FIGURE 5.

Isopeptide cross-links observed in the MUC2 mucin isolated from intracellular granulae of LS174T cells. a, insoluble MUC2 complex was isolated from confluent LS174T cells, reduced-alkylated, separated by AgPAGE, and stained with zinc imidazole. X-O represents the oligomeric MUC2; X-D represents the dimeric, and M represents the monomeric isoform of MUC2. A representative separation of three replicates is shown. The separated bands were cut out, treated with chymotrypsin, and followed by analysis by LC-MS. b–h, annotated MS2 fragment spectra of the parent ions of the observed cross-linked peptides. y-ions are labeled in blue; b and a ions are labeled in red. Immonium ions for leucine or isoleucine, asparagine, phenylalanine, tyrosine, and valine are labeled with their one-letter amino acid code, respectively. b, MS2 fragment spectrum of parent ion [M + 2H]²⁺ 617.31. c, MS2 fragment spectrum of parent ion [M + 2H]²⁺ 553.78. d, MS2 fragment spectrum of parent ion [M + 2H]²⁺ 790.84. e, MS2 fragment spectrum of parent ion [M + 2H]²⁺ 469.75. f, MS2 fragment spectrum of parent ion [M + 2H]²⁺ 824.33. g, MS2 fragment spectrum of parent ion [M + H]²⁺ 920.43. h, MS2 fragment spectrum of parent ion [M + 2H]²⁺ 483.25.