Enhanced LC-MS/MS Proteomics Strategies for Detecting the Unfolded Protein Response (UPR) in Melanoma and Human Aging Neurons

Abstract

Proper folding of secretory proteins is essential for cellular and organismal function. Accumulation of misfolded or aggregated proteins in the endoplasmic reticulum (ER), termed ER stress, can result from insults such as redox imbalance, oxidative stress, and calcium dysregulation. To restore ER proteostasis and prevent secretion of misfolded proteins, cells activate the Unfolded Protein Response (UPR), a tripartite signaling pathway (ATF6, IRE1, PERK) that upregulates proteins involved in folding, trafficking, and degradation. Unlike transcriptomics or western blotting, liquid chromatography–mass spectrometry (LC–MS) proteomics enables quantitative, high-throughput analysis of multiple UPR effectors. This dissertation expands the repertoire of proteomic targets for monitoring the UPR, enabling branch-specific assessment in global samples. Compared to data-dependent acquisition (DDA), data-independent acquisition (DIA) provided the most comprehensive detection of UPR targets. Transcriptomic and proteomic branch-specific targets diverged, underscoring transcriptional regulation of the response and the importance of functional, protein-level measurements. To accelerate sample preparation, we developed automated workflows for unlabeled and tandem-mass-tag peptides on a Biomek i5 (Beckman Coulter) liquid handler using SP3 beads for capture, cleanup, and digestion. These methodologies were applied to drug-tolerant BRAF-mutant melanoma and aging human neurons, two contexts in which UPR activation had been suggested by alternative assays. In both, cell type–specific differences in UPR utilization were observed, demonstrating the ability of these methods to deconvolute UPR activity across biological systems. Finally, in collaboration with Minsoo Kim, Ph.D., we applied DIA proteomics and automated sample preparation to investigate Deficiency of Adenosine Deaminase 2 (DADA2). Quantitative steady-state and time-resolved interactomics revealed secretion-deficient variants engage more extensively with ER proteostasis and autophagy–lysosome components. We also used an automated siRNA screen to show how knockdown of proteostasis factors can modulate ADA2 variant activity and secretion.