Abstract
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.
Highlights
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
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
The extracellular Hb1 from the marine polychaete Tylorrhynchus heterochaetus is a typical HBL Hb found in annelids and vestimentiferans (l-3).lts molecular mass of3.38 MDa (4) and molecular dimensions obtained by a scanning transmission electron microscope and small angle x-ray scattering (5) are similar to other HBL Hbs
Summary
The Hb was prepared as described previously (7), dialyzed against distilled, deionized water, lyophilized, and stored at -70 "C. ESI-MS data were acquired on a VG Quattro II electrospray mass spectrometer (VG Organic, Altrincham, Cheshire, UK), using sample concentrations of 0.5 iLg/iLl in 50% aqueous acetonitrile containing 0.2% formic acid. ESI-MS produces a series of multiply charged ions on the m/z scale from each protein in the sample. On this scale, m / z = (M + nH)/n, where M is the mass of the protein, H is the mass of the proton, and n is a series of consecutive integers. Since the Hb data arise from several proteins, each producing a series of 5-10 multiply charged ions, they were processed in order to condense each series into a single peak on a true molecular mass scale.
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