Quantification of Insoluble Protein Aggregation in Caenorhabditis elegans during Aging with a Novel Data-Independent Acquisition Workflow

NOTE: For a better understanding of the experimental procedure, see Figure 1 for a schematic of the workflow.

1. Mass culture of synchronized aging C. elegans

1. Plate preparation
   1. Pour 30 mL of NGM agar with or without 50 mM fluorodeoxyuridine (FUdR) into each 90 mm plate and let the plates dry for 2 days in culture hoods.
   2. On the day before seeding the NGM agar plate, inoculate a 50 µL aliquot of frozen glycerol stock of the OP50 bacterial strain into 1 L of pre-autoclaved LB medium in a 2 L flask.
   3. Grow the bacteria at 37 °C in a shaking incubator at 250 rpm for 16 h.
   4. After 16 h, centrifuge the bacterial cultures at a speed of 5,000 x g for 10 min.
   5. Discard the supernatant and re-suspend the bacterial pellet completely in 40 mL of autoclaved S-basal solution (containing 50 mM K₃PO₄, 100 mM NaCl in water) to generate the 25x concentrated OP50 E. coli culture.
   6. Dispense 2 mL of the 25x concentrated bacterial culture onto each NGM agar plate and spread evenly so that the bacterial culture covers the entire agar surface.
   7. Remove the lids from the plates in a bacterial culture hood for 2–3 h until the plates are properly dried.
      NOTE: Make sure to evenly distribute the bacteria by swirling the plates every 15 min. This is important as bacteria tend to concentrate on one side of the plate resulting in regions with low bacterial density.
   8. After the plates are dried, close the lids and leave the plates at room temperature for 48 h to ensure complete drying of the OP50 E. coli food source.
      NOTE: Plates can be prepared 2 weeks in advance and stored at 4 °C before use.
2. Maintenance and preparation of synchronized cohorts of C. elegans
   1. To obtain the required input of protein lysate (1.0–1.5 mg) for isolation of the SDS-insoluble fraction, use approximately 3,000 synchronized worms per sample.
   2. Transfer ~100 N2 wild-type gravid adult worms to a NGM 25x OP50 E. coli-seeded plate and maintain the worms at 20 °C for ~72 h until the plate is full with gravid adult worms.
   3. Pipette ~10 mL of S-basal solution onto each plate and collect all the worms into a 15 mL tube.
      NOTE: It is important to use low-retention tips or Pasteur pipettes during this and all future steps involving worm collection to prevent any loss of worms due to attachment to tip surfaces.
   4. Spin the sample at 520 x g for 30 s. Remove the supernatant after spinning. To eliminate any OP50 E. coli, add 10 mL of S-basal solution to the pellet and spin again followed by removal of the supernatant. Repeat this step.
   5. Add 10 mL of sodium hypochlorite bleaching solution (containing 0.5 M KOH and 0.48% sodium hypochlorite in water) to the worm pellet in a 15 mL conical tube and vortex vigorously for 2 min at room temperature.
   6. Centrifuge at 520 x g for 30 s at room temperature to spin down any incompletely dissolved worm bodies and eggs. Then remove the supernatant.
   7. Repeat steps 1.2.5 and 1.2.6 to ensure complete removal of all worm bodies and release of eggs (view tube under microscope to assure).
   8. Wash the egg pellet at least 4 times with 10 mL of S-basal solution to ensure complete removal of bleach solution.
      NOTE: Complete removal of bleach is important as remaining bleach may hinder the hatching of eggs.
   9. Leave eggs resuspended in 5 mL of S-basal media in a 15 mL tube and place in rotor at 20 °C for 24 h to allow eggs to hatch.
   10. After 24 h, take 10 µL of S-basal larvae solution to count the number of worms in it. Use at least three separate aliquots of 10 µL of S-basal larvae solution for replication of the worm count. Calculate the average of the three larval concentrations. Based on the average concentration, dilute the worm solution to obtain ~3,000 larvae per 200 µL of solution.
       NOTE: Make sure to shake the tube gently by inverting as the larvae tend to settle if left undisturbed and that can affect the larva count.
   11. Mix well and add 200 µL of S-basal larvae solution onto each of the 90-mm NGM bacteria-seeded plates.
       NOTE: Add drops of S-basal larvae solution throughout the plate for even distribution of larvae to avoid bacterial depletion from certain spots.
   12. Allow the S-basal solution to dry and then place plates inverted in a 20 °C incubator for 48 h.
   13. After 48 h, collect the worms from the plate in S-basal solution and transfer them to a fresh NGM-seeded plate containing 50 mM FUDR and incubate plates at 20 °C.
       NOTE: It is important to transfer worms early in the L4 stage to ensure FUDR efficiency.
   14. Collect day-2 young adult samples after 48 h by adding 10 mL of S-basal solution onto the plate and transferring the solution into a 15 mL tube. Allow worms to settle by gravity and remove the S-basal solution after it becomes clear.
   15. Wash the worm pellet 2-3x in 5 mL of S-basal solution to remove any attached bacteria. Remove as much S-basal solution as possible and freeze the worm pellet (in 15 mL tubes) in a dry ice/ethanol bath. Frozen samples are stored at -80 °C.
   16. Transfer sample worms to a fresh NGM OP50 E. coli plate containing FUDR every alternate day until day 10. Collect and freeze worms as described in steps 1.2.14 and 1.2.15.
       NOTE: Start with a higher number of worms for day-10 samples to account for any loss in worm number due to transfer and burrowing.

2. Extraction of SDS-insoluble fraction from worms

1. Thaw the frozen worm pellet on ice in the presence of 900 µL of ice-cold worm lysis buffer cocktail containing 20 mM Tris base, pH 7.4, 100 mM NaCl, 1 mM MgCl₂ and EDTA-free protease inhibitor. Briefly vortex samples to ensure complete resuspension of worm pellet in the lysis buffer.
2. Place the 15 mL tubes in the sonication bath and set for 10 sonication cycles (30 s on and 30 s off for each cycle) at high intensity. Repeat for up to five cycles (make sure worms are fully lysed).
3. Spin the lysate at 3,000 x g for 4 min in the cold room. After centrifugation, discard the pellet containing any worm debris and carefully transfer the supernatant to fresh pre-cooled 1.5 mL centrifuge tubes on ice.
4. Quantify the protein concentration using the BCA assay. Transfer lysate aliquots, typically containing about 1 mg of protein, into fresh pre-cooled 1.5 mL centrifuge tubes.
5. Centrifuge the protein lysate for 15 min at 20,000 x g in the cold room.
6. Without disturbing the pellet, transfer the supernatant into a fresh 1.5 mL centrifuge tube and save it as the aqueous-soluble protein fraction.
7. Wash the pellet from the previous step in 500 µL of worm lysis buffer containing 1% SDS at room temperature and centrifuge at 20,000 x g for 15 min at room temperature. Remove the supernatant and save as the SDS-soluble fraction. Repeat this washing step two times to remove any SDS-soluble fraction. The remaining pellet after the third round of washing is defined as the 1% SDS-insoluble protein fraction.
8. Resuspend the SDS-insoluble protein pellet in 60 µL of 70% formic acid and vortex vigorously to dissolve the proteins. Repeat vigorous vortexing as many times as necessary to dissolve the pellet.
9. Sonicate the pellet for 30 min in an ultrasonicator water bath at room temperature.
   NOTE: The SDS- insoluble pellet is typically hard to dissolve in the buffer. However, in the end the entire pellet should be dissolved in this step.
10. Dry the samples in a vacuum concentrator for 1 h to completely remove the formic acid solution.
11. Add 40 µL of 1x LDS sample gel buffer to the dried pellet and heat the sample to 95 °C for 10 min. Briefly vortex and spin down the samples. Load 13 µL onto a 4-12% NUPAGE Bis-Tris gel and run the gel. Stain the gel with a fluorescent protein stain for imaging. Save the rest of the sample for mass spectrometry (MS) analysis.
12. Load the remaining sample into a 4-12% Bis-Tris gel and run for about 20 min for MS in-gel digestion.

3. In-gel digestion with trypsin protease to isolate proteins for MS analysis

1. Prepare the following solutions (fresh): 25 mM NH₄HCO₃ (pH 7–8), 25 mM NH₄HCO₃ in 50% acetonitrile (ACN, pH 7–8), and 50% ACN in 5% formic acid.
2. Dice each gel slice into small pieces (typically <1 mm²) and place these into 0.65 mL siliconized tubes.
3. Add about 100 µL (or enough to cover) of a 25 mM NH₄HCO₃/50% ACN solution and vortex at room temp for 10 min. Extract the supernatant and transfer to a separate tube (to be discarded). Repeat this step two more times.
4. Dry the gel pieces completely in a vacuum concentrator (~20 min).
5. Prepare fresh solutions and add ~100 µL (or enough to cover) of 10 mM DTT in 25 mM NH₄HCO₃ to the dried gel pieces (use freshly prepared NH₄HCO₃). Vortex and briefly spin. Allow the reaction to proceed at 56 °C for 1 h and with shaking at 1400 rpm on a mixer.
6. Remove the supernatant and add 100 µL of 55 mM iodoacetamide (IAA) in 25 mM NH₄HCO₃ to the gel pieces. Vortex and briefly spin. Allow reaction to proceed in the dark for 45 min at RT.
7. Remove the supernatant and discard. Wash the gel pieces by adding ~100 µL of 25mM NH₄HCO₃ and vortexing for 10 min. Briefly spin and remove the supernatant, discarding the latter.
8. Dehydrate the gel pieces by adding ~100 µL (or enough to cover) of 25 mM NH₄HCO₃ in 50% ACN to the gel pieces and vortex for 10 min. Then briefly spin to remove and discard the supernatant. Repeat this step twice or even a third time if the gel pieces are not quite dry as indicated by smaller size of the gel pieces and cloudy white color.
9. Dry the gel pieces completely in a vacuum concentrator (~20 min).
10. Initially add 15 µL of trypsin solution (250 ng trypsin) to each sample, and then add enough 25 mM NH₄HCO₃ to cover the gel pieces (~100 µL). Vortex for 10 min. Then briefly spin and incubate at 4 °C in the cold room for 30 min without mixing.
11. Add 25 mM NH₄HCO₃ as needed to completely cover the gel pieces. Spin and incubate at 37 °C overnight for 16–20 h at 1400 rpm on a mixer.
12. The next day, briefly vortex and spin the digestion. Add about 100 µL of HPLC grade water, spin, parafilm the tube and sonicate continuously for 10 min. Spin briefly after sonication.
13. Transfer the digestion solution, representing the aqueous extraction, into a clean 0.65 mL siliconized tube.
14. Add ~100 µL of 50% ACN/5% formic acid to the gel pieces (enough to cover them), vortex 10 min at room temperature, spin briefly and collect the solution, and repeat once. Pool all solutions containing the extracted peptides from this step and the previous step 3.13 together into a single tube.
15. Vortex the extracted digestions. Dry the peptides completely in a vacuum concentrator (~2 h).
16. Add 30 µL of 0.2% formic acid to resuspend the peptides on a mixer in the cold room for 10 min.
17. Spin the samples at 1850 x g at room temperature for 5 min. Aspirate the peptide solution and move into a new clean 0.65 mL siliconized tube. Then desalt the peptides solution using C18 desalting tips (see below).

4. Desalting digested peptides with C18 desalting tip

1. Set the pipette to 10 µL and attach the C18 desalting tip. Wet the desalting tip by pipetting up 10 µL of 100% ACN and then discarding it. Repeat this step 2x.
2. Wash the C18 desalting tip by pipetting up 10 µL of 50% ACN, 49.8% water, and 0.2% FA and then discarding it. Repeat this step 2x.
3. Equilibrate the C18 desalting tip by pipetting up 10 µL of 0.2% FA in water and then discarding it. Repeat this step 2x.
4. Set the pipet to 10 µL and load the peptides from the solution to the resin by pipetting the digested peptides through the resin up and down 15x.
   NOTE: The repeated action will ensure binding of all peptides to the resin of the desalting tips.
5. Desalt the digested peptides bound to the resin by pipetting up 10 µL of 0.2% FA in water and then discarding it. Repeat this step 4x to complete the desalting step.
6. In a new tube, elute the peptides with 10 µL of 50% ACN, 49.8% water, and 0.2% FA by pipetting up and down 10x. Repeat this step once to elute the protein digest for a second time in the same tube.
7. Dry the desalted peptides completely in a vacuum concentrator (~20 min).
8. Resuspend peptides in 15 µL of 0.2% FA + 1 µL iRT (indexed Retention Time) peptides. Voxtex for 10 min, centrifuge at 12,000 x g for 2 min, and then transfer to an autosampler vial for MS analysis (see below).

5. Mass spectrometry analysis of digested peptides using DDA and DIA

NOTE: Samples can be analyzed using either DDA or DIA LC-MS/MS methods. In this study, the samples were analyzed using a nano-LC 2D HPLC system coupled to a high-resolution mass spectrometer.

1. Use a HPLC system combined with a chip-based HPLC system directly connected to a quadrupole time-of-flight mass spectrometer (other LC-MS configurations and systems can also be used).
2. Analyze samples using reverse-phase HPLC-ESI-MS/MS.
   1. Build the chromatography protocol to load peptides sample onto a C18 pre-column chip. Wash and desalt the loaded peptides with loading solvent (0.1% formic acid) for 10 min at a flow rate of 2 µL/min.
   2. Transfer the peptides to an analytical column C18 chip and elute at a flow rate of 300 nL/min with a 3 hour gradient using mobile phase A (2% acetonitrile, 0.1% formic acid) and B (98% acetonitrile, 0.1% formic acid). The first elution step consists of a linear gradient from 5% B to 35% B over a period of 80 min.
   3. Ramp the mobile phase B to 80% over 5 min, and then maintain the mobile phase B at 80% for 8 min before changing into 5% B to re-equilibrate the column for 25 min.
3. Set up a MS instrument method for DDA and define the instrument parameters as following.
   1. Use the following parameters for Experiment 1:MS1 precursor ion scan from m/z 400–1500 (accumulation time of 250 ms); set the intensity threshold to trigger MS/MS scans for ions of charge states 2–5 to 200 counts; set the dynamic exclusion of precursor ions to 60 s).
   2. Use the following parameters for Experiment 2: MS/MS product ion scans with a MS2 scan range from m/z 100–1500 (accumulation time of 100 ms per each of the 30 product ion scans per cycle); set the collision energy spread to CES=5, and checkmark ‘high sensitivity product ion scan mode’.
      NOTE: The DDA acquisition method is used to build spectral libraries as described in step 6.3 (see below). Here, it will acquire MS/MS spectra for the 30 most abundant precursor ions after each MS1 scan per cycle; the total cycle time is about 3.3 s.
4. Obtain DIA. Set up a MS instrument method for DIA and define the instrumental parameters as follows.
   1. Use the following parameters for Experiment 1: MS1 precursor ion scan from m/z 400-1250 (accumulation time of 250 ms).
   2. Use the following parameters for Experiment 2: MS/MS product ion scans for 64 variable SWATH segments with a MS2 scan range from m/z 100-1500 (accumulation time of 45 msec per each of the 64 product ion scans per cycle); set the collision energy spread to CES=10, checkmark ‘high sensitivity product ion scan mode’.
      NOTE: Use the 64-variable window DIA/SWATH acquisition method as described by Schilling et al.¹³ to perform label-free quantification with a total cycle time of about 3.2 s. For DDA acquisition, instead of using the Q1 quadrupole to transmit precursor ion of a narrow mass range to the collision cell, a range of variable window width (for example m/z 5–90) is used to incrementally step over the full m/z range (m/z 400–1250) with 64 SWATH segments, 45 ms accumulation time for each segment, resulting in a cycle time of 3.2 sec, which includes one MS1 scan with 250 ms accumulation time. The variable window width is adjusted according to the complexity of the typical MS1 ion current observed within a certain m/z range using a SCIEX ‘variable window calculator’ algorithm¹³ (narrower windows are applied to ‘busy’ m/z ranges, wider windows are applied to m/z ranges with few eluting precursor ions). On other MS platforms, alternative DIA window selection strategies can also be implemented.

6. Data analysis

NOTE: Certain data analysis settings should be tailored to specific experimental conditions. For example, the protein database (fasta file) selected will depend on the species that the sample was prepared from (in this protocol we used C. elegans).

1. Use a MS database search engine to analyze DDA acquisitions and identify proteins. Generate a darabase search engine method as follows:
   1. For Sample Description Parameters, select Identification; under Sample Type, select Iodoacetic Acid; under Cysteine Alkylation, select Trypsin; under Digestion (assuming C-terminal cleavage at lysine and arginine), select the name of the mass spectrometer under Instrument; and select Caenorhabditis elegans under Species.
   2. For Specific Processing parameters, check Biological modifications; under ID Focus, select SwissProt; under Database, check Thorough ID; under Search Effort, select 0.05 (10%) under Detected Protein Threshold; and check Run False Discovery Rate Analysis under Results Quality. Save the search engine method and submit the MS raw files to the database search engine for processing, using the generated method.
      NOTE: In an iterative process, all MS and MS/MS scans were automatically recalibrated by the search engine based on initial annotations and results.
2. Click on Export Peptide Summary upon completion of the search and filter all peptide identification results by the confidence threshold of 99 in Excel (false discovery rate (FDR) = 1%).
3. Build MS/MS spectral libraries for further processing of the DIA raw data file, and for further relative data quantification.
   1. Open the DIA Quantitative Analysis Software. Select Library tab and then (at the bottom of the page) click Generate Spectral Library from “Database Search Engine” and open a “Database Search Engine” FDR report (the *FDR.xlsx spreadsheet file) that was automatically generated as part of the DDA raw data file database search process.
   2. Click next, select the Library Settings Schema and click Next. Select Uniprot_Caenorhabditis elegans _proteome as the database then click Finish, the spectral library will be generated.
4. Use the DIA Quantitative Analysis Software to analyze DIA acquisitions for comprehensive relative protein quantification. DIA data analysis pipeline is described as follows.
   1. To analyze and quantify peptides, open the DIA Quantitative Analysis Software to set up an analysis schema by using the template schema. The template schema is available in the software by clicking Settings, then DIA Analysis, and finally BGS Factory Settings.
   2. Set up the parameters in the template schema as the following:
      1) under identification select PTM localization (probability cutoff = 0.75).
      2) under quantification, select Major Group Quantity as Sum peptide quantity, set up Major Group N as Max 7 and Min 1, select Minor Group Quantity as Sum precursor quantity, and set up Minor Group N as Max 10 and Min 1, and selected Data Filtering as Qvalue sparse and do not select Cross Run Normalization.
5. Perform Relative Quantification Analysis.
   1. Select the Pipeline tab, click Set up a DIA Analysis from File, open the MS DIA raw files of interest (for example: sample and control), click Assign Spectral Library, select the library that was generated in 6.3, click Load, and lastly click Next.
   2. Select the analysis schema set up in 6.5.1 and click Next. Select the appropriate database fasta file (in this protocol: Uniprot_Caenorhabditis elegans_proteome) and click Next. Define the condition setup, and assign the different conditions according to the samples and click Next.
   3. Review the analysis overview (summary of the experiment set-up) and browse to the Output Directory and click Finish. Finally click Run Pipeline to perform the label-free quantitative analysis.
      NOTE: Statistical modules in the ‘DIA Quantitative Analysis Software’ automatically perform FDR analysis, generate heat maps and volcano plots comparing the different conditions, generate lists of identified and quantified peptides and proteins and provide Q-values along with relative fold-changes comparing different conditions. Here sample conditions set-up included Day 10 aged worms with two biological replicates and Day 2 young worms with two biological replicates as control.