PepSlide® Peptide Array Design and Analysis Software

If you use peptide arrays then employ PepSlide® software to ease your work, save your time, and discover more from the peptide array data. The software provides a complete set of intuitive and easy-to-use tools for your peptide array applications.

The analysis tool - PepSlide® Analyzer - supports microarray image analysis and quantification of microarray data. This includes automatic batch processing of multiple microarray images. You can identify influential factors of your study and possible relationship between the peptides and the samples with available data mining tools. Furthermore, you can benefit from analyses that directly support peptide array applications such as epitope mapping, biomarker discovery, and characterization or optimization of peptides. PepSlide® Analyzer works natively on both Windows and Mac OS X platforms.

The design tool - PepSlide® Designer - is a Windows application, facilitating peptide library design and graphical array editor.

For the sake of further improvement and maintenance, the analysis tool (PepSlide® Analyzer) is integrated into Spotxel®. The array design tool, PepSlide® Designer, can be downloaded here and used for free without any serial number.

PepSlide® Analyzer - Peptide Array Analysis Tool

The basic module supports microarray image analysis and quantification of microarray data. Utilizing the powerful spot finding methods, the software is able to align the array (or grids) with the image by itself. You can thus setup a batch for automatically aligning and quantifying a large number of microarray images.

The data mining tools help you identify factors that influence your study and discover the relationship between the peptides and the samples. In addition, PepSlide® Analyzer is able to provide meaningful analyses that support peptide array applications such as epitope mapping and peptide optimization.

Image Analysis & Quantification of Microarray Data

Quantification of microarray data with PepSlide® Analyzer is straightforward. All you need to do are:

  • Load the microarray image(s) in TIFF format.
  • Load the array file in PSF or GAL format.
  • Align the array (grids) with the spots in the image (can be done automatically for GAL format).
  • Click the Quantify button or choose that command from the context menu.

Tools that ease your work and save your time

  • You can save the quantified data, the aligned array file, and the path to the image into a project file. When working on this analysis again, simply open the project and all the data will be loaded.
  • Automatic array alignment: Finds the spots in the image and aligns the array with them.
  • Automatic contrast adjustment: Adjust contrast to maximize the spots’ visibility.

Properties of Peptide Library, Block, or Spot - Image & Array Rotation

  • Check properties of a spot family (peptide library), a block, or a spot by hovering mouse over the object.
  • Flip images and rotate them at angles of 90°, 180°, or 270°.
  • Rotate selected spot families, blocks, or the array at an arbitrary angle. The degree change can be as small as 0.01°.

Spot Finding

The software supports two spot finding methods:

  • Fixed-Spot: The software uses the spot’s shape and area as specified in the array file to calculate the spot’s intensity value.
  • Flex-Spot: This powerful algorithm can detect the spot even though its shape and position are not in accordance with the specification in the array file.

In the picture on the right, the spots’ position and shape specified by the array file are depicted by the white dashed circle. Note that the binding signal, shown in red, has different shape and position from its specification. Despite that fact, the spots can be correctly detected by the Flex-Spot method with border highlighted in blue.

Noise Processing

The microarray images may contain noises that mislead the spot detection procedure and result in wrong quantified data. The noise can be background noise that span across the whole slide (the following picture on the left). It can also be foreground noise like the two large red bands (the following picture on the right). In the case of background noise, we want to “remove” the background layer so that only the meaningful signal remains. Foreground noise like the two red bands should not be part of a valid spot’s signal.

The software can effectively handle this task. The results can be seen in the same pictures: only valid spots’ signal are highlighted with a blue border (by the Flex-Spot method).

Background Correction

A consistent background correction can be applied to all spots in the entire array or a block. It can be done automatically with local background correction method or finely using background controls.

  • Local background correction is the default option. Simply quantify the array data; all spots at the selected level - block or array - will have the same background value.
  • By creating a background control located at the image region which best represents the background signal, you can define the background value for the entire array or a block.

Scatter Plot

The scatter plot depicts the features on a two-dimensional chart according to their quantified values. In addition, you can conveniently examine and select features of interest with the following tools.

  • Use the lower and upper threshold bars to select features.
  • Clicking on a feature on the plot will show its spot image as well as its annotation data.
  • Export selected features or a classified group of features to a CSV file.

Array Alignment and Batch Processing

The Array Alignment and Batch Processing functions automate the time consuming tasks when processing a large number of peptide microarray images – the array alignment and the data quantification - and thus free the user from this tedious work.

Given a scanned image and the array annotation file of a peptide microarray, the Array Alignment function finds the spots in the image and automatically aligns the array (or grid) to them.

The Batch Processing function enables the user to specify a set of peptide microarray images and their array file, and then let the software perform the array alignment and data quantification for all the images. The batch processing is fully automated; it does not need any user’s involvement during execution and can be run e.g. at night or during the weekend.

Please note that the current version only supports array alignment and batch processing for GAL array files.

Batch Running Modes

You can configure a batch to perform only the array alignment, or the quantification of the array data, or both of them. Furthermore, PepSlide® Analyzer let you run the batch in two modes:

  • Process all images continuously: The batch is processed continuously from the first image to the last one.
  • Stop and review after each image: You can review the processing results for image before proceeding to the next one.

Batch Results

The software creates three data files for each microarray images.

  • an array file whose layout is aligned with the spots in the image,
  • a CSV file containing only the quantified data, and
  • a project file containing the analyzed data for this image. Opening this project file will load the image, the aligned array, and the quantified data.

The software saves the batch data - containing the list of images, the original array file, and running options - to an XML file. The tasks and time points are tracked in a batch log also in the XML format.

Data Mining Tools

Data mining tools assist you to discover useful information from complex data sets. PepSlide® Analyzer supports K-means Clustering, Hierarchical Clustering Analysis, and Principal Component Analysis.

Assume that you have k microarray images as the result of testing the peptide microarray with k samples. The above batch processing thus generates k project files. You can then directly load these project files and use the data mining tool to discover features that influences your peptide microarray study and the relationship between them. The software also supports data sets stored in a CSV file or compiled from a list of GPR files.

Principal Component Analysis

  • Find features and samples that likely determine the variance of the data.
  • Search for views that are likely to separate the data and show the variance direction.
  • Reduce the dimension of the data set and identify new influential variables.

Hierarchical Clustering Analysis

  • Find possible relationships between features or samples.
  • Identify similarity between features or samples.

K-Means Clustering Analysis

Peptide Array Application Support

By correlating the quantified data and the peptide library design, PepSlide® Analyzer is able to provide meaningful analyses that support peptide array applications.

Epitope Mapping

Epitope mapping is directly supported by means of overlapping peptide libraries. The design tool translates a target protein or antigen into overlapping peptides. The analysis tool assists you to select peptides of interest. Furthermore, it can suggest a grouped list of peptides which may represent an epitope.

The software also support a feature useful for screening. You can search for antigens and proteins that contain peptides on the array. This can be done for selected peptides in a library or all peptides on the array.

Biomarker Discovery

Identification of peptide targets and discovery of biomarker are assisted with overlapping peptide libraries and random peptide libraries. The analysis associated with each library assists you to select peptides with strong binding signal.

If the libraries are screened with a number of samples, data mining tools are useful for revealing meaningful information. You can employ the Principal Component Analysis to find influential factors of the assay. The Hierarchical Clustering Analysis supports you to identify correlation between peptides and samples.

Peptide Characterization and Optimization

Substitution peptide libraries (also called positional or permutation scan) generate variants of a peptide by substituting its residues with certain amino acids. You can use these libraries to characterize an epitope or to optimize a known peptide. The design tool can perform a full replacement for all residues or only at some specific positions. The analysis software provides intuitive tools to examine all generated variants and select those of interest.

The following picture shows all variants of the peptide NYGKY and their “response”. Here, the (P)-Axis lists residues of the peptide and the (S)-Axis contains 20 amino acids for substituting, thus a “point” in the (PS)-plane features a variant. The “response” of a variant is represented by the height of the corresponding bar in the third dimension. You can conveniently use this three-dimensional chart to examine the variants and export the selection to a file for further analysis.

PepSlide® Designer - Peptide Array Design Tool

PepSlide® Designer (abbreviated PSD) facilitates interactive peptide library design and graphical array layout. Completed array designs can then be exported to array files in the suitable format required by your peptide array spotter and analysis software.

Peptide Library Design

The software supports design and generation of Overlapping, Substitution (also called positional or permutation scan), and Random peptide libraries. In addition, you can reuse a collection of peptides by means of the Import library. For quality control, you can choose to replicate peptide spots - double or triple - during the creation of peptide libraries.

Overlapping Peptide Library

This peptide library consists of overlapping peptides translated from a protein. Given a peptide length and an overlapping shift step, the protein is broken into peptides of equal length.

Substitution Peptide Library

This peptide library generates variants of a peptide by substituting residues at specified positions with certain amino acids. This is also called Permutation or Positional Scanning. In its simplest form - replacing residues with only Alanine - this library is similar to an Alanine Scanning.

PSD supports two modes of generating variants of a peptide, the so-called parent peptide:

  • Full Substitution: Suppose that you configure PSD with 20 amino acids. For each residue of the parent peptide, the tool generates 20 peptides by replacing the residue with 20 amino acids.
  • Partial Substitution: You can specify what residue will participate in the substitution and for each residue, an individual list of amino acids for replacing.

Random Peptide Library

You can conveniently generate a large number of random peptides given a peptide length. Simply define the list of amino acids constituting the peptides and for each amino acid, its occurring probability in the generated peptides.

Just click on the image on the right to see how easy to select amino acids and specify their probability in the random library.

Import Library

You can import a collection of peptides into an array design by means of the Import library. The peptide collection is stored in a CSV file with one peptide per line.

You can also import a mixed collection of proteins and peptides. Suppose that the peptide length you set for this peptide library is 12. If the length of a protein in the collection is larger than 12, that protein will be “broken” into overlapping peptides having 12 amino acids with shift step 1.

Graphical Array Editor

PepSlide® Designer facilitates two interfaces to edit a peptide array design. The Array View is a graphical array editor. Here you can create peptide libraries and controls. The editor facilitates visual layout of design objects with drag-and-drops and data replication with copy-and-pastes.

Replicating a design object is possible within an array design or between array designs. In addition, you can conveniently replicate an array design with just a few clicks.

In addition to peptide libraries, you can create marker controls which are useful for the positioning and alignment of the array during analysis. PSD also supports background controls which is an effective way to estimate the degree of nonspecific binding during quantification of peptide array data.

For peptide spots on the array, the Sequence View provides access to their amino acids, as either a whole sequence or separately at each array layer. You can regard this as a view to the third dimension of the array.

Configuration of Supported Amino Acids

The list of supported amino acids can be configured and used automatically during generation of peptide libraries. By default, this list consists of 20 standard amino acids.

You can save, import, or export this list. This enables the software to work with multiple configurations of the peptide array spotter.

In the case of chemical compound libraries, instead of amino acids you simply define the list of supported chemical building blocks.

Peptide Array Templates

PSD employs the concept of array template to define common properties of a peptide array. These include the array’s size, the spot’s morphology, and distances between spots.

PSD supports two types of spot’s morphology:

  • Spot-based: Circular spots generated by the SPOT technology.
  • Pixel-based: Rectangular spots generated by the laser printer technology.

You can combine array templates with lists of supported amino acids to define individual configurations of the peptide array spotter and array designs.

Transferring Array Designs to Peptide Array Spotter

PepSlide® Designer can export the data of the array design, including spots and their annotations, to file formats used by your peptide array spotter to synthesize the peptide arrays. This module needs customization to the peptide array spotter. Please contact us for further information of the customization.

Product Updates

01 Nov 2021 - Spotxel® Reader for Windows computers is available on the Microsoft Store.

14 Jan 2021 – Spotxel® Reader supports in-app help and loading assay setups from a CSV file.

04 May 2020 - Spotxel® supports TIFF taken by cameras, HuProt GAL formats, detachable spot viewer...