Fig. 1.

Identification of TFIIS by quantitative MS analysis of promoter DNA-purified Pol II PICs. (A) Schematic of the quantitative MS approach for the analysis of Pol II PICs. (B) Histogram of abundance ratios (+rTBP:−TBP) for 252 quantified proteins. The distributions were modeled by using a mixture model expectation–maximization algorithm. The model predicts five clusters: four clusters of TBP-stimulated proteins and one cluster of unstimulated proteins. TFIIS is a component of cluster 4. Cluster 5 (highest degree of enrichment) is not shown. (C) Quantification of isotopically labeled peptides derived from TFIIS. A SEQUEST database search (52) matched the MS/MS spectrum of an ion with mass-to-charge ratio (m/z) of 898.1 to the indicated ICAT-labeled peptide sequence corresponding to TFIIS. Relative quantification of the isotopically heavy (Lower) and normal (Upper) ICAT-labeled peptides was determined from the summed signal intensities for each peptide ion during the elution time with XPRESS software (53). The abundance ratio is the average from three independent measurements. P is the ProteinProphet score that describes the probability that the identification is correct (54).