FIGURE 1.

Principle behind isotope-assisted chemical cross-linking for determination of intra- versus intermolecular cross-links in oligomerized proteins. Recombinant proteins produced with either (i) ¹⁴N amino acids (green) or (ii) ¹⁵N amino acids (red) are thoroughly mixed at a 1:1 mass ratio under denaturing conditions and then allowed to reassociate, resulting in the four combinations shown on the left. The proteins are then cross-linked and fully digested with trypsin. For an intermolecular cross-link (shown in black) between two peptides (A and B), there are four possible mass combinations: ¹⁴A to ¹⁴B, ¹⁵A to ¹⁵B, and the mixed forms ¹⁴A to ¹⁵B and ¹⁴B to ¹⁵A. Thus, intermolecular cross-links will exhibit four mass peaks of approximately equal intensity as shown in the mass spectrum on the right. On the other hand, intramolecular cross-links (shown in yellow) only have two possibilities: ¹⁴A to ¹⁴B and ¹⁵A to ¹⁵B, allowing for an unambiguous determination of the cross-link molecular span.