Amino acid isomerization is a spontaneous chemical modification potentially related to the underlying causes of Alzheimer’s disease (AD). We demonstrate that data-independent acquisition mass spectrometry can be used to characterize isomerization in complex protein mixtures. Examination of a large cohort of brain samples revealed a striking relationship between isomerization of tau and AD status. Surprisingly, isomerization was found to be more abundant in both autosomal dominant and sporadic AD samples relative to controls. We hypothesize that lower autophagic flux in AD brains accounts for these results. Additional data strongly support this hypothesis, including quantitative analysis of proteins related to autophagy. For example, isomerization of tau is positively correlated with levels of p62, a recognized indicator of autophagic inhibition. In sum, the data suggest strong ties between isomerization and autophagic flux, which may therefore represent a promising target for future investigations into the therapy and prevention of AD.

Two 25 μm cryo slices (“curls”) of brain tissue was resuspended in 120 μl of lysis buffer of 5% SDS, 50mM Triethylammonium bicarbonate (TEAB), 2mM MgCl2, 1X HALT phosphatase and protease inhibitors, vortexed and and briefly sonicated at setting 3 for 10 s with a Fisher sonic dismembrator model 100. A microtube was loaded with 30 μl of lysate and capped with a micropestle for homogenization with a Barocycler 2320EXT (Pressure Biosciences Inc.) for a total of 20 minutes at 35°C with 30 cycles of 20 seconds at 45,000 psi followed by 10 seconds at atmospheric pressure. Protein concentration was measured with a BCA assay. Homogenate of 50 μg was added to a process control of 800 ng of yeast enolase protein (Sigma) which was then reduced with 20 mM DTT and alkylated with 40 mM IAA. Lysates were then prepared for S-trap column (Protifi) binding by the addition of 1.2% phosphoric acid and 350 μl of binding buffer (90% Methanol, 100 mM TEAB). The acidified lysate was bound to column incrementally, followed by 3 wash steps with binding buffer to remove SDS and 3 wash steps with 50:50 methanol:chloroform to remove lipids and a final wash step with binding buffer. Trypsin (1:10) in 50mM TEAB was then added to the S-trap column for digestion at 47°C for one hour. Hydrophilic peptides were then eluted with 50 mM TEAB and hydrophobic peptides were eluted with a solution of 50% acetonitrile in 0.2% formic acid. Elutions were pooled, speed vacuumed and resuspended in 0.1% formic acid. Injection of samples are one ug of total protein (16 ng of enolase process control) and 150 fmol of a heavy labeled Peptide Retention Time Calibrant (PRTC) mixture (Pierce). The PRTC is used as a peptide process control. Library pools are an equivalent amount of every sample (including references) in the batch. For example, a batch library pool consists of the 14 samples from the batch and two references. System suitability (QC) injections are 150 fmol of PRTC and BSA. One ug of each sample with 150 femtomole of PRTC was loaded onto a 30 cm fused silica picofrit (New Objective) 75 µm column and 3.5 cm 150 µm fused silica Kasil1 (PQ Corporation) frit trap loaded with 3 µm Reprosil-Pur C18 (Dr. Maisch) reverse-phase resin analyzed with a Thermo Easy-nLC 1200. The PRTC mixture is used to assess system suitability before and during analysis. Four of these system suitability runs are analyzed prior to any sample analysis and then after every six sample runs another system suitability run is analyzed. Buffer A was 0.1% formic acid in water and buffer B was 0.1% formic acid in 80% acetonitrile. The 40-minute system suitability gradient consists of a 0 to 16% B in 5 minutes, 16 to 35% B in 20 minutes, 35 to 75% B in 1 minute, 75 to 100% B in 5 minutes, followed by a wash of 9 minutes and a 30 minute column equilibration. The 110-minute sample LC gradient consists of a 2 to 7% for 1 minutes, 7 to 14% B in 35 minutes, 14 to 40% B in 55 minutes, 40 to 60% B in 5 minutes, 60 to 98% B in 5 minutes, followed by a 9 minute wash and a 30 minute column equilibration. Peptides were eluted from the column with a 50°C heated source (CorSolutions) and electrosprayed into a Thermo Orbitrap Fusion Lumos Mass Spectrometer with the application of a distal 3 kV spray voltage. For the system suitability analysis, a cycle of one 120,000 resolution full-scan mass spectrum (350-2000 m/z) followed by a data-independent MS/MS spectra on the loop count of 76 data-independent MS/MS spectra using an inclusion list at 15,000 resolution, AGC target of 4e5, 20 sec maximum injection time, 33% normalized collision energy with a 8 m/z isolation window. For the sample digest, first a chromatogram library of 6 independent injections is analyzed from a pool of all samples within a batch. For each injection a cycle of one 120,000 resolution full-scan mass spectrum with a mass range of 100 m/z (400-500 m/z, 500-600 m/z…900-1000 m/z) followed by a data-independent MS/MS spectra on the loop count of 26 at 30,000 resolution, AGC target of 4e5, 60 sec maximum injection time, 33% normalized collision energy with a 4 m/z overlapping isolation window. The chromatogram library data is used to quantify proteins from individual sample runs. These individual runs consist of a cycle of one 120,000 resolution full-scan mass spectrum with a mass range of 350-2000 m/z, AGC target of 4e5, 100 ms maximum injection time followed by a data-independent MS/MS spectra on the loop count of 76 at 15,000 resolution, AGC target of 4e5, 20 sec maximum injection time, 33% normalized collision energy with an overlapping 8 m/z isolation window. Application of the mass spectrometer and LC solvent gradients are controlled by the ThermoFisher XCalibur (version 3.1.2412.24) data system. Thermo RAW files were converted to mzML format using Proteowizard (version 3.0.20064) using vendor peak picking and demultiplexing. Chromatogram spectral libraries were created using default settings (10 ppm tolerances, trypsin digestion, HCD b- and y-ions) of of EncyclopeDIA (version 0.9.5) using a Prosit predicted spectra library based the Uniprot human canonical FASTA. Quantitative spectral libraries were created by mapping spectra to the chromatogram spectral library using EncyclopeDIA requiring a minimum of 3 quantitative ions and filtering peptides at a 1% FDR using Percolator 3.01. The quantitative spectral library is imported into Skyline (daily version 20.1.9.234) with the human uniprot FASTA as the background proteome to map peptides to proteins. Transition retention time filtering settings set to “use only scans within 4 minutes of MS/MS IDs” for all batches and then removed all peptides/proteins from Skyline document except APP (Uniprot Accession P05067) and Tau (Uniprot Accession P10636). To confirm the identity of isomers in the clinical results, we collected LC-MS/MS data for a set of known synthetic tau isomer standards. Comparison of relative elution times confirms that 386 TDHGAEIVYK 395 from Tau is isomerized at Asp387, in agreement with known amino acid propensities for isomerization and previous examination of tau. Synthetic standards also confirm the initial assignment of the native L-Asp form as the most abundant peak, followed by L-isoAsp. This observation is in agreement with previous results showing that L-isoAsp is the primary product of Asp isomerization.

Brain tissue samples were stratified into 4 groups based on clinical, pathological and genetic data. Cognitive status was determined as dementia (AD) or no dementia (Controls) by DSM-IVR criteria; Controls were from the Adult Changes in Thought (ACT) study and were included only if the last evaluation was within 2 years of death and the last CASI score was >90 (upper quartile for Controls in the ACT cohort). Controls with no or low Alzheimer’s disease neuropathologic change (ADNC) were designated “Control Low Neuropathology” and those with intermediate or high ADNC were designated “Control High Neuropathology''. All cases with dementia had intermediate or high level ADNC and were classified as AD, and were further subclassified as sporadic (“Sporadic AD”) or caused by a dominantly inherited (DI) mutation in PSEN1 or PSEN2 (“Autosomal Dominant AD”). “Sporadic AD” cases were from ACT and the University of Washington (UW) AD Research Center (ADRC), and “Autosomal Dominant AD” cases were from the UW ADRC and the Dominantly Inherited Alzheimer Network (DIAN). Cases with Lewy body disease other than involving amygdala, territorial infarcts, more than 2 cerebral microinfarcts, or hippocampal sclerosis were excluded. Time from death to cryopreservation of tissue, post mortem interval (PMI), was <8 hr in all cases except for those in the “Autosomal Dominant AD”. Each region of human brain tissue was divided into batches of 14 individual samples and 2 pooled references for a total of 16. The first batch of each region was also used to create a region-specific reference pool to be used as a “common reference” and/or single point calibrant, which was homogenized, aliquoted, frozen, and used to compare between batches within a brain region. Human cerebellum and occipital lobe tissue was homogenized, pooled, aliquoted and frozen to be used as a “batch reference” for comparison between batches and other brain regions. Batch design was randomly balanced based on group ratios. For example, batches from the SMTG brain region contained 5 “Sporadic AD”, 4 “Autosomal Dominant AD”, 2 “Control Low Neuropathology”, and 3 “Control High Neuropathology” samples.