Researchers have developed a method for detecting condition-dependent changes in protein conformations in biological samples such as cell lysates. The method paves the way for the discovery of protein biomarkers for diagnosing diseases and other applications on the basis of changes in protein structure in addition to changes in abundance. Paola Picotti of ETH Zurich and coworkers achieve this with a two-step protein digestion coupled to mass spectrometry (Nat. Biotechnol. 2014, DOI: 10.1038/nbt.2999). The first digestion is performed on proteins in their native state by selecting and using a protease enzyme that cleaves proteins on the basis of their structure. In the second step, those pieces are digested further under denaturing conditions by trypsin, a protease that cleaves proteins at sequence-specific sites. Variations in the resulting peptide patterns before and after changing conditions, such as adding a drug, reveal proteins that undergo conformational changes. The researchers showed they can detect drastic or subtle conformational changes even in the complex chemical makeup of a cell extract. For example, they detected the transformation of α-synuclein from a single unfolded protein to a β-sheet-rich fibril of proteins and the unfolding of a single α-helix of myoglobin that has released its heme group.