RESUMEN
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 â¼7000 proteins in each sample batch, of which â¼70% 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.
Asunto(s)
Proteínas , Proteómica , Ratones , Animales , Espectrometría de Masas/métodos , Proteómica/métodos , Marcaje Isotópico/métodos , Control de Calidad , Proteoma/análisisRESUMEN
The identification of thousands of proteins and their relative levels of expression has furthered understanding of biological processes and disease and stimulated new systems biology hypotheses. Quantitative proteomics workflows that rely on analytical assays such as mass spectrometry have facilitated high-throughput measurements of proteins partially due to multiplexing. Multiplexing allows proteome differences across multiple samples to be measured simultaneously, resulting in more accurate quantitation, increased statistical robustness, reduced analysis times, and lower experimental costs. The number of samples that can be multiplexed has evolved from as few as two to more than 50, with studies involving more than 10 samples being denoted as enhanced multiplexing or hyperplexing. In this review, we give an update on emerging multiplexing proteomics techniques and highlight advantages and limitations for enhanced multiplexing strategies.
Asunto(s)
Refuerzo Biomédico , Proteómica , Bioensayo , Espectrometría de Masas , ProteomaRESUMEN
Aging is a process that occurs in tissues and across species, leading to the degradation of many biological processes. We previously demonstrated that rabbits are a feasible model for studying aging due to their genetic homology and relatively short lifespan in comparison to humans. We utilized a cPILOT multiplexing strategy to identify proteomic changes in spleen tissues of young, middle, and old aged rabbits. We identified 63 proteins that change significantly (p < 0.05) with age and notably these proteins relate to nucleotide and RNA binding, DNA repair, actin regulation, and immune system pathways. Here, we explore the implications of aging in the spleen and demonstrate the utility of a rabbit model to understand aging processes.