Proteome-wide identification of metamorphic protein candidates using mass spectrometry

How protein folds remodel at low temperature remains poorly mapped at the proteome scale. We combine brief, temperature-controlled proteolysis with LC-MS/MS to chart cold-dependent conformational changes in Escherichia coli lysate. Methodological validation with the metamorphic protein Sa1V90T--whose /{beta}-plait and 3 states interchange between 30 {degrees}C and 5 {degrees}C--showed that a high-protease, short-digestion regime detects temperature-dependent exposure at four peptides, whereas the temperature-insensitive variant Sa1V90TV52D is largely unchanged. Applied to lysate, the workflow identifies >750 proteolytic sites across >250 abundant proteins (>10% of the identified proteome) whose local susceptibility shifts as temperature decreases from 35 {degrees}C to 5 {degrees}C, including five Sa1V90T peptides recapitulating purified-protein behavior. A site-level scoring model yields 338 destabilized peptides (177 proteins) and 426 stabilized peptides (138 proteins). Cold stabilization prominently marks translation machinery (>15 ribosomal proteins), while cooling also exposes regions in chaperones and cell-division factors. Sequence analysis links temperature sensitivity to charge-enriched composition, consistent with thermodynamic expectations. These data provide a proteome-wide view of cold-sensitive folding, implicate translation and cytokinetic systems in low-temperature remodeling, and illuminate a new strategy for identifying metamorphic proteins on an unprecedented scale.