MS-Expedite Quick Start Guide

 

This guide is aimed towards viewing and annotating spectra acquired on an ABI MALDI-TOF/TOF. MS-Expedite will open a variety of file formats such as Mascot generic formatted peaklists (*.mgf), ABI 4700/4800 binaries (*.t2d), generic mass/intensity pairs (*.txt), etc.

 

 

Getting the NRPP’s MS-Expedite:

 

  • Java 1.5 must be installed for MS-Expedite to run. Go to www.java.com to download.
  • To download/run MS-Expedite, go to www.proteomecommons.org and click on the link for Tools, which will go to the tools section of the website:

 

 

  • In the Tools section, click on Visualization Tools:

 

 

 

 

 

  • MS-Expedite will run using Java WebStart without the need to install it. This insures you are always accessing the most recent version.
  • A default config file will be downloaded the first time the application is run. User preferences will be saved to this file. If a new algorithm is implemented such as changing smoothing or peak detection, it may be necessary to go into the preferences and change some settings.

MS-Expedite Basics:

 

  • The viewer can open a variety of files:
    • ISB XML (.mzxml)
    • Sequest DTA (.dta)
    • MZDATA XML (.mzdata)
    • Applied Biosystems TOF/TOF (.T2D)
    • Mass/intensity pairs (*.txt)
    • Mascot generic format peaklist (.mgf)
    • NRPP Annotation (.anot)
    • .wiff
    • .tiff
  • Opening spectra:

1.      Click on the File…Open (or the Open button on the toolbar), browse to and select the file to open. Select MS or MS/MS when prompted.

2.      Use the folder browsing pane on the left. If not visible, select View….Files from the file menu. Repeat to hide the folder pane. If 4700 or 4800 instruments are available, MS-Expedite can be configured to connect to them directly.

 

To configure MS-Expedite to connect directly to a 4700/4800 database, open the config.ini file located under C:\Documents and Settings\Your logon\NRPP\MSViewer\

 

 

Add/edit the following parameters for each instrument. Instruments are numbered beginning with “0”. If you do not have an instrument, any lines beginning with “T2” should be commented out by preceding the entry with a “#” symbol.

  • T2_alias_0=ak080  – Friendly instrument name.
  • T2_ip_0=192.168.1.209  – IP address of the instrument
  • T2_port_0=1521  – Oracle port number
  • T2_show_spectrum_0=true  – MS-Expedite will display spectra in the browse pane
  • T2_show_peaks_0=false  – MS-Expedite will display peaklists in the browse pane.

 


·        MS-Expedite toolbar:

 

 

 

 

  • A tooltip near the cursor indicates the m/z at the location of the cursor, along with the intensity and S/N ratio of the peak at that m/z.

 

 

Viewing spectra

  • Smooth the spectrum by clicking on the filter button on the toolbar () and selecting Gaussian Filter.
  • Label the peaks by selecting “Peak Labeling” from the menu and clicking on “Label Peaks”.

 

 

 

 

 

 

  • The default parameters typically work well for TOF/TOF spectra. To change parameters, such as the signal to noise ratio for peak picking, click on the preferences button () on the toolbar.
  • Select the Peak Detection tab and adjust the parameters for MS or MS/MS as needed. The parameters shown are typical and should work in most cases.

 

 

 

  • To zoom in on a spectrum, click on the zoom button on the toolbar (), then left click and drag across the desired region of the spectrum. Right click to zoom out.

 

 

 

 

 

  • The viewer supports files that contain multiple spectra/peaklists, such as concatenated Mascot generic formatted peaklists (.mgf).
  • When an .mgf file containing concatenated peaklists is opened, arrows will appear on the toolbar which will allow cycling through the peaklists in the file.
  • Alternatively, a number can be entered into the box and MS-Expedite will jump to that entry.

 

 

 

 

 

 

Using the Annotation Feature to Assist in de novo Sequencing or Labeling of MS/MS Spectra:

 

  • Zoom to the region of interest.
  • Click on the annotation button () and select “Manual”.
  • Left click and drag across the peak of interest, anchoring the cursor on this peak.
  • A colored tooltip will indicate the mass difference between the anchor point and the cursor. This tooltip also displays the amino acid residue (and its mass) with the MW closest to the mass difference.

 

 

 

 

  1. As peaks are selected, the gaps are marked with the amino acid residue and the mass difference between the peaks. This allows for verification of the residue match.
  2. The cursor stays anchored at the most recently selected peak, allowing for sequencing across the spectrum.
  3. The spectrum will scroll if the cursor approaches the sides of the screen.
  4. Hit “escape” to leave the current annotation. At this point, a different peak can be selected to work with or the zoom feature can be used to choose a different portion of the spectrum.
  5. Select manual annotation to re-enter annotation mode. If the previous peak is still anchored, use the escape key and select a new peak.
  6. If a new peak is selected, the new annotation will appear on a different level than the first, allowing for multiple sequences to be annotated (overlapping peptides, b/y sequences).
  7. Use ctrl-Z to undo the previous annotation.
  8. Click on the annotation button and select “clear annotation” to remove all annotation.
  9. The spectrum can be saved along with the annotation (*.anot), or it can be saved as a *.tiff image file.

 

 

 

Adding User Defined PTMs or Amino Acids

 

The viewer by default only considers the 20 typical amino acids and their dipeptide combinations. To add user defined residues:

 

  1. Select PTM from the Edit menu. A tab will open up on the right side of the viewer window.

 

 

 

 

  1. Enter the name for the PTM or residue and it’s molecular weight.
  2. Click on the save button. This will save the entries to a configuration file.
  3. Only entries with the “Consider” box checked will be used in annotating spectra.

 

Editing an Existing Annotation

 

  1. If multiple possibilities (amino acid and dipeptides) exist for a given delta mass, the viewer by default inserts the amino acid residue.
  2. Any of the annotations can be edited to display either a dipeptide matching that mass, or a user defined annotation.
  3. From the annotation button, select “Edit” and right click on the annotation to edit which will bring up the Annotation Editor window.

 

 

 

 

  1. In the Annotation Editor window, all possibilities for that delta mass are listed.
  2. Select the desired entry, or type a user defined name in the box.

 

 


Using b/y ion pairs to find the complementary ion sequence:

 

  1. To view the b/y ion pairs corresponding to the annotated peaks, select View….YB Pair under the annotation button.
  2. Enter the mass of the peptide in the box. The corresponding b/y ion pairs for the annotated ions will be displayed.
  3. Use the complementary ions to annotate the corresponding sequence.