Ion mobility-mass spectrometry study of folded ubiquitin conformers induced by treatment with cis-[Pd(en)(H2O2]2+.

Ion mobility-mass spectrometry is used to study the new conformers of bovine ubiquitin (Ub) and the palladium(II) binding sites after the incubation with cis-Pd(en)(H(2)O)(2) where en = ethylenediamine. Palladium(II) complexes are potentially useful proteomic reagents because they selectively bind to the side groups of methionine and histidine and hydrolytically cleave the peptide bond. Incubating 1.0 mM solution of Ub with 10.0 molar excess of cis-Pd(en)(H(2)O)(2) results with one to four Pd(2+) or Pd(en)(2+) being attached to intact Ub and two conformer families at each of the 4+ to 11+ charge states. The 4+ and 5+ species exhibit a compact form, which is also observed in untreated Ub, and a new highly folded conformer. The 6+ to 10+ exhibit an elongated form, also observed in Ub, and a new partially folded conformer. The new conformers are shown to be more stable if they contain at least one Pd(2+), rather than all Pd(en)(2+). IM-MS/MS of UbPd(2)en+5H shows that both the partially folded and elongated conformers first lose the en ligand, followed by dissociating into product ions that indicate that Met1, Glu51/Asp52, His68, and Glu16 are binding sites for Pd(2+). These results suggest that Pd(2+) is simultaneously binding to multiple side groups across different regions of Ub. This type of sequestering of Pd(2+) probably reduces the efficiency of Pd(2+) ions to selectively cleave Ub because it prevents Pd(2+) anchoring to only Met or His and to an adjacent backbone amide nitrogen and forming the "activated complex" necessary for specific peptide bond cleavage.