Characterization of the disulfide in isolated U24 protein. A) Illustration of isolated U24 protein, demonstrating the formation of a single intramolecular disulfide bond between the only two cysteines in the molecule (i). N-ethylmaleimide (NEM) does not react with the oxidized, disulfide bonded protein (ii) and NEM only modifies cysteines in the reduced form (+125 Da per cysteine) once they are reduced by tris(2-carboxyethyl)phosphine (TCEP) (iii). Use of glutamyl endoproteinase (GluC), which cleaves peptide bonds primarily after glutamic acid under these conditions, yielded two fragments if U24 contained an intramolecular disulfide (iv). Using dithiothreitol (DTT) to reduce the disulfide in U24 (v), GluC cleavage then gave three proteolytic fragments for U24 (vi). B) Tris-Tricine SDS-PAGE results of U24 (isolated from SHuffle) ± DTT and GluC-digested. M: molecular markers; oxidized U24 ((i), 10.2 kDa) shifts to a lower mass once cleaved ((iv), 8.6 kDa) and U24 reduced by DTT ((v), 10.2 kDa) shifts to even lower mass when cleaved ((vi), 6.1 kDa). C) MALDI TOF MS analysis of U24 modified with NEM ± TCEP, and cleaved by GluC ± DTT. Tabulated theoretical masses represent U24 species indicated in A) (i-vi). Experimental masses that were detected are ± 0.2% the theoretical mass.