Surface induced dissociation yields quaternary substructure of refractory noncovalent phosphorylase B and glutamate dehydrogenase complexes.
Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.
Department of Chemistry and Biochemistry, The Ohio State University, 876 Biological Sciences Building, 484 W 12th Ave, Columbus, OH, 43210, USA.
Xin
X
Ma
Mowei
M
Zhou
Vicki H
VH
Wysocki
D000818:Animals; D002417:Cattle; D005969:Glutamate Dehydrogenase / Q000737:chemistry*; D046912:Multiprotein Complexes / Q000737:chemistry*; D010763:Phosphorylase b / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D011817:Rabbits; D053719:Tandem Mass Spectrometry / Q000379:methods
D046912:Multiprotein Complexes; D005969:Glutamate Dehydrogenase; D010763:Phosphorylase b