Fig. 2

Identification of nucleic acid-associated proteins from mass spectrometry data. a. Diagram demonstrating data analysis of mass spectrometry data. Three hundred and twenty-five protein groups were identified in at least two of the three immunoprecipitations, 222 of these were identified as high confidence protein groups and were not identified in any of the control immunoprecipitations. Multiple sample testing with an FDR of 0.05 and a fold change of two was used to identify 63 low confidence proteins from 103 possible low confidence proteins. Uniprot protein annotations were mapped to MSMEG database annotations, removing Uniport annotations that matched to MSMEI or LJ100 database annotations (assigned during automated database searching), resulting in the identification of 220 high confidence and 61 low confidence proteins. Following the manual inspection of spectra for all proteins identified with a minimum of 2 unique peptides, a total of 275 proteins were identified, of which 214 were high confidence proteins and 61 were low confidence proteins. b. Principal component analysis revealed separate clustering of replicate anti-RNAP immunoprecipitations and control immunoprecipitations for the 503 protein groups with two unique peptides. Anti-RNAP immunoprecipitations are displayed in red blocks on the right with protein G Dynabead control immunoprecipitations displayed in green and anti-MYH7 control immunoprecipitations displayed in blue on the left. c. The heatmap shows the clustering of the anti-RNA polymerase immunoprecipitation on the left and control immunoprecipitations right and center. The red colour is indicative of a higher abundance and the blue of a lower abundance of a protein within an immunoprecipitated sample. The grey colouring within the heatmap is representative of the absence of a protein within that immunoprecipitation