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Discovery of dehydroamino acids and their crosslinks in Alzheimer's Disease

ABSTRACT: Alzheimer’s disease is characterized by the accumulation of protein aggregates. Dehydroamino acids are rarely observed posttranslational modifications that contain an electrophilic alkene capable of forming protein-protein crosslinks, which may lead to protein aggregation. We used mass spectrometry-based proteomics to discover 412 sites of dehydroamino acid formation in 184 proteins from protein aggregate-enriched human brain samples, an order of magnitude more than observed in the soluble protein fractions. We further report 11 dehydroamino acid-mediated crosslinks, including 3 in the Tau protein that are 10-fold more abundant in Alzheimer’s disease samples compared to age-matched controls. Dehydroamino acids are prevalent modifications in the Alzheimer’s disease brain proteome and give rise to protein crosslinks that may contribute to protein aggregation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Brain

DISEASE(S): Alzheimer's Disease

SUBMITTER: Samuel Markovich

LAB HEAD: Lloyd M

PROVIDER: PXD053814 | Pride | 2025-03-06

REPOSITORIES: Pride

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Dehydroamino acids and their crosslinks in Alzheimer's disease aggregates.

Markovich Samuel W SW Frey Brian L BL Scalf Mark M Shortreed Michael R MR Smith Lloyd M LM

Brain communications 20250116 1

Alzheimer's disease (AD) is characterized by the accumulation of protein aggregates, which are thought to be influenced by posttranslational modifications (PTMs). Dehydroamino acids (DHAAs) are rarely observed PTMs that contain an electrophilic alkene capable of forming protein-protein crosslinks, which may lead to protein aggregation. We report here the discovery of DHAAs in the protein aggregates from AD, constituting an unknown and previously unsuspected source of extensive proteomic complexi ...[more]

PMID: 39882022

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Project description:Sample preparation: P-AMPK+AMP, P-AMPK+ATPγS, and AMPK+ATPγS protein samples were crosslinked in HBST buffer (25 mM HEPES pH 7.5 at room temperature, 200 mM NaCL, and 1 mM TCEP). Non-crosslinked negative controls were generated using the same procedure without the addition of DSSO. Reactions were initiated by spiking 1 uL of 75 mM DSSO (Thermo-Fisher) in DMSO into a 50 uL solution containing 10 uM protein and mixing. The final molar ratio of crosslinker:protein was 150:1. The reactions were incubated at 25°C for 45 minutes prior to being quenched by the addition of Tris pH 8.0 to a final concentration of 50 mM. Each crosslink reaction was done in triplicate and the reaction replicates were pooled after quenching. 10 μL samples of the pooled samples were loaded for SDS-PAGE analysis with Coomassie staining to confirm the presence of crosslinked protein. The remaining 140 μL of crosslinked and non-crosslinked samples were then acetone precipitated at -20°C overnight and the protein was pelleted by centrifugation at 16,000 rcf for 5 minutes at 4°C. After decanting, the pellets were dried for 15 minutes at room temperature before being resuspended in 12.5 uL of resuspension buffer (50 mM ammonium bicarbonate, 8M urea, pH 8.0). ProteaseMAX (Promega) was added to 0.02% and the solutions were mixed on an orbital shaker operating at 400 RPM for 5 minutes. After resuspension, 87.5 uL of digestion buffer (50 mM ammonium bicarbonate, pH 8.0) was added. Cysteines in the protein samples were reduced and akylated as follows. DTT was added to 5 mM final concentration and the protein solutions were incubated at 56°C in an orbital shaker operating at 400 RPM and for 20 minutes. After reduction, 2.7 uL of 550 mM iodoacetamide was added and the solutions were incubated in the dark at room temperature for 15 minutes. Reduced and alkylated protein solutions were digested using trypsin at a ratio of 1:200 (w/w trypsin:protein) and incubating at 37°C. After overnight incubation at 37°C, the samples were acidified by the addition of trifluoroacetic acid (TFA, Thermo-Fischer) to 1%. Samples were then frozen and stored at -20°C until analysis.

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