Electrospray ionization mass spectrometric determination of the complete polypeptide chain composition of Tylorrhynchus heterochaetus hemoglobin.

Electrospray ionization mass spectrometry (ESI-MS) of the native, reduced, and carbamidomethylated forms of the extracellular, 3.38-MDa hemoglobin from the marine polychaete Tylorrhynchus heterochaetus, when combined with a maximum entropy (MaxEnt) analysis, provided a complete description of the polypeptide chain composition. This hemoglobin, a hetero-multimeric complex of approximately 180 polypeptide chains, consisting of globin and linker subunits in an approximately 3:1 mass ratio, is among the largest protein complexes investigated by ESI-MS. The globin subunits consist of a monomer subunit (chain I, 15575.4 Da) and a disulfide-bonded trimer subunit, 50068.4 Da, consisting of globin chains IIA (16601.9 Da), IIB (16680.4 Da), and IIC (16,794.0 Da). Linker subunits L1-L5, 23233.8, 24835.4, 25326.9, 28202.2, and 26317.2 Da, respectively, were found together with a disulfide-bonded dimer of L2, 52609.4 Da. Using the exact masses of the subunits, a plausible model of the hemoglobin consisting of 144 globin chains (36 monomers and 36 trimers) and 36 linker chains provides a calculated mass of 3.42 MDa.