title: Measuring copper and zinc superoxide dismutase from spinal cord tissue using electrospray mass spectrometry.
abstract: Metals are key cofactors for many proteins, yet quantifying the metals bound to specific proteins is a persistent challenge in vivo. We have developed a rapid and sensitive method using electrospray ionization mass spectrometry to measure Cu,Zn superoxide dismutase (SOD1) directly from the spinal cord of SOD1-overexpressing transgenic rats. Metal dyshomeostasis has been implicated in motor neuron death in amyotrophic lateral sclerosis (ALS). Using the assay, SOD1 was directly measured from 100 μg of spinal cord, allowing for anatomical quantitation of apo, metal-deficient, and holo SOD1. SOD1 was bound on a C(4) Ziptip that served as a disposable column, removing interference by physiological salts and lipids. SOD1 was eluted with 30% acetonitrile plus 100 μM formic acid to provide sufficient hydrogen ions to ionize the protein without dislodging metals. SOD1 was quantified by including bovine SOD1 as an internal standard. SOD1 could be measured in subpicomole amounts and resolved to within 2 Da of the predicted parent mass. The methods can be adapted to quantify modifications to other proteins in vivo that can be resolved by mass spectrometry.
authors: Department of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA. rhoadst@onid.orst.edu
authors: Timothy W
authors: TW
authors: Rhoads
authors: Nathan I
authors: NI
authors: Lopez
authors: Daniel R
authors: DR
authors: Zollinger
authors: Jeffrey T
authors: JT
authors: Morré
authors: Brian L
authors: BL
authors: Arbogast
authors: Claudia S
authors: CS
authors: Maier
authors: Linda
authors: L
authors: DeNoyer
authors: Joseph S
authors: JS
authors: Beckman
mesh_terms: D000818:Animals; D003300:Copper / Q000378:metabolism; D051381:Rats; D055647:Rats, Transgenic; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013116:Spinal Cord / Q000201:enzymology*; D013482:Superoxide Dismutase / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D015032:Zinc / Q000378:metabolism
chemical_list: D003300:Copper; C000606292:Sod1 protein, rat; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D015032:Zinc