Integral Quantification Accuracy Estimation for Reporter Ion-based Quantitative Proteomics (iQuARI)

Abstract

With the increasing popularity of comparative studies of complex proteomes, reporter ion-based quantification methods such as iTRAQ and TMT have become commonplace in biological studies. Their appeal derives from simple multiplexing and quantification of several samples at reasonable cost. This advantage yet comes with a known shortcoming: precursors of different species can interfere, thus reducing the quantification accuracy. Recently, two methods were brought to the community alleviating the amount of interference via novel experimental design. Before considering setting up a new workflow, tuning the system, optimizing identification and quantification rates, etc. one legitimately asks: is it really worth the effort, time and money? The question is actually not easy to answer since the interference is heavily sample and system dependent. Moreover, there was to date no method allowing the inline estimation of error rates for reporter quantification. We therefore introduce a method called iQuARI to compute false discovery rates for reporter ion based quantification experiments as easily as Target/Decoy FDR for identification. With it, the scientist can accurately estimate the amount of interference in his sample on his system and eventually consider removing shadows subsequently, a task for which reporter ion quantification might not be the solution of choice.

Dynamic Regulation of a Ribosome Rescue Pathway in Erythroid Cells and Platelets

“…Analysis of these data revealed ~6,700 ribosome-occupied mRNAs in platelets using a cut-off of 0.3 reads per kilobase of coding sequence per million mapped reads (Figure 1A), consistent with the high complexity of the platelet transcriptome (Bray et al, 2013; Gnatenko et al, 2003; Kissopoulou et al, 2013; Rowley et al, 2011) and proteome (Burkhart et al, 2012; Kim et al, 2014; Martens et al, 2005; Vaudel et al, 2012; Wilhelm et al, 2014). Reticulocyte ribosomes predominantly occupied adult hemoglobin (Hb) mRNAs (Figure 1B), consistent with the fact that Hb transcripts make up the majority of the reticulocyte transcriptome (Clissold et al, 1977) and mostly hemoglobin protein (Denton et al, 1975) is made at this stage of differentiation.…”

Slowed decay of mRNAs enhances platelet specific translation

“…67 Platelet transcriptome, translatome, and proteome correlations For a fully integrated view of platelet gene expression, we compared our platelet RNA-Seq and ribosome profiling data to available protein abundance data from mass spectrometry. [58][59][60]68 We included 8801 platelet transcripts and 6809 translated mRNAs from our data along with 4147 quantified proteins (Figure 2A were supported by all 3 data types, we observed a moderate, statistically significant correlation between either platelet mRNA expression level or ribosome footprint density and protein abundance (Spearman r 5 0.39 and r 5 0.37, respectively) ( Figure 2B; supplemental Figure 1A, available on the Blood Web site). As anticipated, ribosome density was slightly higher on transcripts detected by mass spectrometry (supplemental Figure 1B), consistent with detectability bias favoring abundant proteins.…”

Designing biomedical proteomics experiments: state-of-the-art and future perspectives

“…Of course, it is also important to take note of the limitations of these various techniques when selecting the most suitable DDA approach for the study at hand [47]. For example, although multiplexing strategies have many advantages, it remains important to fully understand the ratio compression due to co-isolated and co-fragmented ions and the consequences it has on the obtained data [48,49].…”