Abstract
Eukaryotic protein phosphorylation modulates nearly every major biological process. Phosphorylation regulates protein activity, mediates cellular signal transduction, and manipulates cellular structure. Consequently, the dysregulation of kinase and phosphatase pathways has been linked to a multitude of diseases. Mass spectrometry-based proteomic techniques are increasingly used for the global interrogation of perturbations in phosphorylation-based cellular signaling. Strategies for studying phosphoproteomes require high-specificity enrichment, sensitive detection, and accurate localization of phosphorylation sites with advanced LC-MS/MS techniques and downstream informatics. Sample multiplexing with isobaric tags has also been integral to recent advancements in throughput and sensitivity for phosphoproteomic studies. Each of these facets of phosphoproteomics analysis present distinct challenges and thus opportunities for improvement and innovation. Here, we review current methodologies, explore persistent challenges, and discuss the outlook for isobaric tag-based quantitative phosphoproteomic analysis.