TDP-43-stratified single-cell proteomics of postmortem human spinal motor neurons reveals protein dynamics in amyotrophic lateral sclerosis.

A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases. [Display omitted] • Single-cell proteomic analysis of human ALS motor neurons directly captured by LMD • ALS motor neuron deficiencies in splicing, translation, and vesicular transport machinery • Stratification of single-neuron proteomes based on pathologic TDP-43 inclusions • Utility of trace sample proteomics in augmenting the study of human neurological diseases Guise and Misal et al. report the unbiased single-cell proteomic analysis of ALS motor neurons directly captured from human tissues to explore disease-associated protein dynamics across TDP43-pathological strata. This study highlights both important technical challenges accompanying single-cell omics studies and emerging avenues for exploring human disease biology with nanoPOTS. [ABSTRACT FROM AUTHOR]