted site.
- Select Tools > Internet Options....
- Click the Security tab.
- Click Local Intranet.
- Click the Custom Level... button.
- In the Security Settings dialog, scroll down until you see the Scripting section.
- Under Active Scripting, click the Enable option.
Note: Some sites use a Global Security Policy that is enforced by the network domain, and some computers may
or cookies from a web server unless the web server is added as a trusted site. (The symptom in the Spectrum Mill
workbench is that the drop-down menus appear empty.) To add your Spectrum Mill server as a trusted site, search
Internet Explorer help for trusted site.
To Stop a Process
Processes (such as Data Extractor, Search, and Sherenga de novo Sequencing) that take longer than a
few seconds have a red Stop button that is displayed in the Results window. This Stop button
terminates the main computation process, but not related processes. If you click the Stop button, but your
server still runs slowly, you need to terminate related processes.
To Use Multiple Browser Windows with the Spectrum Mill Workbench
If you want to work with separate data sets, start the browser from the desktop or other shortcut, then go
to the Spectrum Mill home page URL for your server. In this way, each browser window has its own default
session parameters. Note that when you first open the browser and you go to subsequent Spectrum Mill pages, you
will see the parameters that were stored the last time you clicked Save Settings and the data directory
settings that were stored the last you clicked Make Default in the Select Data Directory -- Web
To Create a Shortcut to the Spectrum Mill Workbench on Your Desktop
When Spectrum Mill is installed on the server, a desktop icon is created to launch Internet Explorer and direct
it to the Spectrum Mill home page. Since Spectrum Mill is not installed on client PCs, you will need to create
the shortcut yourself. Having a shortcut makes it easier to launch multiple Spectrum Mill browsers.
Hint: If you make the Spectrum Mill home page your Internet Explorer default home page, then you do not need
To create a new shortcut, start at step 1 below. To modify the existing Internet Explorer shortcut, start at
- Open Windows Explorer and browse to C:\Program Files\Internet Explorer.
- Right-click iexplore.exe and select Create Shortcut.
- Drag the newly created Shortcut to iexplore.exe and drop it onto your desktop.
- Right-click the shortcut icon on your desktop and select Properties.
- In the Shortcut tab, append the name of your Spectrum Mill server to the Target field:
Target: "C:\Program Files\Internet Explorer\iexplore.exe" MySMServer
- Click OK.
- Right-click the shortcut icon and rename the shortcut to say Spectrum Mill - MySMServer.
- To add the icon to your Windows quick start task bar, simply drag and drop the shortcut icon onto the
File naming, directory structure
- Do not use spaces, the plus sign, spaces, or parentheses in file names. The following characters are not permitted:
| , ; % < > + ( ) ? !. Also, a dot or period (.) is allowed only for file suffixes (e.g., myfile.d).
- For best performance, organize your data into a folder hierarchy.
- Note that you may have up to ten directory levels (folders) between msdataSM and your sample
directory, but we recommend shorter path lengths to reduce memory usage, especially for large data sets.
- Do not place data files directly into the msdataSM folder. Always create a subfolder to contain
the data files.
- Spectrum Mill does not recognize data directories unless they contain at least one data file.
If you set up a directory for spectral collections, be sure to include a data file so the File
Collector on the Tool Belt page can recognize the folder.
- When you run longer processes such as Data Extractor, MS/MS Search or Sherenga de novo Sequencing,
you can use your client PC for other tasks while the processes run.
- Unless your data is on an SSD, to maintain top system performance, defragment your database drive each time you download a
database. For the data drive, archive and remove data that is no longer needed; then (if your drive is
not an SSD) defragment
the data drive.
- In general, enable Maximize CPUs to improve performance for
extractions and searches. However, if you select more directories than
the number of CPUs that are configured on the server, it is best to not
enable Maximize CPUs. For Data Extractor, enable
Maximize CPUs only if folders contain multiple data
- If you process large data files, make
sure you have sufficient memory (RAM) installed on the server. A
reasonable estimate is 2 Gb per CPU for larger data files. If you do
not have sufficient memory, you can restrict the number of CPU cores
that Spectrum Mill uses. See the instructions to
limit the number of parallel processes.
- If Hyper-Threading is not enabled, enable it by rebooting and accessing the system BIOS configuration. With Spectrum Mill B.06.00, enabling Hyper-Threading is now recommended, and can improve search performance from 20-30%, provided there is sufficient memory installed. If you enable Hyper-Threading after installing Spectrum Mill, you will need to reconfigure the Spectrum Mill Request Manager Service to take advantage of the extra CPUs. Set the "maxConcurrentTasks" value to 1 less than the total number of (logical) processors, or lower if there is not sufficient RAM to handle the additional parallel tasks. See the instructions to limit the number of parallel processes.
- If you want a link to appear in a separate window, SHIFT-click the link. For example, in Protein/Peptide
Summary, it is sometimes easier to view the detailed hit results in a separate window. To do so, SHIFT-click
the Filename link for that hit. SHIFT-click does not work for links to the Spectrum Viewer, so
click the %SPI link to view the annotated spectrum in a separate window.
- In Spectrum Viewer, it is much easier to see how candidate peptide sequences align with your spectrum
if you select an ion type to highlight. Look for the Highlight drop-down menu at the bottom of
the Spectrum Viewer. Choose an ion type (for example, b or y) and note the correspondence
indicated by the gray lines.
- If you want to compare results of individual sample analyses from separate directories, see the
Frequently Asked Questions.
- The Spectrum Mill workbench will only work properly if you access it through its http address, and
not as a shared drive.
- If you accidentally run MS/MS Search with incorrect settings, when you perform the next search, mark
the check box on the MS/MS Search form to Remove all prior MS/MS Search results. This removes
the invalid results for the dataset, so that they are not included in results summaries.
- There are a number of files in the Spectrum Mill workbench that you can customize. Before you
modify any files, be sure to make copies so you can restore the originals if necessary.
- If you uninstall the software, first make backup copies of any Spectrum Mill workbench files that
you have customized for your lab.
Software Status Bulletin and Software Patches
A list of known problems or issues for the Spectrum Mill workbench, along
with possible solutions, is found in the Agilent Software Status Bulletin.
To view this bulletin,
Troubleshooting - Software Updates
If the software does not appear to update properly
You install a new version of the Spectrum Mill workbench, but the software does not update properly; some of
the software appears to be from the older version. For example, the software revision number is incorrect or some
new features fail to appear.
Some possible causes are:
- When updating, you did not uninstall the previous version of the Spectrum Mill workbench via Add/Remove
- You have not refreshed the Spectrum Mill pages in your browser window.
To ensure that the server was properly updated:
- Be sure to follow the instructions in the Installation Guide to upgrade your Spectrum Mill MS
Proteomics Workbench. It is particularly important to uninstall the previous Spectrum Mill version using
Add/Remove Programs, rather than just deleting the old software files.
- If you just deleted the old version, re-run setup.exe from the old version and choose Repair….
Then uninstall the Spectrum Mill workbench using Add/Remove Programs.
- (This step is usually not necessary.) On the Spectrum Mill server, restart Internet Information
Services (IIS) to make sure that IIS cache is flushed. To do this, go to IIS administration and stop, then
restart the Spectrum Mill Web site (Default Web Site). Consult your IIS help for details. Alternatively,
simply reboot the Spectrum Mill server.
To ensure that all files are refreshed by Internet Explorer, do the following on each client PC:
- Check that pages are refreshed upon each visit. See the section on "stored pages" under
To Configure Client PCs.
- Delete all temporary internet files. (In Internet Explorer, select Tools > Internet Options...
and delete the temporary internet files. Or see the steps below.)
- Close and reopen any browser windows to the Spectrum Mill Web site.
If you would prefer to remove only the temporary internet files that are
associated with the Spectrum Mill server, do the following on each client PC:
- In Internet Explorer, press F12 to access the
- Click the Network tab.
- Rest your mouse cursor over the toolbar icons and select the one that indicates
Clear browser cache... or press Ctrl-R.
- Close the Developer Tools and refresh the Spectrum Mill page.
If the updated software displays errors
The older version of the Spectrum Mill workbench worked fine, but the updated software gives errors.
This problem can occur when the software update requires a new version of either Perl or Java, and the new
version was not installed.
- If required, update Java and Perl on the Spectrum Mill server. See the latest Installation Guide
for details. You can get a compatible version of Java and Perl from your Spectrum Mill installation
- If required, update Java (JRE) on all client PCs. See the latest Installation Guide for details.
You can get a compatible version of JRE from your Spectrum Mill installation CD.
Troubleshooting - Spectrum Viewer
If the Spectrum Viewer shows no content, or does not display properly
The Spectrum Viewer (the bottom of the screen where the spectrum should be displayed) does not display
properly, or does not display at all.
The following may cause this problem:
- You have the wrong Java version. You need JRE 1.8.x. A
compatible version is provided on the Spectrum Mill disk.
- Your security settings for Internet Explorer (IE) are incorrect. Add
your server as a trusted site for IE.
- You lack the necessary file permissions for the data folder in msdataSM.
- A file that is referenced by the link no longer exists. (Some processed data was deleted.)
- If you do not have the correct Java version on your PC, get a compatible version from your Spectrum Mill installation
- If you saw a warning about scripting being denied, make sure Java
scripting is enabled.
If the warning persists, add your Spectrum Mill server as a trusted site. For more information, search the Internet
Explorer help for trusted site.
- Make sure you have as a minimum read permission for the selected data folder. For example, if IIS
on the Spectrum Mill server is configured for Anonymous access, then the IUSR user
must have permission to read the data files. If this is not the case, have your system administrator set the
permissions manually, or run a script to set the permissions.
- See also If you get an error that says "page not found".
If I get an "add-on failed to run" message
in Spectrum Viewer (or MS Isotope)
The Spectrum Viewer (the bottom of the screen where the spectrum should be displayed)
gives an "add-on failed to run" message.
This message occurs when the Spectrum Mill server is not listed as a trusted
site in Internet Explorer.
Add your Spectrum Mill server URL as a trusted site in Internet Explorer. Do
this for all client browsers as well.
Troubleshooting - Selecting Data Files
If you cannot select a data directory
See If some programs work, but others do not (permission denied).
If you select a data directory, but you see "(none selected)"
You select a valid data directory and click OK, but the Data Directory still shows (none selected).
This problem occurs when cookies are not enabled.
See the sections on cookies and stored pages under To Configure Client PCs.
If a one or more data directories are missing from the Select Data Directory
One or more of my data directories fails to appear in the Select Data Directory dialog box, or I click the button
to select a data directory, but no directories appear in the dialog box.
This happens under a number of circumstances:
- A user has copied a processed data folder into a higher level folder. The folder listing will stop if
it detects any of the following, which it uses to recognize the current folder as a data folder:
- A data file
- One of the following folders: cpick_in, fit_batch_in, results_msfit, results_mstag, results_sherenga
(If one of these appears in the msdataSM directory, then no directories will appear in the dialog
A data file exists in a higher level folder. The Spectrum Mill workbench can only find the highest
level of data files in a given subdirectory. For example, given these two data files,
SM will recognize datafile2.d, but not datafile1.d.
File names use spaces or parentheses or another invalid character, such
as a +.
There are no data files in the data directories.
IIS is configured to use Windows Authentication, and the user lacks permission to read the directories.
An IT security policy prevents display of the data directories. You may need to add the Spectrum Mill
server as a trusted site.
After you have identified the numbered cause above, perform the action with the same number below:
- Delete or move any *.pkl files or processed data folders (cpick_in, fit_batch_in, results_msfit, results_mstag,
results_sherenga) that exist outside of a data folder.
- Move all data files to folders that are at equivalent levels within a given subdirectory.
- Rename the data directory to remove the spaces and parentheses.
- Make sure there are data files in the data directories.
- If you have configured IIS to use Windows Authentication, make sure that the user has permission to read
- Within Internet Explorer, add the Spectrum Mill server as a trusted site. For instructions, search Internet
Explorer help for trusted site.
Troubleshooting - Protein/Peptide Summary, Spectrum Summary
See also: Troubleshooting - Spectrum Viewer
If the Summarize button is disabled or grayed out on the Protein/Peptide
Summary or Spectrum Summary forms
The Summarize button is grayed out and/or does not do anything.
Once you click the Summarize button, the button is disabled until the results appear. If there is
an error (for example, you try to summarize data that has not yet been extracted), the Summarize button
may be unavailable (grayed out), even after you select a new data directory.
To re-enable the button, click the Summary Settings button at the top of the page to reload the Protein/Peptide
Summary or Spectrum Summary form.
If you sort by accession number, but the sort is alphabetical rather than numerical
I tried to sort by accession number on the Protein/Peptide Summary form, but the results were sorted alphabetically
Some databases use alpha characters in accession numbers, so "sorting by accession number" is actually an alphabetical
sort. This accomplishes the objective to find hits that have the same accession number.
If you see an error "proteins not same length"
In one of the protein modes, I saw the following error: "Error: Function: addAAcoverageArrays. Proteins
not same length. Most likely cause is duplicate accession numbers (AR) in the protein database."
This problem has two potential causes:
- The database has the wrong name. Note that the prefix in the database name (NCBInr,
TrEMBL, IPI, Genpept, or dbEST ) must reflect the database format, which is usually
dictated by the site from which the database was downloaded. For example, if you download a SwissProt database
from the NCBI site, then the format is NCBI, not SwissProt.
- A database contains duplicate accession numbers and you attempt a summary in one of the protein modes.
If the cause is number 1 above, then give the database the correct name and
reprocess your data.
If the cause is number 2 above, then remove the duplicate accession numbers and rerun the search and summary.
To remove the duplicate accession numbers:
- Note from the error message the duplicate accession number.
- Open the database in a plain text editing program (e.g., Notepad or WordPad), remove the duplicate accession
number, save the file, and remove any .txt extension that has been appended.
- Go to Protein Databases and recreate the indices.
If peptides appear twice in the summary report
A significant number of peptides appear twice in the summary report.
It is normal for peptides to appear twice if they have different charge states (for example, 0 and 2) or if they
are labeled (for example, D0 and D8). If these are not the cause, then you may need to adjust
the settings for Data Extractor. The most common cause is that the merge time is too short.
Try the following in order. Each time, reprocess your data, starting with data extraction, to see if the number
of redundant peptides decreases. Stop when you achieve satisfactory results.
- In Data Extractor, increase the merge time. (Set Merge scans with same precursor m/z to a larger
number of sec.)
- In Data Extractor, increase the m/z merge window. (Set Merge scans with same precursor m/z
to a larger m/z window.) The default merge window of +/-1.4 m/z is generally more than
wide enough, so this usually isn’t a problem. However, if you previously narrowed the window, some scans
may not be merged.
- Ask your system administrator to lower the merge_SPI or merge_num_peaks settings for your
instrument in msparams_mill\instrument.txt. All instruments that generate MS/MS data use the defaults
of merge_SPI = 70 and merge_num_peaks = 25, but if you add an entry to instrument.txt,
that overrides the defaults.
- The format is merge_SPI, followed by a tab, followed by the value.
- Try changing one variable at a time (either merge_SPI or merge_num_peaks) and
decrement that variable in steps of 5.
- Note that when you lower merge_SPI and merge_num_peaks, more spectra are merged,
but there is an increased risk of merging unrelated spectra.
- The defaults of 25 peaks and 70% SPI were chosen because they produced the most protein identifications,
even though there may be a few redundant peptides.
If no results are listed in Protein/Peptide Summary, even though there were hits
My database search produced hits, but I observe no results when I attempt a protein summary.
This happens when:
- The peptide hits pass the criteria for peptide filtering, but the resulting protein scores do not pass
the criteria for protein filtering.
- You improperly set Filter results by. For example, you set the filter to valid when no
results have been validated, or to sequence-not-validated when all the results have been validated
(for the score filters).
- For Filter by protein score, type a smaller number.
- Set Filter results by to the appropriate validation state to show results.
If clicking %SPI and other links shows an
After installing Spectrum Mill on a new system (WS 2008 R2 or WS2012 R2),
clicking on the %SPI link and other links shows an
and the viewer is shown in a separate tab.
This problem only happens when the user is logged in as administrator, and on
This problem is the result of a change introduced with installation of IE11 on a
computer, affecting behavior of User Account Control: Use Admin Approval
Mode for the built-in Administrator account setting within local
security policy. This setting can be configured using the Local Security
Policy snap-in, in Local Policies / Security Options
branch, under Policy item called: "User Account Control: Use Admin
Approval Mode for the built-in Administrator account".
The steps to do the above are:
- From Start->Run (or WS2012 Search), run
- Under Local Policies, select Security.
- Scroll down to find “User Account Control: Behavior of the elevation
prompt for administrators in Admin Approval Mode” and then enable it.
Troubleshooting - Workflows
If requests do not process or refresh (the workflow is stuck)
The workflow fails to progress. Requests are not processed, and the Request Queue does not refresh.
A process may be waiting for a previous process, which has failed to execute.
- Stop the Spectrum Mill Workflow Manager Service.
- If the service does not stop, go to Windows Task Manager and stop the process SRMHostSvc.exe.
- Restart the Spectrum Mill Workflow Manager Service.
Troubleshooting - Other
If you see unusual or blank selection values
The drop-down menu items and other fields in forms do not have any content, or
I observe unusual or blank selection values with the Spectrum Mill workbench, and some fields are "undefined."
The symptoms indicate a problem with cookies. Try the following in order.
- If you updated from a previous version of the Spectrum Mill workbench, delete any cookies associated
with the Spectrum Mill server.
- Make sure cookies are enabled. For instructions to enable cookies, search Internet Explorer help for
- If the problem persists, add the Spectrum Mill server as a trusted site. Search Internet Explorer help
for trusted site.
If you get a warning about need for additional license
My searches give a warning about four CPUs and the need for an additional license, yet I have only a two-CPU server.
Spectrum Mill licensing is based on the number of physical CPUs. Hyper-Threading and multicore CPUs are considered
to be one physical CPU. However, some older BIOS software may report the wrong value to Spectrum Mill if Hyper-Threading
is enabled. If this is the case, you must disable Hyper-Threading in the BIOS. If you disable Hyper-Threading after installing Spectrum Mill, you will need to reconfigure the Spectrum Mill Request Manager Service to reduce the number of concurrent searches. Set the "maxConcurrentTasks" value to 1 less than the total number of processors. See the instructions to limit the number of parallel processes.
If your list of protein database names does not update
When I click Update Database List in the Protein Databases form, my database list does not update.
For the list to be updated, the web users must have write permission for the file.
Set the write permissions as described in the Installation Guide.
If you get different results between Spectrum Mill versions
My results with the current Spectrum Mill version are different from those with a previous version.
Improvements to the extraction, search, and protein grouping may result in
differences from prior versions.
If you get an error that says "page not found"
Some Spectrum Mill functions return an error that says "Page not found." Examples might include (but are not limited
to) getting a process list in Tool Belt or displaying a spectrum with the Spectrum Viewer.
The server may not be running.
Verify the Spectrum Mill server is running and that Web site is not stopped.
If some programs work, but others do not (permission denied)
Some Spectrum Mill programs work, but others do not. For example, you cannot index a database, nor select a data
directory, but MS Comp works.
This problem may be caused by a user who is logged onto the Spectrum Mill server and is a member of both the Users
and Administrators groups.
Remove the user from the Users group and retain membership in the Administrators group.
If Peptide Selector seems to ignore settings
Peptide Selector ignores some of my criteria to exclude peptides. For example, the form lists C and M as disallowed
amino acids, but some peptides in the results still include those amino acids.
When you mark the check box for Penalize rather than exclude on the Peptide Selector form, the program
applies a penalty score to peptides rather than excluding them. The settings that use penalties change to green
font. Those settings no longer cause peptides to be excluded.
Clear the check box for Penalize rather than exclude.
If Spectrum Mill performance becomes slow
The Spectrum Mill performance appears to be very slow recently.
The slow performance may occur because one or more RAID disks have failed, and
data is being recovered. Another possibility is that free memory is very low.
If the RAID drives are functioning properly, run Task Manager on the server to
view memory and CPU usage. If free memory is very low (< 1Gb), consider
increasing the amount of memory on your server, or configuring the Spectrum Mill
workflow manager to use fewer CPU cores when running parallel tasks.
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