Quantitative phosphoproteomics by mass spectrometry: past, present, and future

Abstract

Protein phosphorylation-mediated signaling networks regulate much of the cellular response to external stimuli, and dysregulation in these networks has been linked to multiple disease states. Significant advancements have been made over the past decade to enable the analysis and quantification of cellular protein phosphorylation events, but comprehensive analysis of the phosphoproteome is still lacking, as is the ability to monitor signaling at the network level while comprehending the biological implications of each phosphorylation site. In this review we highlight many of the technological advances over the past decade and describe some of the latest applications of these tools to uncover signaling networks in a variety of biological settings. We finish with a concise discussion of the future of the field, including additional advances that are required to link protein phosphorylation analysis with biological insight.

Figure 1

Timeline of selected milestones in quantitative phosphoproteomics during the last decade. Each publication has been selected based on implementation of a new method or application of recently developed methodology to uncover novel aspects of signaling networks.

Figure 2

Example workflow of the quantitative phosphotyrosine analysis experiment using iTRAQ labeling. Proteins are extracted from four biological samples (cell lines, stimulation time points, tissue samples), modified and digested. For quantification, the resulting peptides are labeled with iTRAQ reagent and combined. The mixture is then subjected to two steps of enrichment for tyrosine phosphorylated peptides: IP with antiphosphotyrosine antibodies and IMAC. Phosphorylated peptides eluted from the IMAC column are analyzed by LCMS/ MS, typically on a quadrupole TOF mass spectrometer. Each MS/MS spectrum contains both sequence-specific fragmentation events for identification of the peptide and phosphorylation site as well as the low-mass iTRAQ marker ions to quantify phosphorylation across the four samples. iTRAQ quantification is normalized to the supernatant to eliminate variation resulting from the preparation process.

Figure 3

Signaling network analysis by mass spectrometry, drug-target discovery, and kinase inhibition. Mass spectrometry-based phosphoproteomics is used to quantify signaling networks in various cell states resulting from multiple perturbations (e.g., cytokine stimulation, RNAi, small molecule inhibitors). Computational modeling is used to link this information to quantitative phenotypic measurements, thereby identifying key nodes in the signaling network. To validate this model, compounds are developed to target these nodes, and the phenotypic effect is monitored. The specificity of the compounds can then be tested by the Kinobead assay to quantify on- and off-target effects. Changes in the signaling network can then be quantified to establish kinase–substrate relationships given the established targets of the compound.