In our presentation at the ASMS meeting, we discussed two highly complementary phosphopeptide enrichment strategies and contrasted their differences,” said Charles L. Farnsworth, Ph.D., scientist at Cell Signaling Technology (CST). Post-translational modifications are key regulators of protein function, and they play fundamental roles in development, disease, and homeostasis, but because they often exist at low levels, their identification and the analysis of the cellular processes that they regulate is challenging.
In this approach, Dr. Farnsworth and colleagues used two enrichment strategies. The first was immunoaffinity enrichment using several classes of antibodies directed against phosphorylated amino acid sequence motifs; the second, immobilized metal affinity chromatography (IMAC), which is a charge-based metal affinity approach.
The immunoaffinity LC-MS method was performed using PTMScan, CST’s proteomic technology. It allowed CST scientists, said Dr. Farnsworth, to enrich for and quantitate phosphopeptides that would not have been detectable by IMAC alone. Dr. Farnsworth and colleagues identified and profiled over 20,000 phosphopeptides from kinase inhibitor-treated gastric carcinoma cells, with a low false-positive discovery rate, demonstrating the strength of these two complementary approaches in capturing post-translational protein modifications for comprehensive phosphopeptide profiling.
These data helped generate a global view of the phosphorylation in cells, and allowed the annotation of an MAPK pathway and a tyrosine kinase pathway, key steps toward unveiling potential therapeutic targets related to the dysregulation of signaling in these pathways. The same strategy can also be used for other post-translational modifications, such as acetylation, succinylation, methylation, and ubiquitination. In addition, the strategy helped identify new drug targets, providing a platform to characterize substrates of specific signaling proteins, assess the effects of candidate therapeutic agents, and guide the design of subsequent studies.
Moreover, the use of multiple types of antibodies helps dissect the crosstalk between various types of post-translational modifications and clarify their interplay as they orchestrate various cellular processes. A comparative analysis revealed very little overlap between the phosphopeptides that were identified by PTMScan and the ones identified by IMAC, indicating that these two distinct methodologies enrich for different classes of phosphopeptides and present a high degree of complementarity to one another.
“Ideally, as we move ahead with this work, we would like to reduce sample quantity and develop a high-throughput platform to make our analysis more amenable to robotics, to profile many different types of tissues and gather information from larger cohorts and larger studies,” offered Jeffrey Silva, Ph.D., a group leader at CST.
In the course of these experiments, Dr. Farnsworth and colleagues also revealed that this approach can be used even when the nature of the post-translational modifications is not known prior to the experiment. In such a case, a panel of the PTM motif antibodies can be used to perform Western blot analyses to screen for the antibodies best suited for the particular project of interest.
In addition to the motif antibodies used for discovery-based PTM proteomics, CST has also developed other immunoaffinity reagents for LC-MS applications to monitor the activity of specific cellular pathways. “This is different from the motif antibody reagents described earlier because it allows investigators to focus solely on specific signaling pathways that are critical to the underlying biology of their research interests,” noted Dr. Silva.