Fragmentation of 819 knots 1–3 under CID-MS experiments. (A) CID-MS of knot 1. (B) MS-MS of [M+2H]2+ (m/z = 1,690.75) from knot 1, only type I fragmentation is observed. (C) CID-MS of knot 2. (D) MS-MS of [M+2H]2+ (m/z = 1859.00) from knot 2, both types of fragmentations are observed. (E) CID-MS of knot 3. (F) MS-MS of [M + 2H]2+ (m/z = 2,027.09) of knot 3, only type II fragmentation is observed. No fragment ions corresponding to macrocycles, which could arise from molecular links (e.g., a [2]catenane or Solomon link) upon fragmentation of one ring (17), was observed over a range of collision energies, further demonstrating that only knots are present. (G and H) Two different fragmentation pathways (type I, G; type II, H) for the 819 knots corresponding to the MS-MS results shown in B, D, and E; bond cleavage is indicated with a red wavy line, the residues lost in the first step are colored purple, and those lost in the second step are colored blue.