Resources‎ > ‎Microarray‎ > ‎Software‎ > ‎

Protein array profiler

Protein array is used to analyze protein expressions by screening simultaneously several protein-molecule interactions such as protein-protein and protein-DNA interactions. In most cases, the detection of interactions leads to an image containing numerous lines of spots that will be analyzed by comparing tables of intensity values. To describe the observed different patterns of expression, users generally show histograms with the original associated images [1]. The “Protein Array Analyzer” gives a friendly way to exploit this type of analysis, thus allowing quantification, image modeling and comparative analysis of patterns.

The Protein Array Analyzer, which was programmed in ImageJ’s macro language, is an extention of the Dot Blot Analyzer, [2], [3] a graphically interfaced tool that greatly simplifying analysis of dot arrays.
Manuals
The analysis of a set of protein array images must be performed in two steps. The first step consists in analysing every single array, and the second one, into building master tables of measured values and the associated master modelled images grouping the data of the single analysis. It is then possible to input some internal references to normalize the tables and visual representations. A set of demo images and a table are provided through the internet resources to test the program (see the "On Line Documentation & Demo" below).
  • "Array Analysis Menu"

    Regroups analysis functions of single protein array images:
    • "Array Analysis" displays the graphical interface required to analyse an opened protein array image.
    • "Save Results Documents" saves every document concerning one analysis (model, tabs, images) into a folder at the user choiced master repertory.
    • "Save Results Documents at Image Location" saves every document concerning one analysis (model, tabs, images) into a folder placed at the protein array image repertory.
    • "Close Results Documents" closes every document concerning one analysis (models, tabs, images).
  • "Group Pattern Menu"

    Regroups tabs and analysed images management functions:
    • "Masterize from Analysis Repertories" allows to user to choose a repertory containing some protein array analysis folders which will be summarized into master table and modelled images.
    • "Import Master Table" imports a user selected, previously saved master table, and builds the corresponding master modelled image.
    • "Draw "Master - Initial"" draws the master modelled image corresponding to an opened master table, using the maximum value of each array as normalization (it corresponds to the initial aspect returned by the "Array Analysis" item).
    • "Draw "Master - Normalized"" calculates the normalized master table and draws the master modelled image corresponding, from an opened master table, using the current normalization options.
    • "Set Internal Control and References" opens a dialog box to set up to 4 controls and 4 references. When more than one control or reference are choosen, the mean value is used for normalization.
    • "Master Options Settings" menu:
      • "Number of columns for the master images" sets the number of columns in which are arranged the modelled protein arrays. The initial setting optimizes this value to the size of the screen and the protein array number.
    • "Normalization method" allows to choose between 3 methods of normalization:
    • "Normalize according to the min/max of the master".
    • "References used as global normalization of the master": normalizes using specified spots in the master choosen by the "Set Internal Control and References" menu. Specified spots are used for all the master values. Normalized dots display is optimized in the color scale according to the min/max value of the normalized master.
    • "References used for normalization of each array of the master": normalizes using specified spots in the master choosen by the "Set Internal Control and References" menu. Selected spots are used in each individual modelled protein array. Normalized dots display is optimized in the color scale according to the min/max value of the normalized master.
    • "Show master with max array normalization (initial representation)" activates-deactivates the systematic display of a master image presenting the protein array models in their initial aspects (individual normalization according the max value of each array).
    • "Show dots limits on master" activates-deactivates the automatic entourage of spots by a grey circle.
    • "Show internal references on master" activates-deactivates the labelling of the choosen internal control and references in cyan (control(s)) and green (reference(s)).
    • "Save Master Documents" saves every document concerning one analysis (master tables and images) into a folder at the user choiced repertory.
    • "Save Master Documents at Parent Folder Location" saves every document concerning one analysis (master tables and images) into a folder placed at the parent repertory of the masterized protein array analysis.
    • "Close Master Documents" closes all documents concerning one analysis (master tables and images).
  • "Abort Process"

    Click on this ImageJ tool bar icon to cancel demo downloading or erase the palette’s cursor.
  • "On Line Documentation & Demo Menu"

    On line ressources: documentation, demo images of protein arrays and dot blot from different sizes and a master table for training (see "Tutorial" section).
  • "About"

    Click on this ImageJ tool bar icon to get a short off line documentation.
  • "Version and Update Infos"

    Click on this ImageJ tool bar icon to look for new versions.

Tutorials

  • Get the demo images:

    This section shows how to analyse a set of protein array, using the demo images available online for training.
    • Download the demo Images by the "On Line Documentation & Demo Menu" -> "Download/Open a Set of 6 Demo Protein Array Images" menu.The tool downloads and opens 6 images stored on the hard disk at the ".../ImageJ-64/Downloaded Demo Images/ProteinArrayDemo" repertory.
  • Step 1 - single image analysis:

    • Choose one of the 6 images, and start an analysis using the "Array Analysis Menu" -> "Array Analysis" function.This action proposes a method of background subtraction (keep default settings), and builds a graphical interface for the dot matrix analysis.
    • Optimize the visualization by activating some options available from the graphical interface (as examples see the white oval marks on the figure below). For more details on the graphical interface functions, see this web page.Above: from left to right, the selected functions perform an autosetting of the cursor’s diameter by object analysis, a strech of the preview histogram, and finally, the third one applies the "Fire" Look Up Table (LUT).
    • Set the three required dots forming angle (hight-left, hight-right and low-left) using the three green cross hair icones (circled in white in the figure below). To set a mark, click on a cross hair icone and drag the cursor keeping clicked. Marks can be adjusted, by re-selecting the appropriate cross hair symbol (red cross hair: mark to set, green cross hair: set mark, yellow cross hair: mark in setting).Above: the hight-right mark is set, the hight-left is in adjusting mode. Once the third marks is set, a grid is drawed, and the matrix of selections is measured automatically as a table of values like shown below. It may be necessary to change size of the cursor (use +/-) if the "auto-size" is not compatible with the number of lines and columns. Click on "Model" area (white marked) to get a modelled image of the array.End the analysis by clicking on the area "End Quit", save the result documents at the image location, and close the documents (see screen shots below).The analyzer records every document concerning the analysis in a folder (named with the suffix "Results Documents") placed in the parent repertory of the original image. The tab file with the suffix "Grid Measurements Table" will be used in the next step for the building of the master images and tables grouping the single analysis results (figure below).Proceed in the same manner for the other images.
  • Step 2 - masterize single array analyses:

    Once arrays are individually analyzed using the "Array Analysis" tools, the "Group Pattern" menu then allows to obtain a global view of a set of arrays.
    • Select the parent folder containing the array analyses by the "Masterize from Analysis Repertories" function:Below: the selected folder containing the demo array analysis. This function looks for result tables coming from the "Array Analysis" functions, in the parent folder chosen by the user. The tool explores any sub-levels, and builds a master image, or pattern, associated to a master table prensenting all the found results. It is so possible to obtain a global panel of all the arrays of a set of experiments by a simple click.The program exibits two default master representations:The default master pattern (above left) presents the arrays as they came from the analysis, with the visualization scaled between zero and the maximum values encountered in each array. The initial normalized pattern (above right) presents a normalization between zero and the maximum value found in the master. This representation gives the most natural aspect of the modelled pattern compared to the initial images. It can be used to estimate visually the global intensity of an array among the group.
    • Normalize the masters using the internal references provided by the manufacturer by using the "Group Pattern Menu" -> "Set Internal Control and References" function.For this demonstration, "RayBio Mouse Cytokine Array 3" [4] were used. These membranes allow the detection of 62 cytokines. For the normalization of this demonstration analysis we will use as positive dots the A1, A2, B1, B2, and as blank or control the dots L9, L10, M9, M10.Above, the dialog box allows to enter or supress some internal references for the normalization. When more than one control or reference are input, the mean value is used for calculations. Each value is normalized following this formula : Dot Value norm = (Dot Value - mean(Controls))/mean(References). The master pattern is refreshed each time the references are changed. Below, the aspect when all the choosen references are taken into account. Note that the representation puts in evidence the highest relative intensity in the array number 5 (right below).Save the documents by using the "Group Pattern Menu" -> "Save Master Documents at Parent Folder Location" function.Above: a folder with master images and tables is recorded at the parent analysis repertory. The table files are compatible with your favorite spreadsheet software. For OpenOffice, during the opening process, select all the values from the preview and affect the "Standard" type (see below).The window is ready for calculations and histograms.

      Citing this work:

      You can use a formula similar to that found by Rahimi et al. in the "Material and Methods" of this article [2], or use in references a formula like "Carpentier G. Protein Array Analyze for ImageJ (2010) available online: http ://rsb.info.nih.gov/ij/macros/toolsets/Protein Array Analyzer.txt"
Comments