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.
Highlights
From the ‡Stowers Institute for Medical Research, Kansas City, MO 64110; §Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202; ¶Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160
We show that quantitation using either the distributed normalized spectral abundance factor or raw spectral counting is highly reproducible across biological replicates
We focused on the analysis of two highly enriched complexes found in association with RNA polymerase II, DRB sensitivity inducing factor complex (DSIF) and transcription factor II F (TFIIF)
Summary
Tandem Affinity Purification (TAP)—All yeast strains used in this study were obtained from the Open Biosystems collection produced by Ghememaghami et al [11]. In order to focus on biological and not technical variation, the three technical replicates for each biological replicate (9 MudPIT runs in total) were merged using DTASelect prior to assembly of the entire data set using CONTRAST In this data set, spectrum/peptide matches only passed filtering if they were at least seven amino acids in length and fully tryptic. The second data set used for comparison was a merged data set containing peptide identifications for all three Rpb8-TAP purifications For this analysis, protein identifications were only used for analysis if they were supported by at least 200 spectral counts as suggested in the APEX protocol [23]. APEX values were calculated as shown below with the total number of spectral counts (n) and protein probabilities (p) obtained from the ProteinProphet output
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