Spectrum Mill Tool Belt

Table of Contents

• Introduction
• To Stop a Process
• To Create a Saved Results File
• To Create MS/MS Search Summary File
• To View a Parameter Table
• To List Modifications Details
• To Create Reporter Ion Correction Factors
• To Apply Reporter Ion Correction Factors
• To Calculate Discriminant Scoring Coefficients
• To Use the File Collector
• To Export PepXML Files
• To Convert Spectra
  

Introduction

The Tool Belt is a collection of utilities that makes it more efficient to process data from MS and MS/MS analyses of protein digests.  For an overview of these tools, see the introduction to each section in this document.

To return to default settings on the Tool Belt page, click the Spectrum Mill button to go to the Spectrum Mill home page.  Then click the link on the home page to go back to the Tool Belt page.

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 (usually a perl script), but not related processes (such as cgi processes). If you click the Stop button to terminate the main process, go to to Tool Belt to terminate related processes:

1. Navigate to the Tool Belt page.
2. Under Select a Tool, click the Stop process option. (This is the default.)
3. Click List Processes to find out which Spectrum Mill-related processes are running.
4. Select one or more processes to stop.  Avoid terminating processes initiated from another client.
5. Click the Stop Process button.
6. To confirm that the process has stopped, see the message near the bottom of the screen.

Warning: If multiple users invoke the same processes (e.g., data extraction or search) on the same server, there is no way to identify which processes belong to you, so in that case let the cgi processes run to completion.

To Create a Saved Results File

The Spectrum Mill workbench allows you to save validated hits from one search program and then search them with another program. For example, you can save a set of validated hits from an identity mode MS/MS Search and then search this set using variable modifications or homology mode MS/MS Search. This saves time because the modified peptides you identify in variable modifications or homology mode are often derived from the unmodified proteins you identified in identity mode.  Both the saved hits and the spectra you search must be in the same data directory.

To save hits from MS/MS Search:

1. Make sure you have already run MS/MS Search and have either autovalidated the good hits (Autovalidation form) or manually validated them (Protein/Peptide Summary).
2. Navigate to the Tool Belt page and click the option to Create save results file.
3. Select the Database you previously searched.
4. Select the Data Directory where the search results reside. This is the same data directory where the data files reside.
5. Click the Create File button.

Note: Starting with Spectrum Mill version A.03.03, you do not need to go to the Tool Belt to create a saved results file. When you mark the check box to Search previous hits in MS/MS Search, if you have not created a saved results file, then the search page automatically creates one for you. (On the form, be sure to select the database that you searched previously.)

To Create MS/MS Search Summary File

If your MS/MS Search terminated abnormally, you can still create an MS/MS Search Summary File so that you can review partial results on the Protein/Peptide Summary page. To create this file:

1. Navigate to the Tool Belt page and click the option to Create MS/MS search summary file.
2. Select the Data Directory for which you want to create a summary file.
3. Click the Create File button.

To View a Parameter Table

The Spectrum Mill workbench keeps a cumulative record of the parameters you set in programs such as Data Extractor, MS/MS Search, and Sherenga de novo sequencing. To view previously-used parameters:

1. Navigate to the Tool Belt page and click the option to View parameter table.
2. Select the Program for which you want to summarize parameters.
3. Select the Data Directory for which you want to summarize parameters.
4. Click the View button.

To List Modifications Details

Use this utility to list details for the amino acid modifications that are included in three xml configuration files.

1. Navigate to the Tool Belt page and click the List modifications details option.
2. Select an smconfig file:
• smconfig.xml - includes all the modifications that are currently displayed in the Spectrum Mill forms. The file smconfig.xml is created automatically by merging smconfig.std.xml and smconfig.custom.xml (which is a configuration file your server administrator optionally creates).
• smconfig.std.xml - includes all of the modifications that are available when you first install the Spectrum Mill workbench
• smconfig.misc.xml - includes additional modification definitions that Agilent provides "as is". They are provided as a convenience and can be copied into smconfig.custom.xml, but they have not been rigorously tested at Agilent.
3. Click the List button.

The listed details include:

• Modification - the name you see in the Spectrum Mill forms
• Internal ID - the name used by the Spectrum Mill programs
• Type - the modification type
• fixed
• variable
• fixed or variable (XML attribute type="any")
• cyclic - mix or other modifications that invoke multiple search cycles
• Site(s) - the amino acid or peptide-terminal location(s) for the modification
• Delta formula - the difference in chemical composition between the modified and unmodified site
• Delta mass (Da) - the monoisotopic mass difference between the modified and unmodified site

Note: You can also view details for modifications that are currently displayed on your server by clicking the Details button in the Choose Modifications dialog.

To Create Reporter Ion Correction Factors

Use this utility to incorporate the correction factors from an iTRAQ or TMT certificate of analysis into a file within the Spectrum Mill workbench. Once you have created the file with the correction factors, you can apply it to reporter ion calculations for multiple samples. The file can store correction factors from multiple batches of reagents and is appended every time you type correction factors for a new batch number. 

For iTRAQ, only iTRAQ 4-plex quantitation allows the creation of correction factors. iTRAQ 8-plex correction factors do not vary by batch lot, so you do not need to enter them.

You can have TMT correction factors for 2-plex, 6-plex, and 10-plex calculations.

To use this tool:

1. Navigate to the Tool Belt page and click Create Reporter Ion correction factors.
2. Fill in the form. Refer to your certificate of analysis for the correct values.
3. Click the Create button.

To apply the correction factors from a particular batch to the calculations for your sample, see the next section.

To Apply Reporter Ion Correction Factors

After you have stored reporter ion correction factors in a file (as described in the previous section), then you can apply them to your data file(s) so that the Spectrum Mill workbench can do the final calculations.

1. Navigate to the Tool Belt page and click Apply Reporter Ion correction factors.
2. For Batch, select the batch number for the reagents you used with the samples.
3. Select the Data Directory that contains your data files.
4. Click the Apply button.

When the software performs iTRAQ or TMT calculations in Protein/Peptide Summary, it automatically applies the correction factors. Be sure to check the report for a message about the batch number of the correction factors that the software used.

To Calculate Discriminant Scoring Coefficients

1. Collect a representative set of data for your study.
2. Use one of the validation methods (including manual validation) to caarefully validate and review the data set.
3. Create the coefficients based on that data set.
   

1. Navigate to the Tool Belt page and click Calculate discriminant scoring coefficients.
2. In the Name field, type a name for the new set of coefficients.
   
3. Under Data Directory, click Select to choose the directory containing valid data results.
   
4. Click Create.
   
   At the bottom of the page you see the calculations as they are performed.  After they have completed, the name of the new set of coefficients appears in the Discriminant Scoring selection list on the MS/MS Search page. 

   The calculations can take some time. Wait for the calculations to complete before you leave or refresh the page. The Create button will be re-enabled when the calculations are complete, and the new set will be listed in the existing sets.

1. Navigate to the Tool Belt page and click Calculate discriminant scoring coefficients.
2. In the Existing Coefficient Sets list, select the coefficients you intend to update.
   The name appears in the Name field.
3. Mark the Update check box.
4. Under Data Directory, click Select to choose the directory containing valid data results.
   
5. Click Create.

To Use the File Collector

Use this tool to copy files from multiple directories to a single collection directory. It is typically used to consolidate exported summary files (.ssv) to one folder.

1. Navigate to the Tool Belt page and click the File collector option.
2. Select the From directories.
3. Select the To directory. Note that you cannot select a directory unless it contains a data file.
4. Under Files, modify the list to specify the files to copy in the From directories. Wildcards (*) are supported. 
5. Click the Copy Files button.

When the files are copied to the To directory, they are renamed with a prefix that includes the name of the From directory.

To Export PepXML Files

PepXML format is a results exchange format that is supported by the Trans-Proteomic Pipeline from the Institute for Systems Biology and that can be imported into other software packages, such as Skyline, an MRM data analysis package from the University of Washington. To convert search results to this format:

1. Navigate to the Tool Belt page and click Export PepXML.
2. For Validation filter, select valid or all.
3. Select any Required AAs or Disallowed AAs.
4. If you want to include the top-ranked reversed hits, mark the check box for Integrate rank 1 reversed hits as pseudo <search hit> entries.
5. If you want to exclude the protein names, mark the check box for Omit protein names.
6. Select the Data Directories that contain the summary files you want to export. You may select one or more data directories.
7. Select the Export directory to which you intend to export the PepXML-formatted data. If you do not select an Export directory, the program will export to the same folder as the results.
8. Click the Export button.
   Note that the program converts the search summary file(s) to PepXML format, and places the output file(s) in the selected Export directory for which they apply. The output files are of the form DataFileName.pep.xml.
9. To access these files, do one of the following:
• Click the link(s) from the output frame, under Spectrum Mill - Convert to PepXML.
• Navigate to the data folder and click to open the pep.xml file.

To Convert Spectra

You use this tool to convert mzXML files to pkl files and pkl files to mzXML files.

As of Spectrum Mill B.04.01, the data extractors create mzXML files instead of pkl files. You can use this tool to convert the mzXML files to the pkl files required by other software packages.

If you have files that were extracted to pkl using prior versions of the Spectrum Mill workbench, you can convert pkl files to mzXML files with this tool.    

1. Navigate to the Tool Belt page and click Convert Spectra.
2. Choose either Convert pkl to mzXML or Convert mzXML to pkl.
3. For the former conversion, select the Validation filter (either all or valid).
4. Under Data Directories, click the top Select button to choose the directory that contains the spectra you intend to convert.
5. If you want to convert pkl files to mzXML files, click the bottom Select button to choose the Export directory. (If you want to convert mzXML files to pkl files, you do not need to specify a directory. The pkl files go automatically to cpick_in under the corresponding data directory.)
6. Click Convert.