. Author manuscript; available in PMC: 2020 Jan 5.

Published in final edited form as: Nat Protoc. 2019 Jul 5;14(8):2344–2369. doi: 10.1038/s41596-019-0182-2

Table 1 |.

Spin labels for in-situ PELDOR experiment of proteins/peptides

Label	Linkage	Advantages/disadvantages	Ref.

(A) Nitroxide labels
MTSL (R1)	–S–S– (disulfide) bond via cysteines	small size, high specificity and reactivity, and very well-studied label	35,36,46,47
MTSL (R1)	–S–S– (disulfide) bond via cysteines	reduction of nitroxide label and the –S–S– bond under in-situ conditions
3-maleimido-PROXYL
	–C–S– (thioether) bond via cysteine	stable covalent attachment to the protein less specific and irreversible linkage, may react with Arg or Lys, bulkier than MTSL, and limited literature data	12
M-TETPO
	–C–S– (thioether) bond via cysteine	very stable under reducing conditions and covalent attachment to the protein	38
		disadvantages similar to 3-maleimido-PROXYL
(B) Gadolinium(Gd³⁺) labels
Gd(III)-DOTA-M	–C–S– (thioether) bond via cysteine	stable under reducing conditions and higher sensitivity bulky and less specific, endogenous Mn²⁺ may interfere with in-situ experiments, requires higher frequency (94 GHz) for optimal sensitivity, thiol exchange with glutathione or hydrolysis of the succinimide ring may take place in-situ	14,40
Gd-PyMTA
	–C–S– (thioether) bond via cysteine	smaller linker and higher cysteine specificity, lower affinity than DOTA limited literature data	39
GdL
	–C–S– (thioether) bond via cysteine	smaller linker and higher affinity (for Gd³⁺) and reactivity to cysteines limited literature data	41
(C) Trityl labels
TAM1
	–S–S– (disulfide) bond via cysteines	stable under reducing conditions and higher sensitivity bulky, low water solubility, and tendency for aggregation	44,45 (for the latter, the linker is shorter by one bond)