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Nat Protoc. 2014 Nov;9(11):2539-54. doi: 10.1038/nprot.2014.164. Epub 2014 Oct 2.

Affinity purification-mass spectrometry and network analysis to understand protein-protein interactions.

Author information

1
Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.
2
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.
3
1] Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA. [2] Graduate Group in Bioinformatics, University of California, San Francisco, San Francisco, California, USA.
4
School of Medicine, University of California, San Francisco, San Francisco, California, USA.
5
Gladstone Institutes, University of California, San Francisco, San Francisco, California, USA.

Abstract

By determining protein-protein interactions in normal, diseased and infected cells, we can improve our understanding of cellular systems and their reaction to various perturbations. In this protocol, we discuss how to use data obtained in affinity purification-mass spectrometry (AP-MS) experiments to generate meaningful interaction networks and effective figures. We begin with an overview of common epitope tagging, expression and AP practices, followed by liquid chromatography-MS (LC-MS) data collection. We then provide a detailed procedure covering a pipeline approach to (i) pre-processing the data by filtering against contaminant lists such as the Contaminant Repository for Affinity Purification (CRAPome) and normalization using the spectral index (SIN) or normalized spectral abundance factor (NSAF); (ii) scoring via methods such as MiST, SAInt and CompPASS; and (iii) testing the resulting scores. Data formats familiar to MS practitioners are then transformed to those most useful for network-based analyses. The protocol also explores methods available in Cytoscape to visualize and analyze these types of interaction data. The scoring pipeline can take anywhere from 1 d to 1 week, depending on one's familiarity with the tools and data peculiarities. Similarly, the network analysis and visualization protocol in Cytoscape takes 2-4 h to complete with the provided sample data, but we recommend taking days or even weeks to explore one's data and find the right questions.

PMID:
25275790
PMCID:
PMC4332878
DOI:
10.1038/nprot.2014.164
[Indexed for MEDLINE]
Free PMC Article

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