Highly reproducible label free quantitative proteomic analysis of RNA polymerase complexes

Abstract

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports.

Fig. 1.

RNA Polymerase I, II, and III Proteins and Reproducibility. A, Silver stained gels for each of the specific RNAP purifications. The common subunits are indicated by arrows to the right of the panel. B, Venn diagram illustrating the shared nature of different RNAP subunits. Subunits common to all three complexes are indicated in orange text; those common to RNAPI and RNAPIII are shown in green; RNAPII specific subunits are shown in blue; RNAPI specific subunits are shown in black; and RNAPIII specific subunits are shown in red. C, Results from the ANOVA F-test and Tukey's test. ANOVA was performed to test the reproducibility of the dNSAF values between the three biological replicate for each preparation as indicated. The Tukey's test analyzed pair-wise comparisons of reproducibility as indicated.

Fig. 2.

Binary and Quantitative Clustering. A, Cluster analysis of the binary values (0 = absent (black), 1 = present (yellow) for RNA polymerase subunit detection by MudPIT analysis. B, Cluster analysis of the dNSAF values from each RNA polymerase preparation to determine the relatedness of the different purifications. The yellow color intensity is indicative of the dNSAF value according to the scale shown to the right of B. For both figures, the dendrogram at the top of the cluster represents the relationship between the baits whereas the dendrogram to the left represents the relationship between the purified proteins (preys).

Fig. 3.

dNSAF Analysis of RNA polymerase I, II, and III. Relative abundance determined using label-free spectral counting for the 12-core subunits of RNAPII obtained from the Rpb3-TAP preparation (A), the 14-core subunits of RNAPI obtained from the Rpa190-TAP preparation (B), and the 17-core subunits of RNAPIII obtained from the Ret1-TAP preparation (C). Data are expressed as average dNSAF values ± standard deviations. The average dNSAF value for the RNAP subunits for each purification is indicated as a dashed line on the graph.

Fig. 4.

Label-free quantitation at the protein level is highly reproducible. Pearson correlation coefficients (r values) were calculated to determine the reproducibility of spectral counting for each unique peptide across biological replicates. A, Analysis of the reproducibility of the raw spectral counting for all peptides identified in the RNA polymerase data set. B, Analysis of the reproducibility of all unique peptides from all 31 RNA polymerase subunits. The cyan intensity represents the r value obtained from Pearson as indicated on the scale to the right of the figure. C, Analysis of the reproducibility of all unique peptides from the 5 RNA polymerase subunits found in all three complexes (Rpb5, Rpb6, Rpb8, Rpb10, and Rpb12). The r value scale for these calculations is given to the right of the figure.

Fig. 5.

Analysis of the average number of spectral counts observed per unique peptide identification. Data are expressed as the average spectral counts/peptide for each subunit of RNA polymerase II as indicated. These data are representative of the three biological replicates although only data from biological replicate 1 of Rpb3-TAP are shown. At the top of the graph, the maximum number of spectral counts observed for a unique peptide (Maximum), the total number of proteotypic peptides (Proteotyptic), and the total number of amino acids (Total Length) for each subunit is indicated.

Fig. 6.

Comparison of NSAF, APEX, and Protein Prophet. A,Comparison of cNSAF values using the spectral count values obtained from either threshold-based filtering obtained from Contrast (n = 3) or probability-based filtering obtained from Protein Prophet (n = 1) using the criteria described in the methods section. B, Comparison of cNSAF values (n = 3) to the APEX values (n = 1) obtained following analysis of Rpb3-TAP replicate 1 using the APEX protocol. Data from MudPIT analysis of a TAP lysate was used for model creation as described. Use of the APEX protocol is not able to adjust the higher average sampling observed for Rpb3, Rpb4, Rpb5, or Rpb10. (C) Comparison of cNSAF values (n = 3) to the APEX values (n = 1) obtained when using the merged Rpb8-TAP data set for model creation.

Fig. 7.

The elongation factor complexes DSIF and TFIIF associate with RNAPII in a dynamic manner. A, Analysis of GO term enrichment using GOstat. The graph shows the p value for each GO category indicated at the top of the figure from GOstat analysis of the proteins identified in the three biological replicates of Rpb3-TAP. B, dNSAF values from Rpb3- and Rpb11-TAP purifications. The average and standard deviations were calculated from the analysis of at least three technical replicate MudPIT runs from each biological sample. C, dNSAF values obtained from Rpb3-TAP comparing the Tfg1 and Tfg2 subunits of TFIIF to their homologs in RNA polymerase I, Rpa49 and Rpa34 (identified in the Rpa190-TAP purifications).