TitleUsing theoretical protein isotopic distributions to parse small-mass-difference post-translational modifications via mass spectrometry.
Publication TypeJournal Article
Year of Publication2013
AuthorsRhoads TW, Williams JR, Lopez NI, Morré JT, C Bradford S, Beckman JS
JournalJ Am Soc Mass Spectrom
Date Published2013 Jan
KeywordsAnimals, Cattle, Chickens, Copper, Isotopes, Mice, Mice, Transgenic, Molecular Weight, Oxidation-Reduction, Protein Processing, Post-Translational, Proteins, Spectrometry, Mass, Electrospray Ionization, Superoxide Dismutase, Superoxide Dismutase-1, Zinc

Small-mass-difference modifications to proteins are obscured in mass spectrometry by the natural abundance of stable isotopes such as (13)C that broaden the isotopic distribution of an intact protein. Using a ZipTip (Millipore, Billerica, MA, USA) to remove salt from proteins in preparation for high-resolution mass spectrometry, the theoretical isotopic distribution intensities calculated from the protein's empirical formula could be fit to experimentally acquired data and used to differentiate between multiple low-mass modifications to proteins. We could readily distinguish copper from zinc bound to a single-metal superoxide dismutase (SOD1) species; copper and zinc only differ by an average mass of 1.8 Da and have overlapping stable isotope patterns. In addition, proteins could be directly modified while bound to the ZipTip. For example, washing 11 mM S-methyl methanethiosulfonate over the ZipTip allowed the number of free cysteines on proteins to be detected as S-methyl adducts. Alternatively, washing with the sulfhydryl oxidant diamide could quickly reestablish disulfide bridges. Using these methods, we could resolve the relative contributions of copper and zinc binding, as well as disulfide reduction to intact SOD1 protein present from <100 μg of the lumbar spinal cord of a transgenic, SOD1 overexpressing mouse. Although techniques like ICP-MS can measure total metal in solution, this is the first method able to assess the metal-binding and sulfhydryl reduction of SOD1 at the individual subunit level and is applicable to many other proteins.

Alternate JournalJ. Am. Soc. Mass Spectrom.
PubMed ID23247967
PubMed Central IDPMC4119010
Grant ListR01 NS058628 / NS / NINDS NIH HHS / United States
P30ES000210 / ES / NIEHS NIH HHS / United States
P01AT002034 / AT / NCCIH NIH HHS / United States
R01NS058628A / NS / NINDS NIH HHS / United States
P01 AT002034 / AT / NCCIH NIH HHS / United States
P30 ES000210 / ES / NIEHS NIH HHS / United States