Aggregation and disaggregation features of the human proteome

The aim of the project was to quantify protein aggregation and disaggregation in human cells after transient non-lethal heat shock and during recovery. In addition, the non-aggregating proteins were analyzed by two-dimensional proteome profiling to detect changes in thermal stability upon heat shock. For aggregation/disaggregation study, K562 cells were grown in light SILAC medium which was changed to heavy medium 90 minutes before heat treatment (10 minutes at 44C). After heat shock, cells were let to recover at 37C. Samples were collected before and after the heat shock as well as on multiple time points during recovery (one, two, three and five hours). Protein intensities from soluble fraction (extracted with mild detergent - NP40) was compared to a control samples that on parallel were treated with mock shock (10 minutes at +37C). Samples were labelled with TMT labels and pooled. The medium switch prior to heat shock also allowed to monitor changes in protein synthesis caused by the heat shock. For control, an analysis of total protein amount (extracted with strong detergent - SDS) was conducted. For two-dimensional proteome profiling, aliquots of heat shocked and mock shocked samples were exposed to a temperature gradient (from 37.0C to 66.3C). Samples from two adjacent temperatures were labelled with TMT and pooled. Proteins from soluble fractions were quantified and heat shock-induced changes in thermal stability were analyzed.