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Sep 1, 2014 (Vol. 34, No. 15)

Protein Profiling Plumbs Hidden Depths

  • “We have not reached solutions; we have only begun to discover how to ask questions”—these words, set down decades ago by Lewis Thomas, remain as true as ever, inspiring ever more profound (and varied) scientific pursuits.

    One such pursuit is protein profiling, or the analysis of proteins at the proteomic scale.

    While it provides comprehensive information about protein structures and functions, protein profiling is not an end in itself. It is, rather, a means of visualizing cellular pathways and networks. And even this layer of information is not yet the end. Yes, the proteome and the signalome are interesting, but what really matters is their role in health and disease.

    Protein profiling is a way to expose the processes that orchestrate development and maintain (or disturb) tissue homeostasis. Accordingly, protein profiling may drive the development of novel therapeutics. But even if it were to become a commonplace drug development tool, protein profiling would hardly exhaust its potential.

    Protein profiling is not merely factual. It is also conceptual. And the concepts engendered by protein profiling, like all concepts, are developed, refined, and reshaped over time. One of the key lessons that science has provided is that even topics thought to be thoroughly understood may need to be revisited frequently. So it may prove with protein profiling. If so, the subject’s conceptual progress will likely rely on technological advances—but these are already emerging.

  • Proteoform Characterization

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    Researchers from the University of Minnesota and Thermo Fisher Scientific rapidly profiled proteomes and subproteomes with the Orbitrap Fusion Tribrid mass spectrometer. They focused on lysine acetylation, a key post-translational modification.

    Summing up the legacy of decades of research, Steven R. Danielson, Ph.D., chemist at Thermo Fisher Scientific, stated, “The details of how biology works are more complex than we ever imagined.” To make sense of these details, Dr. Danielson insisted, it is “vital to develop high-performance instruments and software.”

    These comments were made, appropriately, after the recent American Society for Mass Spectrometry (ASMS) meeting in Baltimore. At this meeting, Dr. Danielson and colleagues presented work performed in collaboration with colleagues from the University of Minnesota using the Orbitrap Fusion Tribrid mass spectrometer, an instrument that was released just one year earlier. “The Orbitrap Fusion provides investigators the opportunity to collect very high-coverage proteomic data,” said Dr. Danielson.

    This work stemmed from the acute need to develop a sensitive and rapid approach to survey post-translational changes at the proteomic scale. Focusing on lysine acetylation, a key post-translational modification that shapes many cellular processes, including gene expression, transcription regulation, and the function of cytoskeletal proteins, Dr. Danielson and colleagues characterized peptides from Escherichia coli and Caenorhabditis elegans tryptic lysates using a combination of rapid immunoaffinity enrichment and mass spectrometry.

    This approach helped identify 3,452 unique proteins from the C. elegans lysate, representing at least 17% of the organism’s proteome, and 1,666 acetyl-lysine-modified peptides from the E. coli lysate. These data were obtained after 55-minute and 45-minute liquid chromatography runs, respectively. These figures suggest that the approach is both sensitive and speedy.

    A research effort at Thermo Fisher Scientific was launched recently to develop top-down proteomics approaches. The idea is to use the Orbitrap Fusion platform to characterize the spectrum of individual proteoforms of a specific protein.

    “The global pattern of proteoforms is relevant to specific biological states and diseases,” explained Dr. Daneilson. “It involves not just determining that a specific modification is present, but also exploring its relevance to get a more comprehensive look at what happens to a specific protein rather than an isolated view of modifications that are present on specific amino acids.”

  • Phosphopeptide Enrichment

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    Cell Signaling Technology has investigated phosphopetide enrichment using two complementary methods: immunoaffinity enrichment and immobilized metal affinity chromatography (IMAC).

    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.

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