Analysis of Brain Protein Stability Changes in Mouse Models of Normal Aging and α-Synucleinopathy Reveals Age- and Disease-Related Differences.

Here, we utilize the stability of proteins from rates of oxidation (SPROX) technique, to profile the thermodynamic stabilities of proteins in brain tissue cell lysates from Huα-Syn(A53T) transgenic mice at three time points including at 1 month (n = 9), at 6 months (n = 7), and at the time (between 9 and 16 months) a mouse became symptomatic (n = 8). The thermodynamic stability profiles generated here on 332 proteins were compared to thermodynamic stability profiles generated on the same proteins from similarly aged wild-type mice using a two-way unbalanced analysis of variance (ANOVA) analysis. This analysis identified a group of 22 proteins with age-related protein stability changes and a group of 11 proteins that were differentially stabilized in the Huα-Syn(A53T) transgenic mouse model. A total of 9 of the 11 proteins identified here with disease-related stability changes have been previously detected in human cerebral spinal fluid and thus have potential utility as biomarkers of Parkinson's disease (PD). The differential stability observed for one protein, glutamate decarboxylase 2 (Gad2), with an age-related change in stability, was consistent with the differential presence of a known, age-related truncation product of this protein, which is shown here to have a higher folding stability than full-length Gad2. Mass spectrometry data were deposited at ProteomeXchange (PXD016985).

Proteome-Wide Structural Biology: An Emerging Field for the Structural Analysis of Proteins on the Proteomic Scale.

Over the past decade, a suite of new mass-spectrometry-based proteomics methods has been developed that now enables the conformational properties of proteins and protein-ligand complexes to be studied in complex biological mixtures, from cell lysates to intact cells. Highlighted here are seven of the techniques in this new toolbox. These techniques include chemical cross-linking (XL-MS), hydroxyl radical footprinting (HRF), Drug Affinity Responsive Target Stability (DARTS), Limited Proteolysis (LiP), Pulse Proteolysis (PP), Stability of Proteins from Rates of Oxidation (SPROX), and Thermal Proteome Profiling (TPP). The above techniques all rely on conventional bottom-up proteomics strategies for peptide sequencing and protein identification. However, they have required the development of unconventional proteomic data analysis strategies. Discussed here are the current technical challenges associated with these different data analysis strategies as well as the relative analytical capabilities of the different techniques. The new biophysical capabilities that the above techniques bring to bear on proteomic research are also highlighted in the context of several different application areas in which these techniques have been used, including the study of protein ligand binding interactions (e.g., protein target discovery studies and protein interaction network analyses) and the characterization of biological states.