Making Sense of High-Throughput Protein-Protein Interaction Data

2005 ◽  
Vol 3 (1) ◽  
pp. 1-31 ◽  
Author(s):  
Denise Scholtens ◽  
Robert Gentleman
Keyword(s):  
Systems Biology ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Protein Interaction Data ◽  
Local Dynamic ◽  
Graph Theoretic ◽  
Protein Protein Interaction ◽  
Making Sense ◽  
Using Data ◽  
Affinity Purification Mass Spectrometry

Accurate systems biology modeling requires a complete catalog of protein complexes and their constituent proteins. We discuss a graph-theoretic/statistical algorithm for local dynamic modeling of protein complexes using data from affinity purification-mass spectrometry experiments. The algorithm readily accommodates multicomplex membership by individual proteins and dynamic complex composition, two biological realities not accounted for in existing topological descriptions of the overall protein network. A likelihood-based objective function guides the protein complex modeling algorithm. With an accurate complex membership catalog in place, systems biology can proceed with greater precision.

Proteome Organization in a Genome-Reduced Bacterium

Science ◽  
2009 ◽  
Vol 326 (5957) ◽  
pp. 1235-1240 ◽  
Author(s):  
Sebastian Kühner ◽  
Vera van Noort ◽  
Matthew J. Betts ◽  
Alejandra Leo-Macias ◽  
Claire Batisse ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Affinity Purification ◽  
Biological Processes ◽  
Multiprotein Complex ◽  
Data Set ◽  
Basic Principles ◽  
A Genome ◽  
Structural Details ◽  
Cellular Machinery ◽  
Affinity Purification Mass Spectrometry

The genome of Mycoplasma pneumoniae is among the smallest found in self-replicating organisms. To study the basic principles of bacterial proteome organization, we used tandem affinity purification–mass spectrometry (TAP-MS) in a proteome-wide screen. The analysis revealed 62 homomultimeric and 116 heteromultimeric soluble protein complexes, of which the majority are novel. About a third of the heteromultimeric complexes show higher levels of proteome organization, including assembly into larger, multiprotein complex entities, suggesting sequential steps in biological processes, and extensive sharing of components, implying protein multifunctionality. Incorporation of structural models for 484 proteins, single-particle electron microscopy, and cellular electron tomograms provided supporting structural details for this proteome organization. The data set provides a blueprint of the minimal cellular machinery required for life.

scholarly journals Mono- and Biallelic Protein-Truncating Variants in Alpha-Actinin 2 Cause Cardiomyopathy Through Distinct Mechanisms

2021 ◽  
Author(s):  
Malene E. Lindholm ◽  
David Jimenez-Morales ◽  
Han Zhu ◽  
Kinya Seo ◽  
David Amar ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Structural Changes ◽  
Affinity Purification ◽  
Protein Protein Interaction ◽  
Cellular Hypertrophy ◽  
C Terminus ◽  
Associated Proteins ◽  
Cardiac Sarcomeres ◽  
Affinity Purification Mass Spectrometry

Background: ACTN2 (alpha-actinin 2) anchors actin within cardiac sarcomeres. The mechanisms linking ACTN2 mutations to myocardial disease phenotypes are unknown. Here, we characterize patients with novel ACTN2 mutations to reveal insights into the physiological function of ACTN2. Methods: Patients harboring ACTN2 protein-truncating variants were identified using a custom mutation pipeline. In patient-derived iPSC-cardiomyocytes, we investigated transcriptional profiles using RNA sequencing, contractile properties using video-based edge detection, and cellular hypertrophy using immunohistochemistry. Structural changes were analyzed through electron microscopy. For mechanistic studies, we used coimmunoprecipitation for ACTN2, followed by mass-spectrometry to investigate protein-protein interaction, and protein tagging followed by confocal microscopy to investigate introduction of truncated ACTN2 into the sarcomeres. Results: Patient-derived iPSC-cardiomyocytes were hypertrophic, displayed sarcomeric structural disarray, impaired contractility, and aberrant Ca 2+ -signaling. In heterozygous indel cells, the truncated protein incorporates into cardiac sarcomeres, leading to aberrant Z-disc ultrastructure. In homozygous stop-gain cells, affinity-purification mass-spectrometry reveals an intricate ACTN2 interactome with sarcomere and sarcolemma-associated proteins. Loss of the C-terminus of ACTN2 disrupts interaction with ACTN1 and GJA1, 2 sarcolemma-associated proteins, which may contribute to the clinical arrhythmic and relaxation defects. The causality of the stop-gain mutation was verified using CRISPR-Cas9 gene editing. Conclusions: Together, these data advance our understanding of the role of ACTN2 in the human heart and establish recessive inheritance of ACTN2 truncation as causative of disease.

scholarly journals Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

2008 ◽  
Vol 183 (2) ◽  
pp. 223-239 ◽  
Author(s):  
Laura Trinkle-Mulcahy ◽  
Séverine Boulon ◽  
Yun Wah Lam ◽  
Roby Urcia ◽  
François-Michel Boisvert ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Interaction ◽  
Cell Biology ◽  
Protein Complexes ◽  
Affinity Purification ◽  
Specific Protein ◽  
Protein Interaction Data ◽  
Quantitative Mass Spectrometry ◽  
Cell Extracts ◽  
Interaction Partners

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

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