Key Points
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Mass spectrometry-based proteomics is the only method currently available to comprehensively analyse changes in mutant proteomes.
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Several quantitative methods have been introduced to profile mutant proteomes with high sensitivity. However, although full proteome coverage has been achieved by recent studies, proteome profiling still requires substantial experimental efforts.
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Targeted proteome analysis by selected reaction monitoring is a promising analytical concept to overcome the existing limitations in the analysis of low abundance proteins from complex biological samples.
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Several workflows have been introduced for the systematic analysis of posttranslational modifications. The first examples in the field of phosphoproteome analysis have shown how global posttranslational modification profiling can be applied to identify the molecular pathways that are affected in mutant cells.
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Information on protein complexes obtained by affinity purification coupled with mass spectrometry provides important insights into the molecular context of proteins that are encoded by mutated genes.
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A higher-level understanding of complex genotype–phenotype relationships will depend on the proper annotation and accessibility of mass spectrometry-based proteomics data to integrate these data with functional genomics and phenomics data in a biological cyberinfrastructure.
Abstract
The systematic and quantitative molecular analysis of mutant organisms that has been pioneered by studies on mutant metabolomes and transcriptomes holds great promise for improving our understanding of how phenotypes emerge. Unfortunately, owing to the limitations of classical biochemical analysis, proteins have previously been excluded from such studies. Here we review how technical advances in mass spectrometry-based proteomics can be applied to measure changes in protein abundance, posttranslational modifications and protein–protein interactions in mutants at the scale of the proteome. We finally discuss examples that integrate proteomics data with genomic and phenomic information to build network-centred models, which provide a promising route for understanding how phenotypes emerge.
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Acknowledgements
This work was supported by the by the Swiss National Science Foundation grant 31000-10767, by federal funds from the National Heart, Lung and Blood Institute, the National Institutes of Health grant N01-HV-28179 and by SystemsX.ch, the Swiss initiative for systems biology.
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FURTHER INFORMATION
BioGrid protein interaction database
IntAct protein interaction database
MRMAtlas compendium of targeted proteomics assays
PeptideAtlas MS-based peptide data
PeptideSieve tool for prediction of proteotypic peptides
PhosphoPep MS-based data on phosphopeptides
Pride repository for proteomics data
Protein Interaction Network Analysis database (PINA)
Glossary
- Mass spectrometry
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An analytical technique for the identification of the chemical composition of compounds on the basis of the mass to charge ratios of charged particles.
- Affinity purification–mass spectrometry
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A method for the analysis of protein complexes that combines purification of protein complexes using affinity reagents and mass spectrometry.
- Tandem mass spectrometry
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This combines two mass spectrometers: one (MS1) for the detection and selection of precursor ions, which is followed by a second (MS2) for the analysis of fragment ion spectra generated from selected precursor ions after collision-induced fragmentation. The information from the fragment ion spectra is used for peptide identification.
- Dynamic range
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The ratios between the highest and lowest possible ion intensities in a mass spectrum for which accurate masses can be determined by a mass spectrometer.
- Liquid chromatography–tandem mass spectrometry
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Liquid chromatography is used in MS-based proteomics to separate peptides in complex mixtures primarily on the basis of their charge or hydrophobicity using strong cation exchange or reversed-phase chromatography columns.
- Selected reaction monitoring
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This is a sensitive mass spectrometry-based method for targeted proteomics that is based on the measurement of precursor–fragment ion pairs (transitions) of proteotypic peptides.
- Proteotypic peptide
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Proteotypic peptides can be observed by mass spectrometry and uniquely identify a specific protein or a specific isoform of a protein.
- Synthetic genetic array
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This has been primarily applied to yeast and is a technology for the high-throughput analysis of genetic interactions. Yeast deletion strains are crossed with each other to systematically generate double mutant strains. The resulting growth phenotypes are determined based on the size of the resulting double mutant colonies.
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Gstaiger, M., Aebersold, R. Applying mass spectrometry-based proteomics to genetics, genomics and network biology. Nat Rev Genet 10, 617–627 (2009). https://doi.org/10.1038/nrg2633
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DOI: https://doi.org/10.1038/nrg2633
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