Chapter 7
Working with Spot Annotations
Delta2D allows you to place annotations anywhere on a gel image, to annotate spots and to control spot
picking. These annotations, we call them labels, can be independently from spot locations; but you can
also let them snap automatically to the target spot.
Labels can be created individually or automatically. They can be transferred from one gel to the other
with a single click. Delta2D will place them at the corresponding position automatically, following the
defined warpings. Normally labels are collected in a proteome map, but it is also possible to handle
labels on single gels.
You can change label formats according to your preferences. And, for advanced usage, label data and
formats are saved in XML files that can easily be processed by other applications.
| Figure 7.1: | A Dual Channel Image with Labels on the Proteome Map. |
|
7.1 Creating a Label
You can place labels on either of the gel images. Usually, labels for both gel images will be displayed
together in the gel image view, but with a different look.
To start working with labels, select the label tool in the top-left part of the gel image view (see
figure 7.2).
| Figure 7.2: | Delta2D tool panel with activated label tool. |
|
Now click on any point in the gel: a new label will be created where you have clicked (see
figure 7.3).
Newly created labels are placed by default on the master image. If you want to create a label on the
sample image, you can either move it there (see below), or create it directly on the sample image by
holding the Shift-key pressed while clicking on the target place for the label. Now it will be created on
the sample image.
Changing the Text of a Label
To change the text of the label, click once inside the label and
start typing. Press Enter to stop editing. Pressing Escape will discard the changes you have
made.
Moving a Label
You can move a label's text around by dragging it with the mouse. Observe how the
line is placed automatically on the corners or in the middle of the label's border. When the label is
sufficiently near the target, the line will be hidden in order to make the display simpler. When you want
to change the label's target, you can drag the line to move the whole label to the desired
place.
Label Snap
You can adjust a label's target to the nearest spot maximum. Go to the Labeling tab in
the preferences dialog and check the Label snap to spot check box. This makes labels
snap to spot maxima when they are moved around or created. Hold down the Control key
to switch this off temporarily. Alternatively, you can adjust a label by double clicking on
its arrow. Right-click on a label and choose Adjust to make the label point to the spot's
maximum.
Greek Symbols in Labels
You can use greek letters and symbols in labels of spots. Simply press Ctrl
+G while editing a label to switch between normal and greek mode. The greek mode is indicated by a
greek symbol below the label you are editing (Figure 7.4).
| Figure 7.4: | The greek mode |
|
7.2 Labels and Spots
In Delta2D, labels are not bound to spots, i.e. you are free to add a label to anywhere on the gel image
before or after spot detection. They won't be altered or removed by (re-)detection of spots. However, if
there are labels pointing to spots, which are detected as spots later, Delta2D will assign them to
the respective detected spots automatically. Labels are organized separately for each gel,
so a label can only be assigned to a spot that is on the same image. When a label's arrow
points inside a spot, it will show up in the corresponding label column of the quantitation
table.
The assignment is always kept up to date, for example, when you drag a label into another
spot, it will be shown in that spots label column. When you import a new set of labels,
Delta2D will assign them automatically as well. It is also possible to have more than one label
for a spot, in that case the table will show a drop-down box with all the label texts. Only
one of the labels will be visible; to select another label, you can double click on the table
cell.
Creating and editing labels in the quantitation table
Instead of making a new label by clicking on the
gel image view, you may also create it by labeling a spot in the table. Just double click on the spot's
label field and start typing the name. Press Enter to finish editing. The label will be automatically
placed such that it points to the spots center. You can change its name by double clicking on the table
cell.
Sorting and Searching for Labels
The table can be sorted according to label name. Just click on the
column header to activate sorting. Spots without a label are sorted to the bottom of the table. As with
the other columns, clicking again will reverse the sort order.
Use Search 
Label to search for a label. The first matching entry will be selected. You may search for
any part of the label's text, e.g. searching for "Cit" will find "CitZ" or "CitG", whichever comes
first.
________________________________________________________________________________
-
Note:
- Please remember that the quantitation table is a table of spots. Thus, only labels
associated with spots are shown in the tables and, of course, only those labels can be
searched for.
7.3 Working with Labels
Labels can be operated individually or collectively by using the respective menus:
All Labels on a Gel
The Labels menu in the main menu of the Dual View gathers all things you can do with labels as a
whole: load and save label sets, create and move them around, and aquire information from selected
protein data bases. Table 7.1 explains the menu in detail.
 Delete | Delete labels from the selected gel image (master,
sample, or both) |
 Import | Add labels from a file to the current set of labels on
the master or sample gel. If the label file contains
formatting information, you will be asked whether it
should replace the present formatting. |
 Export | Export labels to a file. Formatting information will
always be saved together with the label data. |
 Move | Move all labels from one gel to the other. Label
positions will be adapted according to the match
map. |
 Copy | Copy all labels from one gel to the other. Label
positions will be adapted according to the match
map. |
 Swap | Swap label sets between master and sample gel.
Label positions will be adapted according to the
match map. |
| Label Selected Spots with Spot
IDs | Create Labels on selected spots containing their ID. |
| Label Selected Spots with
Numbers | Create Labels
on selected spots containing consecutive numbers. If
you need a prefix in the numbered labels, define it in
Preferences Labels |
| Label Unlabeled Spots with Spot
IDs | All spots without any label obtain a label containing
their ID. |
| Label Unlabeled Spots with
Numbers | Create Labels on all unlabeled spots containing
consecutive numbers. If you need a prefix in the
numbered labels, define it in Preferences Labels |
| Translate Label Names | Lets you batch change all Labelnames by providing
a list with the current names in one column and the
replacement names in another. |
| Formats. . . | Edit label formats. |
 Delete scout1 data | Delete data of a specific scout from all spots |
 Fetch scout1 data | Fetch data with a specific scout only for those labels
not containing this set of data |
| Refetch scout1 data | Fetch data with a specific scout for all labels and
override this specific data if already present |
| Table 7.1: | The Labels menu. |
|
|
Individual Labels
To manipulate a label individually, just right-click on it to get its context menu (see
figure 7.5).
| Figure 7.5: | The context menu for a label |
|
Deleting a Label
Select Delete from a label's context menu to delete it.
Adjusting a Label
The menu item Adjust works exactly like label snap (see 7.1). Its function is useful,
if you switched off Label snap because you don't want to use it generally, but want to use it in single
situations.
Moving a Label to Another Image
In the context menu you see whether the label is on the master or on
the sample image. Newly created labels are always placed on the master image. Select Move to
sample to move a label to the sample gel. The label's format will usually change when you move it to
another gel.
The option Copy to sample creates an identical label on the corresponding point of the sample image,
but leaves the label remaining on the master image. Here again, the label's format will adapt to the
sample's label formats.
When both gel images are connected by a match map, the label will be moved according to that
match map. Say your label is placed on the master image and you have loaded a match map for master
and sample image. You then place the label inside a master spot. When you move the label to the
sample gel, Delta2D will move it to the point that corresponds to the master spot. This behavior allows
you to collect complete sets of labels from many different gel images which is especially useful when
you want to produce a proteome map containing protein identifications. See section 7.5 for
details.
Working with Scout Data
Scout1
data of each label is easily accessed from its context menu. Use Edit scout data to view and edit the
scout data attached to the selected label. A dialog will open (figure 7.6), showing the data attached to
this label sorted by scouts. The data is fully editable.
| Figure 7.6: | The scout data attached to one label |
|
To quickly delete one scout's complete data from a label, use Delete scout data and the
respective scout from its context menu. To delete one scout's data from all labels, please use the menu
item Labels Delete scout data from the Gel Image Pair View.
Information at a Glance
At the bottom, each context menu shows basic information concerning to the
label it belongs to:
-
Position
- The position of the label is shown in the format Gelname: x-coordinate/y-coordinate.
-
Functional Category
- If available, the functional category of this protein is shown, labeled
with the color of the scout who retrieved this information.
7.4 Formatting Labels
Depending on the color scheme and the gel contrast sometimes it is necessary to adapt the label format
to ensure optimal visibility. Furthermore dynamic label coloring (section 7.4) is an interesting tool for
the visualisation of protein or spot properties.
The appearance of labels can be changed in various ways. You may define different formats
depending on whether the label is on the master or sample gel, and whether it is displayed on a single
gel image or on a dual channel image. For each of these cases you define a separate label format, using
the formatting dialog (see figure 7.7). And of course, you can also save and load appearance
configurations for labels.
| Figure 7.7: | Editing label formats |
|
The Label Formats dialog can be invoked using the menu entry Labels Formats. . . or by using the
keyboard shortcut Ctrl-F.
The Label Formats Dialog
Managing the Label Formats Dialog
Individual Appearance on each view
To make control of individual appearance of labels in each view easier, the Label Formats
Dialog offers an overview of your settings in the left side of the window. It shows four small
previews:
-
master
- labels on single view and dual view
-
sample
- labels on single view and dual view
Select one of the four small previews to see and adjust the label format settings for this specific
view.
________________________________________________________________________________
-
Note:
- Please note: if you want to define an identical format for every view, you have to make
sure to set it in any single view.
Saving Label Data
Note that whenever you save label data, the current label formats will be saved together with the
data.
Specifying Label Formats
The Elements of a Label
A label consists of two main objects: the label itself and the arrow, indicating to which point on the
image the label points. Both of them can be designed in any detail, such as label text, background,
arrowhead, and -line. The components are mostly self-explanatory, and are introduced briefly
below.
Arrow
-
Head Color
- The color used to fill the head of the label's arrow.
-
Show Head
- A check box next to the above button, indicating whether or not heads should
appear on arrows.
-
Head Fill
- A check box indicating whether the arrow head should be filled, or appear
transparent.
-
Line Color
- The color used for displaying the line portion of an arrow.
-
Show line
- A check box next to the above button, indicating whether or not the line portion of
an arrow should be displayed.
-
Line width
- The width of an arrow's line.
Label
-
Border Color
- The color to use for displaying the outline of the label itself.
-
Show Border
- A check box next to the above button, indicating whether or not the border of a
label should be displayed.
-
Label border width
- The width of a label's border.
-
Background Color
- The color used for filling the background of the label itself.
-
Show Background
- A check box next to the above button, indicating whether the background
of a label should be filled, or whether it should appear transparent.
-
Text Color
- The color used to display the label text itself.
-
Font
- The font used to display the label text itself.
Coloring Labels
There are three basic possibilities to define the appearance for the elements of labels. Click on the
drop down button next to an element you want to recolor and choose between:
Color Click on one of the colors in the directly visible palette to quickly allocate a color to the
aspired element. If this small preselection of colors do not suffice your needs, click on the button 
colors
to have a full featured color chooser.
Automatic Note that several options involving choice of display colors provide an Automatic option.
When the display color for a given component is set to Automatic, the color will be derived from the
spot color for the corresponding gel. For example, if the text color for a label in the master
image is set to Automatic, text for the label will be displayed in the same color as spots
appearing only in the master gel (see Section 5.13 for information on configuring these
colors).
Scouts Dynamic coloring opens up an additional benefit from labels: use them as indicators for e.g.
the isoelectric point or the molecular weight of identified spots, as retrieved by scouts. (For more about
scouts please refer to section 7.7.) Thus you can see at a glance the distribution and also outliers in the
selected property over the complete gel image. (fig. 7.8)
| Figure 7.8: | Labels colored according to isoelectric point, based on Scout data |
|
If you set the color control to Scouts, a new dialog will open (fig. 7.9).
| Figure 7.9: | Adjust details for scout based color coding of label elements. |
|
This dialog lets you configure the details:
-
Scout
- Select the scout, the data of which will be used.
-
Property
- Which data of this scout will be used?
-
Gradients
- Choose a default color gradient to be applied to the range of values. You can define
your own color scheme by clicking on the button
and rename it with 
.
-
Normalized/Absolute Values
- Switch from normalized values, ranging between 0 and 1,
to absolute values, ranging between the smallest and the highest value of the selected
property. This is useful for determining a certain color for an exact value.
-
Slider
- Move the slider to a position corresponding an aspired value or use the
-
Position
- field to type it in directly. Now use the
-
Color Picker
- to set the color for this value.
You can assign as many colors to certain values as you want, there's no limit.
Saving Label Formats
To save the current label format configuration, simply click on the Save. . .
button and supply a file name under which to save the current configuration.
Loading Label Formats
To load a previously saved label format configuration, simply click
on the Load. . . button and select a file containing information about a previously saved
configuration.
7.5 Creating and Using a Proteome Map with Spot Identifications
Labels can be used to record spot identifications. You can do this by e.g. creating one union fused
image per project and work group. In this setting, spots are identified on gels using, for example,
peptide mass fingerprinting. Identifications are then transferred to the proteome map. Later on you can
use the proteome map to identify protein spots by visual comparison.
Just as Delta2D helps you to overlay corresponding spots in the images, it may also transfer labels
from a spot on one gel to the corresponding spot on another gel. Delta2D does this in a very reliable and
efficient way, using the same match map that is used to generate dual channel images. Thus you have
complete control over the accuracy.
To add identifications to the proteome map, follow this procedure:
-
Identify and label spots on sample gel
- Create a label for every identified spot on the
sample gel. Make sure that labels point into the centers of the spots.
-
Load proteome map
- Load the proteome map together with the collection of labels for spots
you already have identified. Sometimes it can be useful to integrate your proteome map
temporally into the current project. To exclude it from statistical analysis, change the
respective setting in the quantitation table properties.
-
Warp sample to proteome map
- Create a match map from the sample to the proteome map.
You may wish to hide labels for this step; use the Overlays rollup to do so.
-
Warp the sample gel exactly
- Label positions on the sample gel will be changed according
to the match map.
-
Copy labels from sample to master
- Use Labels Copy your gel image names to
copy the sample labels onto the proteome map. Effectively, you have now added the new
identifications to your proteome map.
-
Export the proteome map labels
- Now you can save the proteome map labels (e.g. for
backup or exchange purposes) using Labels Export Master
A similar procedure can be used to transfer labels from the master gel to a new sample gel. This may
avoid duplicate identifications, and it is much quicker and more reliable than doing the same thing by
hand.2
Suppose you have a sample gel where you have selected interesting spots, e.g. by looking at the dual
channel image. To transfer spot identifications for these spots from the proteome map to the sample gel,
follow these steps:
-
Load proteome map
- Load the proteome map together with the collection of labels for spots
you have already identified.
-
Warp sample to proteome map
- Create a match map from the sample to the proteome map.
You may wish to hide labels for this step; use the Overlays rollup to do so.
-
Copy selected labels to the sample gel
- Right-click on a proteome map label to bring up
its context menu. Select Copy to your gel image's name to move it to the sample gel.
Repeat this for all labels that you want to add to the sample gel.
-
Save sample labels
- You can use Labels Export Sample to save the sample labels.
The result is a label file that contains identifications for interesting spots on the sample
gel. You can see this by loading the sample alone, together with its newly created label
file.
7.6 Support for Protein Identification by Mass Spectrometry
We have added a number of features that make the data flow from gel images to mass spectrometry and
back to gel images more efficient. Automated labeling of spots lets you create labels for spots that you
selected in the gel image views or in the quantitation table.
Automatically Creating Labels in the Dual View
You can choose to label selected spots using spot-IDs or using consecutive numbers. In the screenshot
7.10, we numbered selected spots with an additional prefix, making labels Spot 01, Spot 02, Spot 03
etc. The numbering can be controlled by the options in the Labeling tab of the Preferences dialog (see
sec.9.5). Using automatic numbering helps to keep pick lists and protein identification results
organized.
| Figure 7.10: | Label selected spots with numbers |
|
Open the Gel Image Pair View, select the spots you want to label and choose from the menu
Labels Label Selected Spots with Spot IDs and choose the gel image you want to create labels
on. Even easier is labelling all unlabeled spots: just click on Labels Label unlabeled Spots with
Spot IDs in the menu. To create labels with ascending numbers select the respetive menu item for
either only selected or all unlabeled spots. You can determine a prefix being added in front of any
number when creating numbered labels: Open the Preferences dialog and switch to the Labeling tab.
Type in any string you want to be prepended to the numbers in the field Prefix for Numbered
Labels
Automatically Create Labels in the Quantitation Tables
Automatic labeling is also possible
from within the Quantitation Table: select the matchings or spots in any table, switch to
the single table of the gel image you want to create the labels on and select the menu item
Labels Label Selected Spots with ..., resp. Labels Label Unlabeled Spots with
....
________________________________________________________________________________
-
Note:
- Automatic Labelling works in single gel image tables only.
Automatically Replace Labels with Names of Identified Proteins
Let us show how to make use
of this in the context of protein identification. Say you have identified a set of interesting
spots (e.g. using the expression ratio) and labeled them with consecutive numbers as in the
image above. These labels are then used to create a pick list, similar to the one you see
here.
| Figure 7.11: | Pick list made from labeled spots |
|
Picked spots are processed in the usual way (digestion, mass spec, and database search). The protein
identification results usually come in the form of a table with label names and corresponding protein
names, like this:
| Figure 7.12: | Label names and corresponding protein names in a spreadsheet |
|
You can use this table to automatically rename the labels Spot 01, Spot 02 etc. to show the names of
the identified proteins. First, save the table as a CSV file. When opened in a text editor, the file should
look similar to this:
"Spot 07","Hag"
"Spot 08","Hag"
"Spot 04","Eno"
"Spot 10",""
"Spot 05","CitC"
"Spot 03","EF-Tu"
"Spot 02",""
"Spot 01","GlnA"
"Spot 06","Hag"
|
Now, in Delta2D, go to the Labels menu and choose Translate Label Names. This will open the
Translate Labels dialog (7.13):
| Figure 7.13: | Translate Labels Dialog |
|
Press the Load button and select the CSV file you saved earlier. The dialog will show a preview with
the original and the translated label names. Figure 7.14 shows the gel image from above with translated
labels.
| Figure 7.14: | Translated Labels |
|
_________________________________________________________________________________________________________________________________________________________
-
Note:
- Note that you can remove labels for which you have no translation (i.e. where protein
identification failed). Note furthermore that the old label names will not be available
anymore, so you should export them to a file if you want to save them.
7.7 Scouts: Finding Information in Web Resources
Delta2D's scouts are little software programs that go out to web resources such as GenBank or KEGG
and come back with useful information about a protein on a gel. Scout data can be protein properties
such as isoelectric point or molecular weight, annotations such as pathway information, sequences, and
much more. The information that was retrieved by scouts is attached to labels. The data is organized
into "aspects" i.e. groups of related data about a protein, such as the biochemistry aspect containing
isoelectric point and molecular weight, or the GenBank aspect containing sequences and accession
numbers. The aspects data are saved into the gel pool so they do not need to be retrieved from the web
again.
|
| | Scout | Data |
|
| | GenBank | Protein sequence, accession number etc. from NCBI GenBank Isoelectric
point, molecular weight, and amino acid statistics are computed from the
sequence using the EMBOSS toolkit. |
|
| | KEGG | Enzyme pathways from KEGG, the Kyoto Encyclopedia of Genes and
Genomes. |
|
| | GenoList | Gene and protein information from some GenoList databases maintained at the
Institut Pasteur:
-
SubtiList
- Bacillus subtilis strain 168
-
TubercuList
- Mycobacterium tuberculosis strain H37Rv
-
SagaList
- Streptococcus agalactiae strain NEM316
-
PhotoList
- Photorhabdus luminescens strain TT01
-
CandidaDB
- Candida albicans strain SC5314
|
|
| | AureoList | Gene and Protein information from the AureoList database maintained at
the Institut Pasteur. |
|
| | Physicochemical
properties | Molecular weight, isoelectric point etc., notes. All data is entered by the
user. |
|
| | |
| Table 7.2: | Scouts and the data they access |
|
_________________________________________________________________________________________________________________________________________________________
-
Note:
- Scouts go out to public web sites when retrieving data. If you want or need scouts that
use in-house resources instead please don't hesitate to contact DECODON's technical
support. We always welcome suggestions for new scouts that should be included with
Delta2D.
Accessing Scout Data
Scout data can be accessed by right-clicking on a label. The bottom of the
context menu shows excerpts from scout data, one line per scout. Use the Edit Scout data menu
item to see and edit the data. You can delete the data using the "Delete Scout Data" menu
item.
Using the GenBank Scout
Open the scout by selecting Edit scout Data > GenBank from a label's
context menu.. Enter the protein name and the organism name, then press the Process button. The scout
will access GenBank and retrieve one or more entries. You can double-click on an entry to open
the corresponding web page. You can now select one of the entries and press the button.
This will send the selected sequence to a server at DECODON where isoelectric point and
molecular weight are computed from the sequence (the actual computation is carried out by the
EMBOSS toolkit). The values are then saved, along with more statistics on the amino acid
composition.
Using the KEGG Scout
In the KEGG scout, you can enter the enzyme code (EC number) of a protein
and press the Get Pathways button to retrieve pathways that are using this enzyme. Double click on an
entry to go directly to a web page for the pathway.
Using the GenoList Scouts
GenoList is a collection of bacterial genome databases for
microorganisms such as Mycobacterium tuberculosis or Bacillus subtilis. The protein name will be
taken from the label name. Choose the organism database on the right hand side and press Get
Data. You can fetch Genolist data for all labels on an image by choosing Labels/Fetch Scout
Data/GenoList data in the dual view. Delta2D will fetch data from the last Genolist database you
selected.
Using the AureoList Scout
The AureoList scout works just like the GenoList scout, except that you
have to select which of the Staphylococcus aureus strains N315 and Mu50 you want to
use.
Using the Physicochemical Properties Scout
This scout does not use any web resources but relies on
data input by the user.
