A protein’s structure determines its function. That structure arises from a protein’s sequence of amino acids, folding patterns, and variations, including post-translational modifications (PTMs), which come from chemical changes, such as phosphorylation and glycosylation. Beyond the intrigue of basic biological mechanisms, PTMs play a role in human health. To explore this wide world of proteins, scientists need an expanding array of analytical techniques, and the options keep increasing.
“The human proteome is understood to be coded by just 20,000 genes, and much of the complexity in human proteins arises from alternative splicing products or PTMs,” says Tom Knapman, global marketing manager, life science research and omics at SCIEX. “There are over 300 known PTMs, which are understood to play an important role in cell signaling.” In fact, disease research often targets enzymes that regulate PTMs.
“PTMs are prevalent with respect to infectious disease,” says Nevan Krogan, director of the Quantitative Biosciences Institute at the University of California San Francisco. “When cells get infected with an infectious agent, a pathogen can have the quickest effect by manipulating proteins already made, which can be done by hijacking enzymes, like kinases, that are responsible for PTMs.” 
For example, HIV has accessory proteins that hijack the ubiquitination pathway, which modify proteins with ubiquitin.
Image: Schematic representation of connections between kinases and the substrates that they target. Image courtesy of Nevan Krogan and Mike Shales.
PTMs play a role in the aggregation and degradation of proteins.
PTMs can also be used in other approaches to biomedical research. “Protein PTMs are emerging as important biomarkers for disease states, such as heart disease, cancer, diabetes, and neurological disorders,” says Shweta Shukradas, bioinformatics product manager at Agilent Technologies. For instance, PTMs play a role in the aggregation and degradation of proteins. Altering those processes, Shukradas says, “can result in aberrant signaling leading to disease.”
Tracking those signals, however, requires a range of analytical tools.
The PTM toolbox
Beyond the variety posed by PTMs themselves, proteins in general create a challenging analytical environment. “Dynamic range and sensitivity pose a critical challenge to accurate quantitation of PTMs, as proteins span a wide range of concentrations as they are naturally expressed,” Shukradas explains. “Understanding the biology of a system may require accurate quantitation of critical PTMs on low-level proteins, in conjunction with the more highly expressed proteins.”
To enhance the odds of finding PTMs, scientists enrich the protein modifications in a sample. Biological and chemical techniques exist for doing this.
To discover new PTMs, scientist often use liquid chromatography followed by mass spectrometry (LC/MS). To precisely analyze levels of known PTMs, researchers might turn to selected reaction monitoring/multiple reaction monitoring (SRM/MRM), a technique that uses LC/triple-quadrupole MS (LC/QQQ) instruments, which provide high sensitivity and dynamic range.
Knapman points out that data-independent methods, such as SCIEX’s SWATH Acquisition, collect MS-MS data on everything—including all modified forms of peptides. “The real beauty of SWATH Acquisition, though, is the retrospective nature of the data, which can be mined over and over again to obtain more and more information,” Knapman explains. “Therefore, one can look for known or expected modifications in a targeted manner, and screen MS-MS data to discover new sites of modification from the same dataset.”
As an example, Knapman notes work by Mark Molloy of Macquarie University in Australia and his colleagues. These researchers used SWATH Acquisition to identify “known glycopeptides using a targeted approach,” Knapman explains. Molloy’s team “subsequently mined the same dataset in a speculative manner and discovered 21 glycoforms of IgG1, including two truncated forms that have been rarely reported.”
Data-dealing options
The sensitivity of MS platforms can produce a long list of PTMs. “The biggest bottleneck is deciding which ones to focus on,” says Krogan. By comparing different PTM datasets, Krogan found several ways to pick out the most relevant PTMs. For example, PTMs in a region involved in protein-protein interactions and PTMs in evolutionarily conserved regions of a protein are more likely to be functionally relevant. Other aspects also matter. “If a phosphorylation site is near a ubiquitination site, they are both more likely to be relevant,” Krogan says. “You see commonalities in PTM patterns between infectious diseases and cancer.” So, this kind of computational analysis promises many opportunities ahead, especially in finding new treatment targets.
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Other experts agree on the value of computational approaches and software. As an example, Shukradas points out that Skyline—software developed in the lab of Michael MacCoss at the University of Washington, Seattle—can be used “to facilitate transition from protein and PTM discovery into targeted analysis to maximize efficiency.” This software is free, open-source, and works with various data formats. “Agilent has developed a tight integration between its mass spectrometry software called MassHunter and Skyline software that enables experimentally collected LC/quadrupole-time of flight data to be used to produce MRM methods, that can then be directly run on an LC/QQQ in a more targeted fashion,” Shukradas explains. “Skyline’s local-client interface allows for rapid analysis and quantitation of the results.”
Giving more scientists an opportunity to explore data also provides an increasingly available option for working with PTMs. Juan Antonio Vizcaíno, proteomics team leader at the European Bioinformatics Institute, says, “Public proteomics data can be used to find non-previously detected PTMs, and there are different ways to do it. For example, Vizcaíno and Lennart Martens, professor of systems biology at Ghent University, wrote that proteomics data can be repurposed to look for novel PTMs. As Vizcaíno explains, “Public data can be re-analyzed with new hypotheses in mind. For instance, maybe someone did phospho-proteomics experiments that were originally analyzed without considering the possible presence of other PTMs, in addition to phosphorylation.” Although phosphorylation is studied widely, other forms of PTMs exist, such as glycosylation. So, Vizcaíno and Martens wrote: “It would be highly beneficial to achieve a closer interaction between existing proteomics and glycomics resources.”
Specific PTMs impact a range of health problems. As Knapman points out: “It has often been observed that many diseases, particularly cancers, can be classified as having a malfunction in kinase activity, and therefore quantitative profiling of protein phosphorylation is often performed in the search for potential biomarkers of a number of cancers.”
Other forms of PTMs also play a role in healthcare. “Cell-surface proteins are almost always glycosylated, and the characteristic patterns of glycosylation play an important role in cellular recognition and immune response,” Knapman says. “As a result, it is perhaps unsurprising that these proteins are often implicated as drug targets due to the specificity of this recognition.”
The expanse of proteoforms and concentrations creates a challenging analytical scenario, and PTMs increase the complexity of connecting structure with function. Given the role of PTMs in basic biology gone right and wrong, it’s worth the effort to learn more about these chemical changes and how they impact biology. By combining hardware and software tools, scientists can consider various pathways to tracking PTMs. To really dig deeply as fast as possible, working together promises new opportunities. For example, a team of scientists from Taiwan developed CruxPTM—a database tool that the developers say “could help scientists narrow the scope of their PTM research and enhance the efficiency of PTM identification in the face of big proteome data.” As scientists combine their knowledge of PTMs, new medical approaches will surely emerge.