Determining the identity of an intact protein by mass spectrometry without first digesting it with an enzyme is called 'top-down' proteomics. Although this approach has been applied to single proteins, the inability to separate a complex protein mixture in a way that is suitable for high-throughput analysis by mass spectrometry has limited the application of the top-down method on a large scale. Tran et al. report a new combination of separation approaches that allowed analysis of >3,000 protein forms produced from 1,043 genes in HeLa S3 cells, a >20-fold increase over previous top-down studies in mammalian cells. In contrast to the current dominant strategy requiring enzymatic digestion, top-down proteomics enables protein variants and complex combinations of post-translational modifications to be identified. This allowed Tran et al. to monitor changes in phosphorylated and methylated protein species and isoforms from splice variants over time in cells exposed to DNA damaging agents. The new separation approach appears to provide throughput and capacity superior to the gold-standard method of two-dimensional gel electrophoresis, and thus may facilitate top-down approaches to better identify biomarkers, understand modifications induced by intracellular signaling and catalog the proteome in its entirety. (Nature published online, doi:10.1038/nature10575, 30 October 2011)