Figure 1.

A simulated PISA assay combining samples incubated at temperatures ranging from 37 to 67 °C. (a) Examples of simulated melting curves. (b) Relationship between simulated ΔSm and ΔTm. Simulated ΔSm and ΔTm are linearly correlated when samples heated at 37 to 67 °C are combined. (c) Relationship between simulated log2 fold changes and ΔTm. Log2 fold changes and ΔTm are positively related, but they do not show good linearity as simulated ΔSm and ΔTm. Dashed boxes highlight hypothetical proteins showing |ΔTm| ≥ 2. (d) Distribution of log2 fold changes of hypothetical proteins showing |ΔTm| ≥ 2. The majority of the log2 fold changes are within a narrow range. (e) Equations describing the relationship among simulated ΔSm, fold change (FC), and ΔTm. Intensity_(treated) means the TMT reporter ion intensity of integral treated samples (combining from multiple samples covering the full melting curve). Intensity_(control) indicates the TMT reporter ion intensity of integral control samples (combining from multiple samples covering the full melting curve). Intensity_{(treated or control at top plateau)} indicates the TMT reporter ion intensity of the individual sample incubated at the top plateau of a melting curve. ΔSm is the difference between integral treated and integral control samples in simulated data. ΔTm is the melting temperature shift between treated and control samples. Fold change (FC) is the ratio of integral treated sample versus integral control sample. (f) Equations describing the relationship between experimental ΔSm and fold change (FC). In a PISA experiment, Intensity_{(treated or control at top plateau)} is not available and experimental ΔSm is approximated by normalizing the difference between integral treated and integral control samples on integral control samples. Experimental fold change (FC) remains the same as simulated fold change. There is a linear relationship between experimental ΔSm and FC (ASm = FC – 1).