Chapter
10
Fragmentation
Fragmenting peptides based on the Roepstorff notation or fragmentation by repeated cleavage of residues from either terminus.
The MS/MS fragmentation can be carried out on peptides selected either in the Peptide window (Chapter 9.4) or on highlighted sequences in a sequence window (Chapter 3.2). Once you have an ms/ms window you can enter a new peptide sequence or change the displayed sequence.
MS/MS fragmentation 10.1
The ms/ms fragmentation can be selected
1. From the main menu when a sequence window has the focus (Cleavage|MS/MS fragmentation). If part of a sequence has been highlighted, it will be shown in the toolbar and be used for fragmentation. Otherwise the whole sequence will be taken as the basis for fragmentation. If the sequence is longer than 50 residues you will be asked if you want to perform ms/ms on the entire sequence.
2. By selecting ms/ms (button or local menu) for a given selected peptide in the peptide window (‘Automatic cleavage’, Ch. 9.4). This displays the peptide in the toolbar of the MS/MS fragmentation window and the fragmentation below.
3. From the local pop-up menu of the mass search result window (Chapter 6.1).
The fragmentation pattern is shown using the Roepstorff notation [P. Roepstorff & J. Fohlman, Biomed. Mass Spectrom. 11, 601 (1984)]. Notice that the Y ions are shown as their double prime fragments (this can be changed in the ms/ms setup, se below).
The title bar of the window shows the elemental composition of the peptide under investigation.
Clicking the label of each column toggles through the three possible prime states (protons added to the base fragment): none, single and double prime.
After the list of fragment ions, the expected immonium ions are listed.
The toolbar at the top of the window contains from left to right:
Ø The peptide being fragmented is shown in the edit line. This line can be edited, and the table is updated whenever a change occurs. Note: if the line contains post-translationally modified residues, the edit line is grayed and cannot be edited.
Ø A panel showing the mass of the intact peptide with a button that toggles between average (blue) and monoisotopic masses (red) (please note that this button determines both the intact mass and the table values)
Ø Setup button. Set the chemical composition and number of fragment ions. See below for more details.
Ø Ms/ms graph button to show a graphical representation of the ms/ms fragmentation. This window is similar to the Ms graph in Chapter 6.1 except that each group of sequence ions can be turned on and off.
Ø
The frames button. When activated, a size-able frame is displayed in
the right hand part of the window listing all fragments from the main table in
a numerically sorted list. The two
tables are linked, so when a value is selected in the sorted frame list, the
corresponding mass in the main table will be highlighted in yellow. The top
part of the frame is an edit box where you may enter a mass to search for. As
you enter the mass, the sorted mass list below will then scroll to the closest
fit.
Ø The button toggles the multiply charged fragment ions on and off.
Ø The drop-down selection box determines the maximum number of charges that will be displayed when the ‘++’ button is activated.
Ø Modification state of the peptide terminals is listed in the right hand panel. You can change the terminals either by double clicking on the panel or by right clicking in the window and choose ‘Set terminals’ from the local menu.
Masses are by default only shown with two decimals, but in the local menu (right-click the mouse) you can toggle 4-decimal mass display on and off.
Printing is done from the local menu, main toolbar or main menu (File|Print). Copy to clipboard is done in a similar manner (menu command: Edit|Copy to clipboard or <Ctrl+C>). Through the pop-up menu you can further select to copy an individual column (e.g. Copy special | C ions).
Posttranslational modifications
You can modify individual residues in the peptide under fragmentation by double clicking on the relevant residue. In the resulting ‘Insert modification’ dialog box, you either enter the modification name and composition or you select the relevant modification from a modification file. For more details see ‘Amino acid modifications’, Chapter 3.6.
When a residue is modified, the sequence edit line in the toolbar will be grayed, and it will not be possible to edit the sequence. Modifications will be displayed in an information line at the bottom of the display.
i Info: If your sequence have modified residues when selected (e.g. from the sequence window) you will get the message “Sequence contains modifications. Edit line not active” which means that you cannot edit the sequence in the edit line (grayed), but the modification will be incorporated in the mass calculations. You should beware of possible fragmentation of the side chain (e.g. loss of the modification).
MS/MS setup
Pressing the setup button in the MS/MS window enables you to edit the ms/ms table, mass of fragment, fragment displayed, and default prime state.
The name column gives the name of the fragment (1 character). In the composition column you enter the composition, the protons columns are the numbers of primes (0, 1 or 2). If the ‘OK’ column is checked the corresponding fragment will be displayed.
References:
P. Roepstorff & J. Fohlman, Biomed. Mass Spectrom., 11, 601 (1984)
Ladder sequencing 10.2
Ladder sequencing shows the sequence and masses of the selected (or manually edited) sequence after sequential removal of residues from either the N- or the C-terminal end. The ladder sequence can show a maximum of 100 residues.
The function is selected in a manner similar to ms/ms fragmentation.
Ø Ladder sequencing can be selected when a sequence window is open by selecting Cleavage|Ladder sequence. If part of the sequence is highlighted (Chapter 3.2), the selected sequence will be displayed in a list with successive removal of a residue from the C-terminus followed by a list of peptides with successive removal of residues from the N-terminus. If no sequence is selected, the display will initially be clear and you will have to enter a sequence manually.
Ø When you have a peptide list (from automatic fragmentation) you select a peptide (highlight) and choose ‘Ladder sequencing’ from the main or the pop-up menu.
The ladder sequence can be edited in the edit box of the local toolbar. Changes are immediately shown on screen.
The first line of the list tells you the mass type (ave./mono.) and charge state of the peptide masses. Then follows the N-terminal sequence ladder followed by the C-terminal ladder.
Toolbar
From left to right the toolbar shows:
Edit box for the base sequence. The ladder sequences are updated whenever changes are made to the base sequence.
Mass of base sequence.
Av./Mo. Toggles between average and monoisotopic masses.
M / MH+ / MH++ / M-H Charge state button. Toggles between no charge, singly charged positive ions, doubly charged positive ions and singly charged negative ions.
.4/.2 toggles between displaying the mass using four and two decimals.
Setup: Enables you to specify whether
the N- and/or C-terminal modification of the parent window should be used when
generating each step of the ladder sequence.
Exit: Close the ladder sequence window.
Printing is selected either from the main toolbar (<Ctrl+P>) or the pop-up menu.
Pop-up menu
The pop-up menu contains, in addition to the commands in the toolbar, the option to set the charge state. The available options are: No charge (default), singly charged (MH+), doubly charged (M2H++) and negative singly charged (MH-). In addition you can select Average/Monoisotopic, 2/4 decimals, Setup and Printing.
i Like for the ms/ms fragmentation above, modified residues in your sequence you will give you the message “Sequence contains modifications. Edit line not active”. This means that you cannot edit the sequence in the edit line (grayed), but the modification will be incorporated in the mass calculations.