Agilent Technologies Spectrum Mill MS G2721AA User manual

Agilent Technologies
Agilent G2721AA Spectrum Mill
MS Proteomics Workbench
Quick Start Guide
A guide to the Spectrum Mill workbench
Use this reference for your first steps with the Spectrum Mill workbench.
What is the Spectrum Mill MS Proteomics Workbench?
The Spectrum Mill software is a collection of tools for high-
throughput processing of MS and MS/MS spectra to provide protein
and peptide identifications and relative quantitation. The
Spectrum Mill workbench can do in minutes what could take you
hours or days to do manually.
This software includes bioinformatics tools for:
•Spectral preprocessing for MS/MS and MS-only data, including
spectral quality filtering and precursor charge state assignment
for MS/MS spectra
•Protein database search, including searches for both MS/MS and
MS-only spectra
•Data review, validation, and comparison for large data sets,
including those from two-dimensional LC/MS/MS analyses and
differential expression studies
•De novo sequencing
•Identification of mutations, post-translational modifications,
and chemical modifications
•Differential expression quantitation
The Spectrum Mill workbench accepts Agilent data, as well as data
from other vendors’ mass spectrometers. The software executes on
a web server, and you access the program via a web browser
window.

2 Spectrum Mill Workbench Quick Start Guide
New features in Spectrum Mill workbench version A.03.03
The following are key changes to the user interface and functionality:
Support for more
instruments and
data types
Now supported:
•Agilent Q-TOF
•Electron transfer dissociation (ETD) data from Agilent ion trap
•MALDI-TOF-TOF data (via *.mgf file, requires license for Generic Data
Extractor)
•Thermo Fisher Scientific LTQ, LTQ FT, and LTQ Orbitrap, in various
dissociation modes (requires a license for Thermo Data Extractor, as well as
co-resident Thermo Fisher Scientific Xcalibur software that is equivalent to or
newer than the version you used to acquire the data)
Data Extractor Ability to disable similarity merging, to improve coverage and to detect more
low-level peptides for simple samples (for example, a single protein)
MS/MS Search Capability to search peptides that have a charge up to +7
New Maximum ambiguous precursor charge, which allows you to choose the
maximum charge state that you want MS/MS Search to consider when the Data
Extractor could not discern the charge state for the peptide
Capability to use protein pI to filter candidate proteins from the database
Improved scoring schemes, including Dynamic peak thresholding, which is a
scoring enhancement that enables identification of more low-abundance and
short-chain peptides
Explicit “mass gap” search that allows you to search for an unexpected or
unknown modification
Ability to automatically create a file of previously validated hits from the MS/MS
Search page, without using the Tool Belt page
MS/MS Autoval-
idation
Addition of +5 charge state to the autovalidation rules
Capability to perform autovalidation across data files in multiple subdirectories,
with all peptides contributing to the protein score
Addition of peptide pI as a validation criterion for samples where the pI is known
(for example, from offgel fractionation)

Spectrum Mill Workbench Quick Start Guide 3
Protein/Peptide
Summary
Spectrum Viewer support for c- and z-ions, which are prominent in ETD spectra
Retention time in peptide summaries
Fragmentation mode in peptide summaries, so you can ascertain the mode for
each spectrum (for example, CID versus ETD)
Enhanced protein grouping based upon shared peptides
Ability to align sequences of proteins within a protein group
Ability to sort and filter by peptide pI
New capability in the Spectrum Viewer to type your own sequence and have it add
to the list of available sequences that the software shows when you click the Rank
arrow buttons
Ability to sort peptides by the numerical position of their first amino acid in the
protein sequence
Capability to distinguish in results tables between true mass errors and mass
differences produced by peptide modifications
Support for more
amino acid modi-
fications
Support for Deamidated (N) and Deamidated (Q) as variable modifications. The
software displays the first by default; system administrators can add the second by
copying a definition from smconfig.misc.xml to smconfig.custom.xml.
Expanded iTRAQ support, to include data from the Generic Extractor and the
*.raw (LCQ/LTQ) Extractor. The latter uses merged MS3spectra.
Support for both original iTRAQ (which assumes complete labeling) and iTRAQ
Partial-mix, which allows you to check for incomplete labeling
Miscellaneous New Easy MS/MS Search form that simplifies use of the software and streamlines
the data processing steps
New Multiple Sequence Aligner utility to align sequences of proteins from a
database and highlight amino acid differences
Support for Web-based interaction with GeneSpring MS
Easier, faster input of mass lists into Manual PMF, with MH+/M selection that
allows you to input either protonated or neutral species (directly compatible with
output from a variety of instrument data systems)
Faster selection of data folders
Sherenga de novo sequencing support for the Agilent Q-TOF and ETD data from
the Agilent ion trap

4 Spectrum Mill Workbench Quick Start Guide
The ability to store iTRAQ correction factors for multiple iTRAQ batches and to
apply them to the iTRAQ calculations for specific data sets
Expanded capabilities for Peptide Selector
Updated instrument selection lists
The default Spectrum Mill font size is now “smaller”, so you may use the default
“medium” size in your browser and the Spectrum Mill software still uses “smaller.”

Spectrum Mill Workbench Quick Start Guide 5
Spectrum Mill manuals and online help
Scientists Familiarization Guide Follow step-by-step instructions to process example data.
Application Guide Learn details to use the software.
Online Help Consult the online help for in-depth information not given in the
Application Guide. To access help, click links on the home page, Help buttons on
Spectrum Mill forms, or blue dividing bars on Spectrum Mill forms.
Quick Reference Card After you are familiar with the software, consult this card
for an overview of the steps to process MS/MS data.
Thermo Ion Trap Data Extractor Quick Start Guide Get a quick overview of the
optional data extractor for Thermo Fisher Scientific ion trap data files.
Generic Data Extractor Quick Start Guide Get a quick overview of the optional
Generic Data Extractor for peak list files.
QSTAR Data Extractor Quick Start Guide Get a quick overview of the optional
Applied Biosystems/MDS Sciex QSTAR Data Extractor.
System
Administrators
Installation Guide Use this guide to install the software on the server.
Application Guide See the following chapters:
•Chapter9:System Administration
Get an overview to install databases and perform other system administration tasks.
• Chapter 10: Files Created during Spectrum Mill Data Processing
Refer to this chapter to troubleshoot data processing, to selectively remove
parts of the processing, or to decide which files to archive.
Online Help From any Help page, click links under For System Administrators:
•ProteinDatabases(link to millhtml\SM_instruct\faman.htm)
Learn details to install databases, create indices, and create subset databases.
• Server Administration (link to millhtml\SM_instruct\servadmn.htm)
Learn details to perform other system administration tasks.

6 Spectrum Mill Workbench Quick Start Guide
Setting up the Spectrum Mill server
See the Spectrum Mill MS Proteomics Workbench Installation Guide. If you wish
to update from a previous version of the Spectrum Mill workbench, see Chapter 2.
Note that the server name cannot have an underscore.
Setting up your client PC
Operating system Check that you have one of the following:
•Windows®2000 Server, SP4
•Windows Server 2003, SP1
•Windows XP, SP2
1Select Start >Run....
2In the Open box, type winver.
Browser Check that your web browser is Internet Explorer 6.0 (IE 6.0) or Internet Explorer
7.0 (IE 7.0).
1Open Internet Explorer.
2Select Help > About Internet Explorer.
Java Check that Internet Explorer on the client is configured for Sun JRE 1.5.x (also
called J2SE 5.0) or Java SE 6. You can get a compatible version of JRE from your
Spectrum Mill installation CD.
1With IE 6.0 or IE 7.0, select Tools > Internet Options....
2Click the Advanced tab.
3Scroll down to the Java (Sun) section to view the version.
Screen resolution If supported on your monitor, use the Control Panel to set screen resolution to
1280 x 1024, with True Color (32 bit).
1In Control Panel, double-click Display.
2Click the Settings tab.
3Adjust the settings if necessary.
NOTE At the time of release, Windows Vista is not yet supported. If you wish to use Vista, please
contact Agilent to check the support status of Vista.

Spectrum Mill Workbench Quick Start Guide 7
Stored pages Disable caching.
1With IE 6.0 or IE 7.0, select Tools > Internet Options....
2Click the General tab.
3Do one of the following:
•If you have IE 6.0, under Temporary internet files, click the Settings...
button. Then click Every visit to the page.
•If you have IE 7.0, under Browsing history, click the Settings... button. Then
click Every time I visit the webpage.
Cookies Enable cookies so you can save form settings.
1With IE 6.0 or IE 7.0, select Tools > Internet Options....
2Click the Privacy tab and set to Low, or click the Advanced button and enable
cookies.
3For more detailed instructions to enable cookies, search Internet Explorer help
for cookies.
4If the drop-down menus in the Spectrum Mill workbench appear empty, you
may need to add the Spectrum Mill server as a trusted site. Search Internet
Explorer help for trusted site.
Active scripting Enable JavaScript (Active Scripting).
1With IE 6.0 or IE 7.0, select Tools > Internet Options....
2Click the Security tab.
3Select Local Intranet.
4Click the Custom Level... button.
5In the Security Settings dialog, scroll down until you see the Scripting section.
6Under Active Scripting, click the Enable option.
Browser text size Especially for lower-resolution monitors (e.g., 1024 x 780), set a smaller text size to
avoid excessive scrolling.
1Do one of the following:
•With IE 6.0, select View > Text Size.
•With IE 7.0, select Page > Text Size.
2Set to Smaller.

8 Spectrum Mill Workbench Quick Start Guide
Running the Spectrum Mill workbench from the client
Open Internet Explorer and point to the URL for the Spectrum Mill server. This
has the general format server/millhome.htm. If you are not sure of the URL, ask your
system administrator.
Setting up Agilent TOF analyses for use with the Spectrum Mill
workbench
When you set up data acquisition parameters on the Agilent TOF, it is critical that
you establish analysis settings that are compatible with Spectrum Mill processing.
For details, see the chapter on processing MS-only data in the Spectrum Mill MS
Proteomics Workbench Application Guide.
Software Status Bulletin and software patches
A list of known problems for the Spectrum Mill workbench, along with possible
solutions, is found on the Agilent Web site.
1Go to http://www.agilent.com/chem/.
2On the left, click Technical Support.
3Under Downloads and Utilities, click Status Bulletins and Patches.
4On the Software Status Bulletins and Patches page, click Spectrum Mill.
5If you are prompted to log in, and you are registered on the Agilent Web site,
type your login name and password, then click Enter. If you are not registered,
click the link to register.
6On the Software Status Bulletins and Patches page, click Spectrum Mill.
7When prompted for your Spectrum Mill password, type specmill (all lower
case) into the text box, then click SUBMIT.
8View the Software Status Bulletin, or download a software update.

Spectrum Mill Workbench Quick Start Guide 9
Spectrum Mill modules
Tool type Module Function ApplicationGuide
Chapter(s)
Spectral
preprocessing
Data Extractor Extract spectra from data files. Processing depends
on data type and may include averaging,
centroiding, filtering by quality, assigning precursor
charge, and calculating spectral features.
1, 6
Interpretation MS/MS Search Search MS/MS spectra against databases. 1
Spectrum Matcher Match MS/MS spectra against other MS/MS
spectra.
1
de novo Sequencing Use Sherenga de novo sequencing to interpret
MS/MS spectra.
1, 3
PMF Search Search MS-only spectra against databases. 6
Result summary Protein/Peptide Summary Summarize, review, filter, and validate results from
MS/MS searches.
1, 2
Spectrum Summary Sort and classify MS/MS spectra based on spectral
characteristics.
1
de novo Summary Summarize and review de novo interpretations. 1, 3
PMF Summary Summarize and review results from MS-only
searches.
6

10 Spectrum Mill Workbench Quick Start Guide
Utilities Tool Belt Use tools to speed processing, examine parameters
and search statistics, stop processes, etc.
7
Protein Databases Create indices for databases so searches run faster,
create subset databases, etc.
9
Build TIC Visualize how much of your sample has been
interpreted. Locate spectra that indicate potential
peptide phosphorylation sites.
1
Peptide Selector Perform theoretical digestion and then select
peptides that meet certain criteria (e.g., likely to
produce high-quality MS/MS spectra).
8
Multiple Sequence Aligner Align amino acid sequences of proteins from a
database and highlight the amino acids that differ
8
MS Digest Perform theoretical enzymatic digestion and
calculate masses of peptides that result.
8
MS Edman Search text fields in protein databases, with or
without peptide mass filtering.
8
MS Product Calculate theoretical fragment ion masses from
peptides.
8
MS Comp Calculate amino acid composition for a peptide,
given peptide mass and immonium ions.
8
MS Isotope Calculate and view isotope patterns of peptides. 8
Tool type Module Function ApplicationGuide
Chapter(s)