Analyzing Protein‐Protein Interactions from Affinity Purification‐Mass Spectrometry Data with SAINT
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Abstract
Significance Analysis of INTeractome (SAINT) is a software package for scoring protein?protein interactions based on label?free quantitative proteomics data (e.g., spectral count or intensity) in affinity purification?mass spectrometry (AP?MS) experiments. SAINT allows bench scientists to select bona fide interactions and remove nonspecific interactions in an unbiased manner. However, there is no ?one?size?fits?all? statistical model for every dataset, since the experimental design varies across studies. Key variables include the number of baits, the number of biological replicates per bait, and control purifications. Here we give a detailed account of input data format, control data, selection of high?confidence interactions, and visualization of filtered data. We explain additional options for customizing the statistical model for optimal filtering in specific datasets. We also discuss a graphical user interface of SAINT in connection to the LIMS system ProHits, which can be installed as a virtual machine on Mac OS X or Windows computers. Curr. Protoc. Bioinform. 39:8.15.1?8.15.23. © 2012 by John Wiley & Sons, Inc.
Keywords: protein?protein interactions; label?free quantitative proteomics; affinity purification?mass spectrometry (AP?MS); statistical model
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Installation and Data Formatting
- Basic Protocol 2: Running SAINT
- Support Protocol 1: Visualization of Network
- Alternate Protocol 1: Running SAINT Through ProHits Interface: Virtual Machine GUI
- Guidelines for Understanding Results
- Commentary
- Literature Cited
- Figures
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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Installation and Data Formatting
Necessary Resources
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PDF or HTML at Wiley Online LibraryFigures
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Figure 8.15.1 Choosing the appropriate version and optional arguments in SAINT. View Image -
Figure 8.15.2 Illustration of input data in the TIP49 dataset. View Image -
Figure 8.15.3 Importing the SAINT result files into Cytoscape for the analysis of the TIP49 dataset. View Image -
Figure 8.15.4 The network visualization of the TIP49 dataset in Cytoscape. View Image -
Figure 8.15.5 Select samples to analyze using the ProHits interface. View Image -
Figure 8.15.6 Define controls and samples and select parameters for file preparation. View Image -
Figure 8.15.7 Select SAINT options and initiate analysis within ProHits. View Image -
Figure 8.15.8 Tracking of the SAINT analysis parameters in ProHits. View Image -
Figure 8.15.9 Graphical user interface to explore SAINT results. View Image -
Figure 8.15.10 Automated Cytoscape generation of SAINT results from ProHits. View Image -
Figure 8.15.11 Comparison of probabilities using different options in the TIP49 dataset. View Image -
Figure 8.15.12 Ideal design of an AP‐MS experiment with negative controls and biological replicates. Hypothetical experiment involving the purification of four different baits (colored circles) and a negative control (gray circle). Each of the biological replicate experiments is performed for each of the baits in a single batch. Different biological replicates are performed on batches of cells harvested at different times and for which purification and proteolysis is done on different days. Notice the randomization of the loading order of the samples on the mass spectrometer to help preventing bias (e.g., carry‐over). View Image
Videos
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