Summary of chemical tools suitable for IDP labeling and modulation, as discussed in the text.

Goal	Strategy	Advantages	Disadvantages	References
Purification of IDPs	Purification under denaturing conditions	• In principle, compatible with all IDP/IDRs	• Denaturant must be removed	27, 28 and 172
			• Refolding required for constructs that contain both folded and IDR domains
	Purification at extreme pH	• Straightforward recovery of target protein	• Applicable to only a small subset of proteins	18 and 22–26
	Purification at extreme temperature	• Straightforward recovery of target protein	• Applicable to only a small subset of proteins	26
Labeling with spectroscopic or imaging probes	Maleimide or iodoacetamide-based probes	• Site-specific at cysteine residues	• Not applicable in cells	36–41
		• Compatible with isotopic labeling for NMR	• Potential labeling at multiple sites
			• Probes may perturb IDP function and LLPS
	N-Hydroxysuccininimide ester-based chemistry	• Site-specific at lysine residues	• Not applicable in cells	42
		• Compatible with isotopic labeling for NMR	• Potential labeling at multiple sites
			• Probes may perturb IDP function and LLPS
	Fusion protein-based approaches	• Genetically encoded	• Large fusion proteins may perturb IDP function and LLPS	See ref. 41 and 183 for comparison of the effects on LLPS of small molecule labeling vs. fusion protein labeling
		• Applicable in cells
	Bioorthogonal chemical approaches and amber suppression	• Genetically encoded	• UAA incorporation may be inefficient and lead to low protein yields	56, 61–67, 70, 184 and 185
		• Applicable in cells	• Probes may perturb IDP function and LLPS
		• Site-specific labeling
		• May be compatible with isotopic labeling for NMR
	Native chemical ligation and expressed protein ligation	• Site-specific labeling	• Segments must be accessible synthetically	77, 79, 116 and 118
		• Multiple probes can be introduced at the same time	• Difficult to label sites away from the N- or C-terminus	Also see relevant references for introducing PTMs through NCL and EPL below
		• Access to a wide range of chemical probes	• Final protein yields may be low
		• May be compatible with isotopic labeling for NMR	• Not applicable in cells
			• Probes may perturb IDP function and LLPS
Segmental labeling for NMR spectroscopy	Cfa GEP	• Trans-splicing can be achieved under denaturing conditions	• Splicing reaction must take place at a cysteine residue	29, 79 and 172
		• High efficiency
		• Robust to a range of extein sequences
		• May be applied in cells
	MCM2	• Salt-inducible splicing can be used to control intein activity	• Splicing cannot be performed under denaturing conditions	83
		• Robust reactivity under high-salt conditions	• May not be applicable in cells
		• Serine-based reaction mechanism provides versatility	• Splicing rate and extein sequence compatibility are poor compared to Cfa
	Sortase	• A possible alternative to intein-based methods	• Requires the insertion of a five/six amino acid scar into the target protein	90–96 and 186
		• May be used to attach proteins to cell surfaces	• Continued reaction between reactants and desired product may reduce yield
Introduction of post-translational modifications	Enzymatic methods	• Straightforward introduction of native PTMs	• Lack of control over stoichiometry and location of PTM installation	100–102
	Genetic encoding of a bioisostere	• Straightforward introduction of PTM mimics	• Scope of PTMs is limited to those that can be mimicked effectively by a bioisostere	23, 46 and 104
		• Applicable in cells
	Amber suppression	• Introduction of native PTMs or PTM mimics	• Scope of PTMs is limited to those that have a tRNA synthetase available	105 and 106
		• Applicable in cells	• UAA incorporation may be inefficient and lead to low protein yields
	Cysteine alkylation	• Efficient and specific introduction of methyl-lysine mimics	• Methyl-lysine mimic may not faithfully reproduce the function of the native PTM	41, 107, 109 and 110
		• Reactions are compatible with denaturing conditions	• Not applicable in cells
	Native chemical ligation and expressed protein ligation	• Site-specific introduction of PTMs	• Segments must be accessible synthetically	125–128, 187 and 188
		• Multiple PTMs can be introduced at the same time	• Difficult to introduce PTMs away from the N- or C-terminus
		• Access to a wide range of modifications	• Final protein yields may be low
			• Not applicable in cells