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Large-scale mapping of human protein-protein interactions by mass spectrometry

Rob M Ewing et al. Mol Syst Biol. 2007.

Abstract

Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

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Figures

Figure 1
Figure 1
Data processing summary. Pie chart showing categorization of all immunoprecipitation experiments by type.
Figure 2
Figure 2
IP-HTMS data analysis pipeline. (A–B) All bands from the lane(s) corresponding to a bait are extracted and MS/MS data acquired. (C) Data from each MS/MS acquisition are searched against a non-redundant human protein sequence database using the Mascot search engine. (D) Data from all bands corresponding to each bait are merged and protein and peptides clustered to generate a non-redundant list of protein identifications. (E) Spurious proteins and promiscuous binding proteins are removed. (F) A data table is produced for each bait protein with all of the scoring information, including scores and ranks by band and experiment. This data table contains all data required for the estimation of bait–prey interaction probability. (G) An interaction confidence score is calculated based upon a partial least squares model trained on the replicated subset of the data.
Figure 3
Figure 3
GO coincidence maps. Coincidence maps showing enrichment of bait–prey GO category combinations. Each bait–prey category combination is represented by a square in the matrix and colored according to the P-value from a pairwise statistical test (Fisher exact test) of association. (A) Bait–prey biological processes. (B) Randomly permuted bait–prey biological processes. (C) Cellular component categories. (D) Randomly permuted bait–prey cellular component categories.
Figure 4
Figure 4
Comparison of interaction data sets to gene co-expression data. Red and green fractions of each bar correspond respectively to the proportions of positive and negative co-expression correlations for each data set. The numbers above each column represent the numbers of co-expression measurements overlapping the respective data set, and the numbers in parentheses represent the ratio of positive co-expression correlations to negative co-expression correlations. (1) The complete set of co-expression correlation measurements (Lee et al, 2004). (2) The set of co-expression gene pairs mapping to one or more IP-HTMS baits. (3) The set of IP-HTMS bait–prey pairs for which a co-expression measurement is available. (4) The set of Y2H (Rual et al, 2005) interactions for which a co-expression measurement is available. (5) The set of known (Ramani et al, 2005) interactions for which a co-expression measurement is available.
Figure 5
Figure 5
LYAR interactors also show strong gene co-expression with LYAR. Box plot showing distribution of P-values for all genes coexpressed (in three or more studies) with LYAR. Red points indicate co-expression P-values for 12 LYAR IP-HTMS interactors. Interactor descriptions include known subcellular localizations in square brackets where available.
Figure 6ab
Figure 6ab
Global and focused views of human interaction map. (A) Complete bait–bait connectivity map for 323 human bait proteins. Baits are represented as nodes in the graph. The size of the node represents the number of prey proteins identified for the bait. The thickness of edges between nodes represents the proportion of preys in common between the baits. Nodes are colored according to a combined disease and biological process classification, and selected classes indicated in the legend. (B) Focused views of selected bait–bait subnetworks (cross-referenced by roman numerals to panel A).
Figure 6c
Figure 6c
(C–F) Complete interaction networks (representing both baits and preys) for selected groups of baits. Nodes are colored according to cellular component or biological process as indicated on each figure. Baits are shown as large, labeled oval shapes, preys as small, labeled oval shapes. Arrow direction indicates a bait–prey relationship and line thickness indicates the interaction confidence score (see legend in panel C). Preys are grouped according to the baits with which they were identified (except panel E where they are grouped according to interaction confidence score). (C) Proteasome baits (corresponds to bait–bait cluster B (panel iv)). (D) Sumoylation pathway (corresponds to bait-bait cluster B (panel vi)). (E) Nek6. (F) Translation initiation and elongation (corresponds to bait–bait cluster B (panel iii)).
Figure 6de
Figure 6de
(C–F) Complete interaction networks (representing both baits and preys) for selected groups of baits. Nodes are colored according to cellular component or biological process as indicated on each figure. Baits are shown as large, labeled oval shapes, preys as small, labeled oval shapes. Arrow direction indicates a bait–prey relationship and line thickness indicates the interaction confidence score (see legend in panel C). Preys are grouped according to the baits with which they were identified (except panel E where they are grouped according to interaction confidence score). (C) Proteasome baits (corresponds to bait–bait cluster B (panel iv)). (D) Sumoylation pathway (corresponds to bait-bait cluster B (panel vi)). (E) Nek6. (F) Translation initiation and elongation (corresponds to bait–bait cluster B (panel iii)).
Figure 6f
Figure 6f
(C–F) Complete interaction networks (representing both baits and preys) for selected groups of baits. Nodes are colored according to cellular component or biological process as indicated on each figure. Baits are shown as large, labeled oval shapes, preys as small, labeled oval shapes. Arrow direction indicates a bait–prey relationship and line thickness indicates the interaction confidence score (see legend in panel C). Preys are grouped according to the baits with which they were identified (except panel E where they are grouped according to interaction confidence score). (C) Proteasome baits (corresponds to bait–bait cluster B (panel iv)). (D) Sumoylation pathway (corresponds to bait-bait cluster B (panel vi)). (E) Nek6. (F) Translation initiation and elongation (corresponds to bait–bait cluster B (panel iii)).
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