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Collagen IV, also called type IV collagen, is the network-forming collagen that provides a major scaffold of basement membranes. Three polypeptide chains assemble into a molecular unit, and units associate through their ends into a sheet-like network. This organization suits a thin supporting layer beneath an epithelium, rather than a long cable made of parallel molecules.^[ref]

The Collagen IV NC1 domain is a noncollagenous end region where adjoining molecular units meet. A sulfilimine bond links a methionine side chain in one unit to a hydroxylysine side chain in another. Thus the scaffold combines molecular association with a chemically specific covalent bond.^[ref]

One can distinguish two questions about a basement membrane: which proteins form its scaffold, and how neighboring units are fastened together. Isolating linked peptides and comparing their mass spectrometry and nuclear magnetic resonance signals answered the second question. The investigation connected a tissue-scale network to the identities of individual amino acid residues.^[ref]

R. Timpl coauthored the 1979 isolation and characterization of laminin, a large glycoprotein associated with basement membranes. The researchers isolated it from a mouse tumour producing abundant basement-membrane material and used antibodies to investigate its distribution.^[ref]

Purification and localization answer complementary questions. Purification establishes a distinguishable molecular component; localization connects that component with a tissue structure. Finding both laminin and type IV collagen in the material helped show that a basement membrane is an organized extracellular assembly, rather than a layer made of one interchangeable protein.

A sulfilimine bond is a covalent sulfur–nitrogen linkage conventionally represented with an $\mathrm{S}=\mathrm{N}$ unit. In type IV collagen, it links the sulfur of a methionine side chain to the nitrogen of a hydroxylysine side chain from a neighboring protein unit. It is chemically different from a disulfide bond, which links two sulfur atoms.^[ref]

The collagen example makes atom identity important. Although hydroxylysine contains a hydroxyl group, the identified interprotein linkage involves its nitrogen, not that oxygen. Linking separate protein units reinforces the type IV collagen network of the basement membrane.

The 2009 identification combined two kinds of evidence. High-accuracy mass spectrometry constrained the composition of isolated crosslinked peptides, while nuclear magnetic resonance supplied information about the local chemical environment. Together these supported the sulfur–nitrogen assignment; mass alone would not uniquely specify how atoms of a given composition are connected.^[ref]

This is a useful example of why a protein crosslink needs a chemical description as well as a mechanical one. Observing that two protein units remain connected can establish a covalent association under the measurement conditions, but identifying the bond requires evidence about the participating atoms and their bonding environment.

Type IV collagen is a network-forming collagen of the basement membrane, the specialized extracellular matrix underlying an epithelium and supporting other tissue interfaces. Triple-helical molecular units associate into a sheet-like scaffold and are reinforced by intermolecular covalent bonds.^[ref]

The chemistry of that reinforcement was identified using mass spectrometry and nuclear magnetic resonance. Researchers isolated linked peptides from adjoining units and identified a sulfilimine bond between a methionine residue and a hydroxylysine residue. Chemical reduction broke the linkage and recovered the constituent peptides, supplying a further test of the proposed bond.^[ref]

The example connects three scales: the identity of an amino acid side chain, a chemical link between proteins, and stabilization of a supporting tissue network. Knowing only that a specimen contains collagen leaves these structural and chemical distinctions unresolved.