Table 1.

Strategies to model the presence and absence of PTMs on proteins

Strategy	Advantages	Limitations	Representative references
Proteins naturally modified in cells under known conditions	• Straightforward to access with few manipulations • The absence of PTM can be readily modeled by mutating the target site or knocking out/down the ‘writer’ protein	• Conditions leading to many PTMs are not known • Degree of endogenous modification is often heterogeneous • Site-specific modification may be difficult to achieve	[12,13]
Use of small synthetic peptides harboring the PTM in its native context	• Readily generated through chemical synthesis • Wide variety of modifications can be accessed • Possible to model multiple PTMs within the same peptide • Can be equipped with photo-crosslinkers • Can be used in higher throughput format such as peptide microarray	• Does not capture the context of the full-length folded protein, which may partially or completely abolish interaction with partners • Cannot be used to capture PTM-triggered PPIs that do not directly involve the modified residue	[14–25]
Use of natural amino acids that mimic a PTM	• Easy to implement through straightforward mutagenesis • Introduces modification at specific site(s)	• Significant structural differences often result in imperfect mimicry • Most PTMs cannot be modeled using a natural amino acid	[12,28–32]
Using the endogenous ‘writer’ protein(s) to install a PTM	• Can be relatively simple to generate using recombinant writers • Engineered writers can be generated to target specific substrate proteins	• Biochemical origin of many PTMs are unknown or hard to reconstitute • Can be difficult to homogeneously modify specific site(s) • Some PTMs are not enzymatically installed	[33–35]
Protein semi-synthesis through expressed protein ligation (EPL)	• Homogeneously modified full-length proteins can be generated • A wide variety of natural/synthetic modifications can be installed • Multiple, different modifications can be installed	• Technically demanding • EPL is performed under denaturing conditions; refolding the resulting protein may be challenging • Typically precludes experiments in living cells • Internal modifications on larger proteins are challenging to access	[36–41]
Genetic code expansion (GCE)	Homogeneously modified full-length proteins can be generated Close structural mimics of PTMs can be incorporated that are resistant to removal Modified proteins can be expressed in living cells The modified residue can be theoretically incorporated into any site of any protein that can be recombinantly expressed	A limited number of PTMs have been genetically encoded Efficiency of incorporation is site-dependent Incorporation of multiple modifications into one proteins can be challenging	[42,43,45–50,52–56]