The enormous diversity of proteoforms produces tremendous complexity within cellular proteomes, facilitates intricate networks of molecular interactions, and constitutes a formidable analytical challenge for biomedical researchers. Currently, quantitative whole-proteome profiling often relies on non-targeted liquid chromatography-mass spectrometry (LC-MS), which samples proteoforms broadly, but can suffer from lower accuracy, sensitivity, and reproducibility compared with targeted LC-MS. Recent advances in bottom-up proteomics using targeted LC-MS have enabled previously unachievable identification and quantification of target proteins and posttranslational modifications within complex samples. Consequently, targeted LC-MS is rapidly advancing biomedical research, especially systems biology research in diverse areas that include proteogenomics, interactomics, kinomics, and biological pathway modeling. With the recent development of targeted LC-MS assays for nearly the entire human proteome, targeted LC-MS is positioned to enable quantitative proteomic profiling of unprecedented quality and accessibility to support fundamental and clinical research. Here we review recent applications of bottom-up proteomics using targeted LC-MS for systems biology research. SIGNIFICANCE: Advances in targeted proteomics are rapidly advancing systems biology research. Recent applications include systems-level investigations focused on posttranslational modifications (such as phosphoproteomics), protein conformation, protein-protein interaction, kinomics, proteogenomics, and metabolic and signaling pathways. Notably, absolute quantification of metabolic and signaling pathway proteins has enabled accurate pathway modeling and engineering. Integration of targeted proteomics with other technologies, such as RNA-seq, has facilitated diverse research such as the identification of hundreds of "missing" human proteins (genes and transcripts that appear to encode proteins but direct experimental evidence was lacking).
