Post-translational modifications (PTMs) provide a rapid mechanism that enables protein phenotypic diversity so that proteins can react to external and internal disturbances and regulate cellular activity. Advanced mass spectrometry (MS) analysis has allowed for the identification of over 600 distinct PTM classes jointly comprising an order of 106 unique sites but the true functional fraction is unknown.1 Major PTM types include phosphorylation, acetylation, glycosylation, succinylation, methylation, malonylation, SUMOylation, and ubiquitination.2
The focus of vertebrate studies has mainly been on canonical phosphorylation, but research indicates that phosphorylation of other non-canonical amino acids also regulates integral aspects of cell biology. According to Cristina Martin-Granados, PhD, cell signaling research area scientific lead at Abcam, Claire Eyers used strong anion exchange-mediated phosphoproteomics to detect non-canonical phosphorylation. The findings indicated that non-canonical phospho-sites account for approximately one-third of the number of observed canonical phospho-sites.3
Non-canonical phosphorylation is highly susceptible to hydrolysis at low pH and/or at elevated temperature, therefore, standard biochemical techniques of phosphoprotein characterization are largely unsuitable for analysis of “atypical” phosphorylated amino acids.
In addition, the discovery of non-lysine ubiquitylation led to the concomitant revelation that non-proteinaceous ubiquitylation substrates such as glycogen exist.4 “This represents a major paradigm shift in our understanding of ubiquitylation and the breadth of biological processes that it regulates,” said Martin-Granados. Over a decade ago, reports highlighting the existence and biological relevance of non-lysine ubiquitylation first appeared.5 The number of studies has since rocketed.6-11
“PTMs are sub-stoichiometric, highly dynamic, transient, and generally labile in nature,” said Martin-Granados. “They are often present in a small subfraction of the protein population making detection by antibody-based approaches difficult.”
Enrichment of a specific PTM can help tackle low stoichiometry challenges. For example, immunoprecipitation can be performed before Western blotting or MS analysis. Ion exchange, immobilized metal ion affinity, and immunoaffinity chromatography are also enrichment techniques that can be used to segregate PTM proteins/peptides from the unmodified pools, to decrease sample heterogenicity while increasing analytic efficiency and reliability.
PTMs can be cell- or tissue-specific and experiments require very stringent controls. The choice of positive and negative experimental controls is essential to correctly interpreting results. “There is evidence that some PTM modifications can block the binding site of the antibody on its target protein leading to a false negative result,” said Martin-Granados. In addition, since many PTMs are the aftermath of enzymatic reactions, sample processing can affect the target if unwanted enzymatic activity is not controlled.
Characterization of PTMs relies heavily on proteomics analyses. Antibodies are essential detection and enrichment tools. “But the development of highly specific antibodies with exquisite binding affinity remains challenging due to the small size of the PTM chemical moieties, similarities in the chemical structure, and poor antigenicity. In addition, specific recognition of the PTM and surrounding sequence or a pan-PTM may be required,” said Martin-Granados. Polycloncal antibodies can present drawbacks in delivering reproducible and reliable data due to strong lot-to-lot variations.
Computational methods for predicting PTMs are attracting considerable attention. The AI program AlphaFold is a valuable tool to predict unsolved protein structures from their amino-acid sequence.12,13
Unfortunately, AlphaFold2 does not consider the impact of PTMs on protein structure, but databases on protein PTMs and computational tools are available, and NMR spectroscopy and MS largely complement the limitations of AlphaFold2.14,15 With time, it may become possible to create and integrate new algorithms into AlphaFold2-generated structures and PTM databases to achieve a comprehensive outlook for PTM prediction.15
Although advances in MS have enabled the mapping of individual ubiquitin modifications that generate the ubiquitin code, the intricate architecture of polyubiquitin signals has remained largely elusive. Ubiquitin-clipping is a novel methodology that has provided insight into ubiquitin chain architecture and can be useful to decipher combinatorial complexity and architecture.16
The discovery of ester-linked ubiquitin linkages also presents an opportunity to design new antibodies against these linkages. “This will be challenging due to the increased ability of the ester bond when compared to the canonical isopeptide linkage,” said Martin-Granados.
Another recent strategy is specific uncaging-assisted biotinylation and mapping of phosphoproteome, SubMAPP, which integrates an activatable proximity labeling enzyme with an orthogonal phosphorylation enrichment scheme and LC-MS/MS. SubMAPP is a highly sensitive method to characterize the subcellular phosphoproteome in living systems with high temporal resolution.17
- Bradley D. The evolution of post-translational modifications. Curr Opin Genet Dev. 2022 Oct;76:101956. doi: 10.1016/j.gde.2022.101956. Epub 2022 Jul 14.
- Ramazi S and Zahiri J. Post-translational modifications in proteins: resources, tools and prediction. Database, Volume 2021, 2021, baab012. doi: 10.1093/database/baab012
- Hardman G, et al. Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation. The EMBO Journal (2019)38:e100847. doi:10.15252/embj.2018100847
- Kelsall IR, et al. HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation. The EMBO Journal (2022)41:e10970. doi:10.15252/embj.2021109700
- Wang X, et al. Ube2j2 ubiquitinates hydroxylated amino acids on ER-associated degradation substrates. J Cell Biol (2009) 187 (5): 655–668. doi:10.1083/jcb.200908036
- Pao KC, et al. Activity-based E3 ligase profiling uncovers an E3 ligase with esterification activity. Nature. 2018 Apr;556(7701):381-385. doi: 10.1038/s41586-018-0026-1. Epub 2018 Apr 11. PMID: 29643511
- Kelsall IR, et al. The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells. PNAS. 116 (27) 13293-13298. doi:10.1073/pnas.1905873116
- Otten EG, et al. Ubiquitylation of lipopolysaccharide by RNF213 during bacterial infection. Nature. 2021 Jun;594(7861):111-116. doi: 10.1038/s41586-021-03566-4. Epub 2021 May 19. PMID: 34012115; PMCID: PMC7610904
- Zhu K, et al. DELTEX E3 ligases ubiquitylate ADP-ribosyl modification on protein substrates. Science Advances, 2022, 8 (40), ff10.1126/sciadv.add4253ff. ffhal-03862525f
- Sakamaki J and Mizushima N. Protocol to purify and detect ubiquitinated phospholipids in budding yeast and human cell lines. STAR Protocols, Volume 4, Issue 1,2023, 101935, ISSN 2666-1667, doi:10.1016/j.xpro.2022.101935
- McCrory EH, Akimov V, Cohen P, Blagoev B. Identification of ester-linked ubiquitylation sites during TLR7 signalling increases the number of inter-ubiquitin linkages from 8 to 12. Biochem J 9 December 2022; 479 (23): 2419–2431. doi:10.1042/BCJ20220510
- Jumper J, et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). doi:10.1038/s41586-021-03819-2
- Varadi M, et al. Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research, Volume 50, Issue D1, 7 January 2022, Pages D439–D444, doi:10.1093/nar/gkab1061
- Laurents DV. AlphaFold 2 and NMR Spectroscopy: Partners to Understand Protein Structure, Dynamics and Function. Frontiers in Molecular Biosciences. Vol 9 2022. doi:10.3389/fmolb.2022.906437
- Biehn SE and Lindert S. Protein Structure Prediction with Mass Spectrometry Data. Annual Review of Physical Chemistry 2022 73:1, 1-19. doi:10.1146/annurev-physchem-082720-123928
- Swatek KN, et al. Insights into ubiquitin chain architecture using Ub-clipping. Nature. 2019 Aug;572(7770):533-537. doi: 10.1038/s41586-019-1482-y. Epub 2019 Aug 15. PMID: 31413367; PMCID: PMC6823057
- Liu Y, et al. Spatiotemporally resolved subcellular phosphoproteomics. 2021 PNAS 118, e2025299118, doi:10.1073/pnas.2025299118