Phosphoproteomics in cancer

Abstract

Reversible protein phosphorylation serves as a basis for regulating a number of cellular processes. Aberrant activation of kinase signaling pathways is commonly associated with several cancers. Recent developments in phosphoprotein/phosphopeptide enrichment strategies and quantitative mass spectrometry have resulted in robust pipelines for high-throughput characterization of phosphorylation in a global fashion. Today, it is possible to profile site-specific phosphorylation events on thousands of proteins in a single experiment. The potential of this approach is already being realized to characterize signaling pathways that govern oncogenesis. In addition, chemical proteomic strategies have been used to unravel targets of kinase inhibitors, which are otherwise difficult to characterize. This review summarizes various approaches used for analysis of the phosphoproteome in general, and protein kinases in particular, highlighting key cancer phosphoproteomic studies.

Figure 1

Applications of quantitative phosphoproteomics in cancer. Quantitative phosphoproteomics can lead to discovery of aberrantly activated signaling pathways and therapeutic targets in cancers. It can also reveal downstream effectors of mutant kinases, thereby providing an opportunity to understand the molecular mechanisms that lead to oncogenesis. Identification of cellular targets of kinase inhibitors in cancers and off‐target effects of kinase inhibitors are some of the other interesting applications of such strategies.

Figure 2

Fractionation and enrichment strategies for phosphoproteomics. Phosphoproteome is a minor fraction of any cellular proteome. The complexity of the cellular proteome hinders the analysis of phosphoproteins. Prior to phosphoproteomic analysis by mass spectrometry, it is therefore essential to reduce the complexity of the proteome using fractionation/enrichment strategies. Tyrosine phosphopeptides are often enriched using immunoaffinity‐based methods involving anti‐phosphotyrosine antibodies while both serine/threonine and tyrosine phosphopeptides can be enriched using immobilized metal affinity chromatography (IMAC) or titanium dioxide. Whereas metabolic labeling using SILAC would be the preferred method to analyze cancer cell lines, in vitro chemical tagging methods such as iTRAQ are preferred for analyzing phosphoproteome from tissue specimens. Relative differences in the extent of phosphorylation are quantified at the MS level in SILAC‐based strategies, while reporter ions released during MS/MS fragmentation are used for quantitation in iTRAQ‐based strategies.

Figure 3

Key milestones in the global analysis of protein kinases and phosphoproteome using mass spectrometry. The past decade has witnessed rapid development of methodologies for global analysis of phosphoproteome. In less than ten years since the initial global phosphoproteomic studies were attempted, the field has evolved from being able to study a few proteins in a single experiment to profiling the dynamics of tens of thousands of phosphorylation sites and almost the entire kinome complement of the cell in a single experiment. Only representative studies are highlighted as even the number of seminal studies is too large to depict here.

Figure 4

Identification of cellular targets of kinase inhibitors. Immobilized kinase inhibitors can be used to identify their cellular targets. The target selectivity of such inhibitors can be determined by incubating lysates from cells labeled using the SILAC method with matrix alone or the small molecule inhibitors bound to the matrix. The relative abundance of non‐specific binders in the two states is unchanged while the relative kinase targets are selectively observed in the context of small molecule inhibitors bound to the matrix. Phosphopeptide enrichment coupled to this strategy will additionally reveal the endogenous phosphorylation status of any enriched kinases.