Highly reproducible label free quantitative proteomic analysis of RNA polymerase complexes.
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.
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Amber L
AL
Mosley
Mihaela E
ME
Sardiu
Samantha G
SG
Pattenden
Jerry L
JL
Workman
Laurence
L
Florens
Michael P
MP
Washburn
D016000:Cluster Analysis; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D015233:Models, Statistical; D010455:Peptides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012313:RNA / Q000737:chemistry; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry; D020539:Sequence Analysis, Protein / Q000379:methods
D010455:Peptides; D011506:Proteins; D029701:Saccharomyces cerevisiae Proteins; D012313:RNA; D012321:DNA-Directed RNA Polymerases