Figure 2.

Quantitative Analysis of pS65-Ub Proteoforms on the MOM in iNeurons using Ub Clipping and Intact Mass Spectrometry

(A) Scheme depicting the products produced by LbPro^∗ on distinct Ub proteoforms.

(B) Workflow for analysis Ub proteoforms in iNeurons. Mitochondria were purified from iNeurons expressing WT or S65A-Parkin with or without depolarization (6 h). Mitochondrial extracts were treated with LbPro^∗ and Ub monomers purified by size exclusion chromatography. Samples were then subjected to LC-intact mass analysis prior to quantification of isotopic clusters for phospho and diGLY-containing Ub^ΔGG proteoforms across all charge state distributions.

(C) Mass spectra for individual Ub^ΔGG proteoforms in response to depolarization of WT iNeurons. Shown below are spectra of the isotopic cluster for the z = 12 precursors.

(D) Relative abundance of Ub^ΔGG proteoforms. Error bars represent SEM; n = 3. n.d., not determined.

See also Figure S2.