TITLE: Proteomics workflow for APP/Aβ TOMAHAQ analysis in endosomal and lysosomal fractions
AUTHORS: Hankum Park, Frances V Hundley, J. Wade Harper

[DESCRIPTION]
The ability to detect processing of APP to the Ab amyloid peptide is challenging. This protocols describes methods for analysis of Ab "half-tryptic" peptides from purified organelles (endosomes and lysosomes). The targeted proteomics approach using TOMAHAQ coupled with Tomahto, which is an API for use on a Thermo orbitrap instrument that facilitates detection of trigger peptides and fragmentation of target peptide reporter ions.

[STEPS]
SECTION: Sample preraration
1. Prepare all samples (unfiltered PNS, Lyso, and Endo; Amicon-filtered PNS_LMW, Lyso_LMW, and Endo_LMW) as described (dx.doi.org/10.17504/protocols.io.byjfpujn).  For full peptide sequences and associated proteomic parameters, see the attached document.
SECTION: Sample preraration
2. Reduce all samples by adding TCEP to 5 mM final and incubate at 25 ºC for 30 min with shaking.
SECTION: Sample preraration
3. Alkylate cysteines by adding iodoacetamide to15 mM final and incubate at 25 ºC for 30 min with shaking and protected from light.
SECTION: Sample preraration
4. Protein precipitation:
SECTION: Sample preraration
4.1. Dilute samples with EPPS buffer for 1.2 M urea final concentration.
SECTION: Sample preraration
4.2. Add 6.1 N TCA solution to 20% final and incubate at 4 ºC for 1.5h. Centrifuge samples at 21,000xg for 15 min at 4 ºC and remove supernatants.  
SECTION: Sample preraration
4.3. Wash twice with ice-cold acetone by centrifuging at 21,000xg for 10 min at 4 ºC. After final wash, briefly dry protein pellets in a SpeedVac. 
SECTION: Sample preraration
5. Resuspend pellets in 10 µL of 8 M urea buffer, sonicate in a water bath sonicator, and dilute urea by adding 10 µL of 200 mM EPPS.
SECTION: Sample preraration
6. Peptide digestion:
SECTION: Sample preraration
6.1. Add 0.3 µg of LysC and incubate at 37 ºC for 2h with shaking. Further dilute urea to 1.6 M final by adding 200 mM EPPS. 
SECTION: Sample preraration
6.2. Further digest peptides by adding 0.4 µg trypsin and incubate at 37 ºC overnight with shaking.
SECTION: Sample preraration
7. The next day, add acetonitrile (ACN) to 30% final, and label peptides by adding 3.6-5.3 µL of TMT 11-plex reagents (10 μg/μL in anhydrous ACN) for 1h at 25 ºC with shaking. Quench labeling reaction by adding hydroxylamine to 0.5% final followed by incubation at room temperature for 15 min. 
SECTION: Sample preraration
8. Dry pooled sample by SpeedVac, and desalt by C18 StageTip. 
SECTION: Sample preraration
9. Add synthetic reference trigger peptides labeled with super-heavy TMTsh as described (dx.doi.org/10.17504/protocols.io.byjcpuiw). The peptides employed and their associated ionization and fragmentation properties are provided in the attached spreadsheet.
SECTION: TOMAHAQ experiment
10. Perform TOMAHAQ experiments using the Tomahto software package on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer coupled to an Easy-nLC 1200 UHPLC system. Load each sample on a C18 column (30 cm, 2.6 μm Accucore [Thermo Fisher], 100 µm I.D.), and eluted using a 150-min method over a gradient from 5% to 38% B (95% ACN/0.125% formic acid). The instrument method only controlls Orbitrap MS1 scans (resolution at 120,000; mass range 300−1500 m/z; automatic gain control (AGC) target 2*105, maximum injection time 50 ms). Peptide targets are imported into Tomahto, and the following decisions are made by Tomahto in real-time.
SECTION: TOMAHAQ experiment
11. Export summed signal-to-noise ratio (S/N) to csv file, and analyze by R 3.6.3. Adjust S/N of each channel using the isotopic impurity table of TMT reagents provided by the vendor. Normalize the adjusted S/N values according to the total reporter values in each channel according to a SPS-MS3 analysis, assuming equal amount of loading. Test a statistical significance between DMSO- and compound-treated group using two-sided Student’s t-test with t_test() function in the rstatix package version 0.7.0.
SECTION: SIM experiment
12. For the absolute quantification of the target peptides, apply selected ion monitoring (SIM) experiments. Perform SIM experiments on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer coupled to an Easy-nLC 1200 UHPLC system. Load each sample on an in-house packed C18 column (30 cm, 2.6 um Accucore [Thermo Fisher], 100 µm I.D.), and elute using a 150-min method over a gradient from 5% to 38% B (95% acetonitrile, 0.125% formic acid). Monitor target peptides within a 50 min window around the scheduled retention time. Isolate a pair of trigger and target peptides, and accumulated separately targeting same AGC value (5*104; resolution at 240,000) and subsequently analyze in a single Orbitrap SIM scan. If two target peptides share similar retention time and same FAIMS CV values, multiplex their detections into a single SIM scan.
SECTION: SIM experiment
13. Import RAW files from the SIM experiments into Skyline, and extract the precursor ion peaks with 10 ppm accuracy. Because the TMT-labeled target peptides and TMTsh-labeled trigger peptides have the same retention time, measure the area under each peak, and calculate the ratio between target and trigger. Derive absolute amount the target peptide by multiplying the ratio by the known amount of the trigger peptide. Divide the absolute amount of target peptide by the relative quantitation from TOMAHAQ to calculate the absolute amount from each channel.