Oct 15, 2021
Version 2
Proteomics workflow for APP/Aβ TOMAHAQ analysis in endosomal and lysosomal fractions V.2
- Hankum Park1,2,
- Frances V Hundley1,2,
- J. Wade Harper1,2
- 1Department of Cell Biology, Harvard Medical School Boston, MA 02115, USA;
- 2Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815, USA
Protocol Citation: Hankum Park, Frances V Hundley, J. Wade Harper 2021. Proteomics workflow for APP/Aβ TOMAHAQ analysis in endosomal and lysosomal fractions. protocols.io https://dx.doi.org/10.17504/protocols.io.bys8pwhwVersion created by hankum_park
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: October 05, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 53824
Keywords: ASAPCRN
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Abstract
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.
Attachments
Materials
| A | B | C | |
| REAGENT or RESOURCE | SOURCE | IDENTIFIER | |
| Antibodies | |||
| a-EEA1 (C45B10) rabbit mAb | Cell Signaling Technology | 3288 | |
| a-RAB5 (C8B1) rabbit mAb | Cell Signaling Technology | 3547 | |
| a-PSEN1 (D39D1) rabbit mAb | Cell Signaling Technology | 5643 | |
| a-PSEN2/AD5 (EP1515Y) rabbit mAb | Abcam | ab51249 | |
| a-LAMP1 (D2D11) rabbit mAb | Cell Signaling Technology | 9091 | |
| a-LAMP2 (D5C2P) rabbit mAb | Cell Signaling Technology | 49067 | |
| a-TMEM192 rabbit pAb | Proteintech | 28263-1-AP | |
| a-HA | Biolegend | 901513 | |
| a-HA (6E2) mouse mAb | Cell Signaling Technology | 2367 | |
| a-FLAG M2 mouse mAb | Sigma-Aldrich | F1804 | |
| a-ZO-1 rabbit pAb | Proteintech | 21773-1-AP | |
| a-Golga1 rabbit pAb | Proteintech | 12640-1-AP | |
| a-Calreticulin rabbit pAb | Proteintech | 10292-1-AP | |
| a-S6K rabbit pAb | Proteintech | 14485-1-AP | |
| a-RAB11 (D4F5) rabbit mAb | Cell Signaling Technology | 5589 | |
| a-Lamin A/C (4C11) mouse mAb | Cell Signaling Technology | 4777 | |
| a-VDAC1/Porin rabbit pAb | Proteintech | 55259-1-AP | |
| a-RAB7 (D95F2) rabbit mAb | Proteintech | 9367 | |
| a-DYKDDDDK tag, mouse mAb (FG4R) | Thermo Fisher Scientific | MA1-91878 | |
| a-GAPDH (D16H11) XP rabbit mAb | Cell Signaling Technology | 5174 | |
| a-APP CTF (C1/6.1) mouse mAb | BioLegend | 802801 | |
| a-APP A4 (22C11) mouse mAb | Sigma | MAB348 | |
| a-PEX19 rabbit pAb | Proteintech | 14713-1-AP | |
| a-CD71/TFR1 (D7G9X) rabbit mAb | Cell Signaling Technology | 13113 | |
| a-HSP90 (3F11C1) mouse mAb | Proteintech | 60318-1-Ig | |
| a-BACE1 (D10E5) rabbit mAb | Cell Signaling Technology | 5606 | |
| IRDye 680RD Goat a-Rabbit IgG secondary antibody | Li-Cor | 926-68071 | |
| IRDye 680RD Goat a-Mouse IgG secondary antibody | Li-Cor | 926-68070 | |
| IRDye 800CW Goat a-Rabbit IgG secondary antibody | Li-Cor | 926-32211 | |
| IRDye 800CW Goat a-Mouse IgG secondary antibody | Li-Cor | 926-32210 | |
| Goat a-Rabbit IgG, HRP-linked antibody | Cell Signaling Technology | 7474P2 | |
| Goat a-Rabbit IgG HRP conjugate | Bio-Rad | 1706515 | |
| Goat a-Mouse IgG HRP conjugate | Bio-Rad | 1706516 | |
| Chemicals, peptides, and recombinant proteins | |||
| a-FLAG M2 magnetic beads | Sigma-Aldrich | M8823 | |
| Pierce a-HA magnetic beads | Thermo Fisher Scientific | 88837 | |
| TMT10plex Isobaric Label Reagent Set plus TMT11-131C Label Reagent | Thermo Fisher Scientific | A34808 | |
| TMTProTM 16Plex Label Reagent set | Thermo Fisher Scientific | A44520 | |
| Super Heavy TMT Label Reagent | Thermo Fisher Scientific | A43073 | |
| Pierce™ High pH Reversed-Phase Peptide Fractionation Kit | Thermo Fisher Scientific | 84868 | |
| HyClone Fetal bovine serum | GE Healthcare | SB30910 | |
| Puromycin | Sigma-Aldrich | P9620 | |
| G418 (Geneticin) | Invivogen | ant-gn-2 | |
| Dulbecco’s MEM (DMEM), high glucose, pyruvate | GIBCO / Invitrogen | 11995 | |
| PhosSTOP | Roche | 04906845001 | |
| Complete EDTA-free protease inhibitor cocktail | Sigma-Aldrich | 11873580001 | |
| Tris(2-carboxyethyl)phosphine hydrochloride solution | Sigma-Aldrich | 646547 | |
| Iodoacetamide | Sigma-Aldrich | I1149 | |
| Trichloroacetic acid solution 6.1 N | Sigma-Aldrich | T0699 | |
| Trifluoroacetic acid | fisher scientific | A11650 | |
| Hydroxylamine solution 50 wt. % | Sigma-Aldrich | 438227 | |
| Formic Acid | Sigma-Aldrich | 5330020050 | |
| Pierce Trypsin Protease, MS grade | Thermo Fisher Scientific | 90305 | |
| Lysyl endopeptidaseR (Lys-C) | Wako | 129-02541 | |
| REVERT 700 total protein stain kit | Li-Cor | 926-11016 | |
| NuPAGE LDS sample buffer (4X) | Thermo Fisher Scientific | NP0007 | |
| NuPAGE sample reducing agent (10X) | Thermo Fisher Scientific | NP0009 | |
| NuPAGE MES SDS Running Buffer (20X) | Thermo Fisher Scientific | NP0002 | |
| Immobilon-FL PVDF Membrane | Millipore | IPFL00010 | |
| WHEATON Dounce Tissue Grinder, 7 mL | DWK Life Sciences | 357542 | |
| KIMBLE KONTES Dounce Tissue Grinder, 2 mL | DWK Life Sciences | 885300-0002 | |
| Nonidet P40 substitute | Sigma-Aldrich | 74385 | |
| Urea | Sigma-Aldrich | U5378 | |
| EPPS 0.2M buffer solution, pH 8.5 | Alfa Aesar | J61476.AE | |
| Empore C18 47 mm Extraction Disc, Model 2215 | 3M | 98060402173 | |
| Sep-Pak C18 1 cc Vac Cartridge | Waters | WAT054955 | |
| Dyngo4a | Cayman Chemical | 29479 | |
| Lanabecestat (AZD3293) | Selleckchem | S8193 | |
| Semagacestat | Cayman Chemical | 16713 | |
| BPN-15606 | MedChemExpress | HY-117482 | |
| RIPA lysis and extraction buffer | Thermo Fisher Scientific | 89900 | |
| Reference peptides for APP/Ab (see Supplemental Data Table S7) | Biomatik Thermo Fisher Scientific | Custom order | |
| Experimental models: Cell lines | |||
| 293 cells | ATCC | CRL-1573 | |
| 293EL-APP-/-: TMEM192-3xHA; APP-/-; FLAG-EEA1 | This study | ||
| 293EL-APP*: TMEM192-3xHA; APP-/-; FLAG-EEA1; APPSw;T700N | This study |
Sample preraration
Sample preraration
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.
Reduce all samples by adding TCEP to 5 mM final and incubate at 25 ºC for 30 min with shaking.
Alkylate cysteines by adding iodoacetamide to15 mM final and incubate at 25 ºC for 30 min with shaking and protected from light.
Protein precipitation:
Dilute samples with EPPS buffer for 1.2 M urea final concentration.
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.
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.
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.
Peptide digestion:
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.
Further digest peptides by adding 0.4 µg trypsin and incubate at 37 ºC overnight with shaking.
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.
Dry pooled sample by SpeedVac, and desalt by C18 StageTip.
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.
TOMAHAQ experiment
TOMAHAQ experiment
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.
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.
SIM experiment
SIM experiment
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.
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.