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Gerald M. Edelman reduced IgG disulfide bonds and used dissociating chemical solutions, including $6\mathrm{mol}{\mathrm{L}}^{-1}$ urea. Ultracentrifugal analysis showed a decrease in molar mass, supporting a multichain protein. In the comparison reproduced in his lecture, starch gel electrophoresis in urea separated serum IgG and a urinary protein from the same patient, alongside their reduced and alkylation-treated preparations. The untreated samples provided the treatment comparison; the urinary material provided a chain-identity comparison. The treated serum sample revealed heavy- and light-chain bands. Two-dimensional peptide mapping compared the urinary protein with the isolated immunoglobulin light chain. Together these readouts supported a shared chain identity.^[ref]

The controls address distinct alternatives. Treatment-dependent separation supports chains joined in the starting protein; a matching gel electrophoresis position alone would not uniquely identify a chain. Agreement of peptide mapping adds an independent chemical comparison. Neither readout gives atomic coordinates, and this patient's preparation does not establish a common protein primary structure for all antibodies.

Reduction targets protein disulfide bonds, while protein denaturation weakens the folded assembly. Neither operation is the proteolysis that cuts peptide bonds. This explains why chain separation and Papain digestion of IgG give different inventories: four intact chain backbones in the first case, three assembled fragments in the second.

Peptide mapping compares separated peptides produced by cleavage of a protein. Matching patterns provide a chemical comparison beyond an intact protein's gel electrophoresis position.

Trypsin is a serine protease: an enzyme that performs proteolysis by catalysing peptide bond hydrolysis. It preferentially cuts on the C-terminus side of lysine and arginine amino acid residues, leaving the selected amino acid residue in the preceding fragment.^[ref]

The bond being broken is the backbone amide linkage, rather than the lysine or arginine amino acid side chain. A formal connectivity equation for hydrolysis isHere $R^1$ and $R^2$ stand for the connected portions of the original peptide. Actual terminal protonation and electric charge depend on pH; the equation displays connectivity. Water supplies the elements needed to form the two new ends, while the enzyme is regenerated.

Selective cutting turns an unwieldy protein into shorter peptides, making peptide mapping useful for comparing proteins. In cell culture, trypsin also helps detach adherent biological cells: the HeLa culture viability experiment provides a concrete example of its use and subsequent stopping of proteolysis. The same enzyme can therefore serve both chemical analysis and preparation of a biological cell suspension.