The correct molecular weight of myoglobin, a common calibrant for mass spectrometry

Abstract

Myoglobins from horse heart muscle, horse skeletal muscle and sperm whale are widely used as calibration standards or test compounds for various mass spectrometric methodologies. In all such cases reported in the literature, a molecular weight value is used (16,950.5 and 17,199, respectively) which is based on the assumption that amino acid 122 in this 153 amino-acid-long protein is asparagine, overlooking a published suggestion that it is aspartic acid instead. Since the mass assignment accuracy for matrix-assisted laser desorption mass spectrometry is reported to be +/- 0.01% and for electrospray ionization +/- 0.0025%, and error of one mass unit in approximately 17,000 would be significant. The mass-to-charge ratio of ions of the tryptic peptide encompassing amino acid 122 derived from commercially available horse heart and horse skeletal myoglobins, the apomyoglobin of the latter, and the tryptic and chymotryptic peptide of sperm whale myoglobin proved that in both proteins amino acid 122 is indeed aspartic acid, rather than asparagine. This finding was further confirmed by the collision-induced dissociation spectra of the [M + H]+ ions of the tryptic peptides from the horse myoglobins and the chymotriptic peptide from sperm whale myoglobin. Thus, the correct molecular weight of horse myoglobin is 16,951.49 and that of the sperm whale protein is 17,199.91.

Capillary isoelectric focusing–mass spectrometry: analysis of protein mixtures from human body fluids

“…Transformation of these data yielded a relative molecular mass (M r ) for myoglobin of 16,951.0 Da [ Fig. 4(b)], which is in very good agreement with the calculated molecular weight of myoglobin (16,951.5 Da;Zaia et al, 1992).…”

Rapid validation of molecular structures of biological samples by electrospray‐mass spectrometry

“…It should perhaps be noted that this sequence had been adopted by the group which determined the 3D structure of horse heart myoglobin using X-ray crystallographic techniques [22]. Additional evidence in support of the correctness of our experimental mean M, value comes from a recent paper [23] which reports a combined enzymatic hydrolysis/FAB-MS dete~ination of the sequence of the relevant peptide (119-133) from horse heart myoglobin.…”

An electrospray ionization mass spectrometric study of the subunit structure of the giant hemoglobin from the leech Nephelopsis oscura

“…Processing used a maximum entropy (MaxEnt) based approach [11,12] incorporated as part of the Micromass MassLynx software suite supplied with the spectrometer. Mass scale calibration employed the multiplycharged series from horse heart myoglobin (M-1882, Sigma Chemical Co., St. Louis, MO), using a calculated mass of 16951.5 Da [13], based on the following atomic weights: C ϭ 12.011, H ϭ 1.00794, N ϭ 14.00674, O ϭ 15.9994, and S ϭ 32.066 [14].…”