PTMs: Uncovering Another Layer in Protein Diversity

Post-translational modifications (PTMs) alter the properties of a translated protein by adding a functional group or protein to at least one of its amino acid residues.¹ PTMs also alter protein phenotypes without changes to coding sequences (CDSs), substantially increasing protein diversity.² According to Ryan Bomgarden, Senior R&D Manager at Thermo Fisher Scientific, “PTMs can provide various chemical modifications that are used to change the charge, solubility, stability, and structure of proteins. The wide variety of PTMs stems from enzymes evolving to modify different amino acids through their unique functional groups.” PTMs have also been implicated in age-related diseases and present as possible targets for treating disease.³

Challenges to PTM detection

PTMs remain difficult to survey due to biological diversity and technical constraints. Each of the PTMs that exist will have distinct chemical properties that require distinct isolation methods.⁴ These modifications can also be expressed at low concentrations. “While PTMs occur on a small fraction of the total protein population, their low occurrence does not equate to physiological irrelevance. PTMs can act as fine-tuning mechanisms to precisely control protein activity, even at low concentrations,” notes Henrick Horita, Marketing and Sales Manager at Cytoskeleton.

Enriching PTM-modified proteins with immunoprecipitation (IP)

The low concentration and high diversity of PTMs necessitate using methods that enrich specific PTMs. IP represents the most common approach for this, where either all PTM species for a given protein or all proteins with a specific PTM can be isolated. Either way, Horita says that “affinity kits such as the ones produced by Cytoskeleton contain affinity matrices comprised of antibodies or binding domains that specifically recognizes the PTM.” The method of PTM enrichment used is also important when assaying PTMs. Mike Knierman, Biopharma Workflow Manager at Agilent Technologies, explains that “the PTM enrichment method must be careful not to skew PTM recovery. For example, a protocol that uses high pH in sample processing can lose phosphate groups by hydrolysis, cause deamidation of asparagine and glutamine, or reshuffle disulfide bonds.”

Western blotting: Initial approaches to PTM identification

After protein enrichment, a series of techniques have been prepared to characterize PTMs further. Among these is the western blot, a foundational analytical technique for separating and detecting proteins. The technique relies on antibodies that specifically label the protein or the PTM within the protein. While they are useful for detecting PTMs in specific proteins, they also face challenges in throughput. “Antibodies that target specific PTMs are difficult to generate because cross-reactivity to the native protein can occur,” Knierman says.

Proximity ligation assays: Identifying protein interactions and improving PTM detection

The reliance on antibodies to bind specifically to the protein or PTM of interest raises the risk of detecting proteins other than the protein of interest.⁵ Proximity ligation assays mitigate this issue by employing an antibody pair conjugated by connector oligonucleotide sequences.⁶ The technique relies on rolling circle amplification (RCA) to generate a single-stranded sequence of the template that can be detected by hybridizing probes in a fluorescence-based assay. The key to this assay, notes Bomgarden, lies in the “additional specificity for antibody target binding as the signal is only generated when the nucleotide-linked antibodies are close enough for ligation.” A series of proximity ligation assays have been developed for fluorescence-based quantification and visualization, including MilliporeSigma’s DuoLink^® Proximity Ligation Assay and Thermo Fisher’s TaqMan^TM Protein Assays Open Kit. According to Bomgarden, TaqMan Kits are “extremely sensitive as RT-PCR is used to amplify low signals and provide more reproducible quantitation of PTMs and proteins.”

In vitro assays: detecting PTM activity

In vitro assays are another useful complement to western blots for characterizing PTMs. These experiments focus on purifying the proteins of interest and determining whether they contain a specific PTM. This is accomplished by incubating proteins with specific enzymes, substrates, co-factors, and energy sources to investigate protein activity. Some assays feature a form of labeling to identify phosphate transfer events. Radioactive labeling of the ³²P isotope from the γ position of ATP into a peptide represents a means to monitor kinase activity for a given protein.⁷ Other assays, such as Time-Resolved Forster Resonance Energy Transfer (TR-FRET), use antibodies that label phosphopeptide or phosphotyrosine residues to detect phosphopeptide accumulation and kinase activity.⁸ Regardless of the assay, fine-tuning the methodological parameters—readout technology, protein purification, kinetic and thermodynamic parameters, and experimental conditions—is essential for yielding biological insights.⁹

Mass spectrometry (MS): High-throughput PTM characterizations

Mass spectrometry helps overcome the challenges inherent in western blot and other in vitro techniques for characterizing PTMs. This approach relies on ionizing molecules and proteins to the gas phase. “Mass spectrometry does not require PTM-specific reagents to identify and quantify multiple PTMs in a single run,” Kniermann adds. “Additionally, mass spectrometry has greater specificity and better quantitation capability than western blotting techniques. It is sometimes useful to isolate all peptide fragments with the PTM, but it is not always necessary.”

Many mass spectrometers can be used to identify PTMs with this approach,¹⁰ including the Agilent 6545XT AdvanceBio Q-TOF spectrometer. Knierman puts it this way, “This spectrometer has the capability for bottom-up and top-down approaches to PTM characterization. The top-down approach can help pinpoint the location of and relationship of PTMs within a protein. Bottom-up approaches provide more complete fragmentation coverage and localization of all PTMs but may lose the context of the PTM’s relationships with each other.”

Conclusions

PTMs play key roles in diverse biological processes. Despite their importance, the study of PTMs faces several challenges, including their high diversity and low concentrations in vivo. Nonetheless, numerous techniques to characterize these PTMs exist. Some of these methods, like western blots and in vitro assays, can detect proteins with a specific PTM or all PTM species within a protein. Other approaches can identify changes in PTM activity or identify protein interactions and modifications. For high-throughput PTM characterization, mass spectrometry remains the method of choice. Which of the protocols is used depends on the research questions being asked. But with the plethora of methods available, we have an unprecedented opportunity to better understand PTMs and their role in human disease and health.

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