Multiplexing enables the monitoring of hundreds to thousands of proteins in quantitative proteomics analyses and increases sample throughput. In most mass-spectrometry-based proteomics workflows, multiplexing is achieved by labeling biological samples with heavy isotopes via precursor isotopic labeling or isobaric tagging. Enhanced multiplexing strategies, such as combined precursor isotopic labeling and isobaric tagging (cPILOT), combine multiple technologies to afford an even higher sample throughput. Critical to enhanced multiplexing analyses is ensuring that analytical performance is optimal and that missingness of sample channels is minimized. Automation of sample preparation steps and use of quality control (QC) metrics can be incorporated into multiplexing analyses and reduce the likelihood of missing information, thus maximizing the amount of usable quantitative data. Here, we implemented QC metrics previously developed in our laboratory to evaluate a 36-plex cPILOT experiment that encompassed 144 mouse samples of various tissue types, time points, genotypes, and biological replicates. The evaluation focuses on the use of a sample pool generated from all samples in the experiment to monitor the daily instrument performance and to provide a means for data normalization across sample batches. Our results show that tracking QC metrics enabled the quantification of ∼7000 proteins in each sample batch, of which ∼70% had minimal missing values across up to 36 sample channels. Implementation of QC metrics for future cPILOT studies as well as other enhanced multiplexing strategies will help yield high-quality data sets.

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