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DiLeu isobaric labeling coupled with limited proteolysis mass spectrometry for high-throughput profiling of protein structural changes in Alzheimers disease

ABSTRACT: High-throughput quantitative analysis of protein conformational changes has a profound impact on our understanding of the pathological mechanisms of Alzheimers disease (AD). To establish an effective workflow enabling quantitative analysis of changes in protein conformation within multiple samples simultaneously, here we report the combination of N,N-dimethyl leucine (DiLeu) isobaric tag labeling with limited-proteolysis mass spectrometry (DiLeu-LiP-MS) for high-throughput structural protein quantitation in serum samples collected from AD patients and control donors. 23 proteins were discovered to undergo structural changes, mapping to 35 unique conformotypic peptides with significant changes between the AD group and the control group. 7 out of 23 proteins, including CO3, CO9, C4BPA, APOA1, APOA4, C1R and APOA, exhibited potential correlation with AD. Moreover, we found that complement proteins (e.g., CO3, CO9 and C4BPA) related to AD exhibited elevated levels in the AD group compared to those in the control group. These results provide evidence that the established DiLeu-LiP-MS method can be used for high-throughput structural protein quantitation, which also showed great potential in achieving large-scale and in-depth quantitative analysis of protein conformational changes in other biological systems.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Lingjun Li

PROVIDER: MSV000091408 | MassIVE | Fri Mar 03 12:33:00 GMT 2023

SECONDARY ACCESSION(S): PXD040585

REPOSITORIES: MassIVE

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Project description:Alzheimer's disease (AD) is a relentlessly progressive, fatal neurodegenerative disorder that results in widespread protein dysfunctions. However, the full extent of aberrant proteomic changes in AD and their impact on cellular physiology remains unknown. This is, in part, because of the challenges of comprehensively measuring the proteome. Here, we used plexDIA, an approach that provides deep proteomic coverage and high throughput by parallelizing the acquisition of peptides and samples, to characterize proteomic changes in AD. Using human dorsolateral prefrontal cortex tissue, we identified 281 differentially abundant proteins in AD. By systematically analyzing compartment and protein complex-specific shifts in protein abundance, we identified an AD-specific decrease in levels of the 20S proteasome, the catalytic core of the cell's primary protein degradation pathway. This alteration was accompanied by widespread decreases in proteasome subunit stoichiometries. Many proteasome substrate proteins were negatively correlated with 20S levels and increased in AD, suggesting that reduced 20S levels leads to abnormal protein accumulation. By analyzing proteins increased in AD, we identify key properties of such proteins: They have fast degradation rates, they contain signal sequences that allow them to be targeted for proteasomal degradation, and they are targeted by quality control pathways that recognize mislocalized proteins. Changes in these gene products at the protein and mRNA levels were highly discordant, providing additional evidence for increased protein abundance driven by impaired clearance. We also identified coherent sets of ubiquitin system enzymes, proteins that target substrates for proteasomal degradation, whose levels robustly discriminate AD from non-AD samples. One subset exhibited consistent increases in AD, while another, which contained the tau E3 ligase Cul5, exhibited consistent decreases, revealing complex changes in the ubiquitin system in AD. Taken together, our results suggest that decreased ubiquitin-proteasome system capacity and impaired clearance of short-lived and mislocalized proteins contribute substantially to proteopathic burden in AD.

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DiLeu isobaric labeling coupled with limited proteolysis mass spectrometry for high-throughput profiling of protein structural changes in Alzheimers disease

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