Agilent Spectrum Mill MS Proteomics Workbench

What's New in This Version




New in B.07.00

The BI.07.00 version of Spectrum Mill eliminates the dependency on Internet Explorer, and can now be used with most web browsers including Google Chrome, Firefox, and Microsoft Edge. Primary development and testing is now done with Google Chrome. This document describes differences between the BI.07.00 and prior versions of Spectrum Mill.

Home Page


Data Extractors


Thermo .RAW Extractor revisions include: Generic Extractor now extracts .MGF files from Bruker timsTOF.

MS/MS Search


MS/MS Autovalidation


Protein/Peptide Summary


Quality Metrics and False Discovery Rate (FDR)


Spectrum Matcher


Spectrum Summary


de novo Sequencing


Protein Databases


Tool Belt


New in B.06.00.200


The B.06.00 version of Agilent Spectrum Mill MS Proteomics Workbench supports 64-bit Windows operating systems and includes enhancements that increase the flexibility of the software and enable greater productivity. This document describes differences between the B.06.00 and prior versions of the Spectrum Mill workbench. Note: If you have upgraded from a prior version, you will see some differences in validated results compared to the earlier version. When comparing data sets, it is best to reprocess (extract, search, autovalidate) them using the B.06.00 version. See Quality Metrics & FDR

Installation and Configuration

The following installation and configuration features are new in the Spectrum Mill workbench version B.06.00. For details, see the Installation Guide and the Site Preparation Guide. Both are on your software disk.

Operating system

Spectrum Mill B.06.00 is supported on the following Microsoft Windows operating systems:

Only 64-bit versions of the above operating systems are supported.

Windows XP and Windows Server 2003 are no longer supported.

If you configure a new system, Agilent recommends that you provide adequate memory (16 GB or more) and adequate disk space (1 to 2 TB for data). For a new system, Agilent recommends 24 to 32 GB of memory.

Perl

ActiveState Perl 5.18.4 is supplied with this release. Install the 64-bit MSI. (If you are updating from Spectrum Mill B.04.01 or earlier, uninstall the prior version first.)

JRE

Java JRE 1.8u111 (1.8.0_111) is included in this release. Install both the 32- and 64-bit JREs, even if you run the 64-bit Internet Explorer (IE 11). Later versions of JRE 8 may be available (see www.java.com), and in general should work with Spectrum Mill.

The JRE is required to support the Spectrum Viewer and other applets.

Be sure to install JREs on all client browsers, as well as the Spectrum Mill server. 

IIS

IIS must be installed before you install the Spectrum Mill workbench.

See the Installation Guide for details on installation and configuration of IIS.

MSFileReader support (for .raw files)

To process Thermo Scientific .raw files, Spectrum Mill B.06.00 can use the 64-bit Thermo Scientific MSFileReader.

If you update your Spectrum Mill installation from B.04.01 or prior, then you have the 32-bit MSFileReader installed, so you must install the 64-bit version. If you update your Spectrum Mill installation from B.05, you already have the 64-bit version.

Web browser support

The following Web browser is supported with this version: If you are upgrading from Spectrum Mill workbench B.04.01 or earlier, note that the prior release required Compatibility View for Internet Explorer 9 and later. For this release you must disable Compatibility View.


MS/MS Search


Quality Metrics and False Discovery Rate (FDR)


Home Page


Protein Databases


Tool Belt


Spectrum Matcher


Protein/Peptide Summary


Spectrum Summary


Data Extractors


New in B.05.00

The B.05.00 version of Agilent Spectrum Mill MS Proteomics Workbench provides the following new features: Note: If you have upgraded from a prior version, due to improvements in data extraction, search scoring, and protein grouping, you will see some differences in validated results compared to the earlier version. When comparing data sets, it is best to reprocess (extract, search, autovalidate) them using the B.05.00 version.

Installation and Configuration

The following installation and configuration features are new in the Spectrum Mill workbench version B.05.00. For details, see the Installation Guide.

Operating system

Spectrum Mill B.05.00 is supported on the following Microsoft Windows operating systems:

Only 64-bit versions of the above operating systems are supported.

If you configure a new system, Agilent recommends that you provide adequate memory (16 GB or more) and adequate disk space (1 to 2 TB for data).

Perl

ActiveState Perl 5.18.4 is supplied with this release. Install the 64-bit MSI. (Uninstall the prior version first.)

JRE

Java JRE 1.8u45 is included in this release. Install both the 32- and 64-bit JREs, even if you run the 64-bit Internet Explorer (IE 11).

Be sure to install JREs on all client browsers, as well as the Spectrum Mill server. 

IIS

IIS must be installed before you install the Spectrum Mill workbench.

See the Installation Guide for details on configuration of IIS.

MSFileReader Support (for .raw files)

To process Thermo Scientific .raw files, Spectrum Mill B.05.00 can use either Xcalibur or the 64-bit Thermo Scientific MSFileReader. If you want to use one of these programs, install it on the server before you install the Spectrum Mill workbench. You can install it afterwards, but you must do some additional steps. (See the Installation Guide for details.)

If you update your Spectrum Mill installation, and you previously had installed MSFileReader, you must install the 64-bit version.

Web Browser Support

The following Web browser is supported with this version:

Internet Explorer Compatibility View

If you are upgrading, note that the prior release required Compatibility View for Internet Explorer 9 and later. For this release you must disable Compatibility View.

Workflow Automation

New workflow automation tools are available.


Tool Belt and Metrics


MS/MS Autovalidation


Protein/Peptide Summary


Data Extractors


MS/MS Search


Utilities



Protein Databases



New in B.04.01

The B.04.01 version of Agilent Spectrum Mill MS Proteomics Workbench provides the following new features:


Data Extraction

The Agilent Q-ToF data extractor for B.04.01 now adjusts the tolerance based on m/z for finding related precurors with SILAC data. The differences are minor, but you will see some differences. The same adjustments may affect merging, where fewer spectra might be merged.

Extract to mzXML

Automation: MS/MS Search supports multiple CPU cores

Spectrum Mill's MS/MS Search can now take advantage of multiple CPU cores. In the MS/MS Search page, select Maximize CPUs, and in the Workflows page, select Max CPUs per search. When enabled, batches from a single data directory will be assigned to available CPUs. The parallel searches are listed in the Request Queue as tasks with a "P". The search progress is indicated as the number of batches completed out of total batches. When using this search mode, it is recommended to increase the batch size to 150 or more, instead of the former default of 81.

To prevent a single user from unfairly monopolizing all CPU's on a Spectrum Mill server, all directories submitted to the queue with Maxmize CPUs enabled compete for CPU access. When multiple directories are submitted to search, they all will share the available CPUs, with processing occurring in parallel. When Maximize CPUs is not enabled, each search on a directory retains exclusive access to a single processor until all of its batches have completed.

When you select a queued Maximize CPUs search to Remove that has begun processing, the Request Queue will continue to show the search in the queue. Wait a little while for any currently executing batch searchs to complete, then click Request Queue to refresh the list.

Protein/Peptide Summary Changes

Review Fields

The Review Fields layout has been changed slightly to accomodate additional fields. The new spectral quality related fields include Prec Av Chi2 (Precursor Averagine Chi2) and Isol Pur (Isolation Purity). The pI field is now Prot pI, and the Peptide pI field is now just Pep pI. The Var mod sequence field is now VML sequence.

Protein Grouping

You may re-enable the grouping mode used in prior versions by selecting the Prot pI review field.

Export results to Mass Profiler Professional (MPP)

The Protein-Protein Comparison Columns and Protein-Protein Comparison Redundant summary modes now support exporting to the MPP generic format. To only report the top group protein, use Protein-Protein Comparison Columns with 1 shared peptide as the Protein grouping method. To report all proteins, use the 1 shared, expand subgroups method. The Protein-Protein Comparison Redundant mode will always report all proteins (the 1 shared, expand subgroups method is not allowed in this mode).

Peptide Mode changes

Protein-Peptide Comparison Columns Mode: Enhanced VML Reporting and Excel Export

The Protein-Peptide Comparison Columns summary mode has been enhanced to enable Excel export and to allow peptide level or modification site level report organization. Specific changes include:

Revised MS/MS Search Scoring

Autovalidation Changes

The Autovalidation Auto thresholds strategy now supports a Min. Sequence Length filter. The default value for this field is 1 so as not to surprise users who upgrade from prior versions, but higher values are recommended.

Note: A value of 6 or higher is recommended for the Min. Sequence Length value. Sequences shorter than 6 tend not to be unique to a single protein.

In addition, the Peptide FDR for validated proteins has been removed from the Auto thresholds Protein polishing strategy. It is only available when using the Auto thresholds - discriminant strategy.

Note: It is recommended that you open and re-save all Autovalidation parameter files that use the Auto thresholds strategy that were created using B.04.00.

Peptide Selector: Export Q-TOF Inclusion lists from results

This feature is only available if Agilent Q-TOF data has been selected.
You can now generate Agilent Q-TOF target lists based on search results. The target list can include peptides from proteins that were unidentified from a prior list of proteins, that contain only one peptide, or a combination of both.

In Peptide Selector, first make sure the Protein(s) to Select From list (bottom left of the form) displays the list of accession numbers that were used to generate the orignal target list. In the Save File Parameters section, mark the Generate inclusion or MRM list file check box, and select MassHunter Q-TOF MS/MS target list selected. Then mark the From results check box to view the Valid Results to Filter selections. Use the Select... button to select the data folders containing the results. Select either Unidentified, Single peptide, or Unidentifed + Single peptide as the proteins to select. Click Select to generate the target list.

Q-Exactive Support

The data extraction and MS/MS Search now support data acquired from a Thermo Q-Exactive and Thermo Q-Exactive Plus instruments. You must either have installed Xcalibur on the Spectrum Mill server, or have downloaded and installed the MSFileReader (you must select the 32-bit version to install). See the Spectrum Mill Installation Guide for details.

TMT Correction Factors and TMT6 changes

The Tool Belt now supports creating and applying correction factors for TMT in additon to iTRAQ. In addition, the TMT modification definition has been changed to account for a prominent unmatched singly charged ion of mass 230 (cleavage of bond between reagent and peptide, with charge staying on the reagent side), and a prominent set of ions at (precursor mass - (155 to 159) ) /(parent_charge -1), from cleavage of the amide bond in the reagent group, with charge-1 staying with the peptide side of the bond.

Important TMT6 Changes

TMT6 reagents with lot numbers that begin with MJ and later modified the 127 and 129 reporter ions masses by almost 50 ppm. This release supports these changes. The prior TMT6 definition has been moved to smconfig.misc.xml and renamed with "pre-MJ" appended. Summaries, including ratios, with older data will still report correctly. However, File Details and Spectrum Summary may indicated different scores and ion labels if the 127 and 129 ions are not matched. If you need to extract and search data that was prepared with TMT6 lots prior to "MJ", please contact your Agilent representative to request a special data extractor and search program that will support the older definitions.

Tool Belt: Enhanced quality metrics reporting

The Tool Belt tool "Report FDR and search statistics" is now "Report FDR and quality metrics", and has been enhanced to provide quality metrics in addition to the search statistics. The new metrics include sample handling, site localization, and isobaric label incorporation.

Protein Databases: Make non-redundant

The Protein Databases Utility page provides a new utility to remove redundant entries. When keeping/removing redundant entries (identical sequence), only the one nearest the top of the FASTA file is kept. However, for UniProt databases, SwissProt entries are preferentially kept over TrEMBL entries regardless of order in the FASTA file.

Build TIC changes

The Build TIC form has changed to provide more filtering options for data that has been extracted to mzXML. The "Neutral loss" selection has been removed, and the "Y axis" filtering selections have been enhanced to allow for building TIC's based on the following:

The data must be extracted with B.04.01 in order to fully support the above options, and only data which has been extracted to mzXML supports all of the above.

The "MS1 Precursor Intensity" corresponds to the former "Neutral loss" of "None". The "MS2 Phosphate Precursor-Neutral loss (% of base peak)" corresponds most closely (but not exactly) to the former "Neutral loss" of "H2PO4". The "MS2 Dissociated Intensity" corresponds most closely (but not exactly) to the former "Neutral loss" of "H2O". These are the only filtering selections that will work with pkl files.


New in B.04.00

The B.04.00 version of Agilent Spectrum Mill MS Proteomics Workbench included extensive new workflow automation capabilities, the ability to use Spectrum Mill identification results to create MRM lists for triple quadrupole instruments, as well as many other enhancements that increase the flexibility of the software and enable greater productivity. This section describes differences between the A.03.03 and the B.04.00 versions of the Spectrum Mill workbench.


Installation and Configuration

The following installation and configuration features are new in the Spectrum Mill workbench version B.04.00. For details, see the Installation Guide.

Operating system

Spectrum Mill B.04.00 is supported on the following operating systems:

Both 32-bit and 64-bit versions of the above operating systems are supported. Spectrum Mill programs run as 32-bit applications but are able to address up to 4Gb of memory per process when running on 64-bit systems.

Windows Server 2000 is no longer supported.

If you configure a new system, Agilent recommends that you install a 64-bit operating system and provide adequate memory (8 GB or more) and adequate disk space (12 TB for data).

Perl

ActiveState Perl 5.14.2.1402 is supplied with this release. Both 32-bit (x86) and 64-bit (x64) versions are provided, so select the one that is appropriate for your operating system. See the Installation Guide for details on installation of Perl and implications for configuration of IIS.

Java (JRE)

Java JRE 1.6.29u is included in this release. Both 32-bit (i586)) and 64-bit (x64) versions of JRE are provided. 

Be sure to install JRE on all client browsers, as well as the Spectrum Mill server. 

IIS

Install IIS before installing Spectrum Mill workbench.

See the Installation Guide for details on installation and configuration of IIS.

MSFileReader Support (for .raw files)

Prior versions of Spectrum Mill required that Xcalibur software be installed to process Thermo Scientific (.raw) data files. You no longer need Xcalibur, provided you  install  the Thermo Scientific MSFileReader, which you can download for free. Xcalibur is still supported.  Refer to the Installation Guide for details.


Process Automation Tools

New process automation tools let you perform an entire data analysis, from spectral extraction to final results summary, in a completely unattended way. This new capability includes:


Creation of MRM Lists

The new MRM Selector streamlines protein quantitation workflows that use identification results to create multiple reaction monitoring (MRM) lists for triple quadrupole instruments.


False Discovery Rate (FDR)

False discovery rate (FDR) is an independent measure of the likelihood that the results are incorrect. Calculation of FDR is important to ensure the validity of results, and is a requirement for publication in some journals. Spectrum Mill workbench now provides tools to calculate FDR in Autovalidation and report FDR's in the Tool Belt at the peptide and spectra level, and protein level. See "What's new" for autovalidation, and the Tool Belt, to learn more about these new options.


Data Extractors

Java is a U.S. trademark of Sun Microsystems, Inc.


MS/MS Search


MS/MS Autovalidation


Protein/Peptide Summary


Tool Belt



Peptide Selector


Protein List to Masses Utility

A new utility is available to calculate the masses and formulas for a set of specified peptides.


Protein Databases


Server Administration


What happened to Easy MS/MS Search?

Easy MS/MS Search has been replaced by new process automation tools that provide more power and flexibility.



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