Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

1. Quantitatively Acetylate and/or Succinylate Proteins Using Isotope-labeled Acetic or Succinic Anhydride

1. Prepare 3 replicates of 100 µg of bovine serum albumin (BSA) protein sample in parallel, at a concentration of 1 µg/µL (total of 100 µL), using urea and triethylammonium bicarbonate (TEAB) solution (8 M urea, 200 mM TEAB, pH 8).
   NOTE: It is important to use amine-free buffer. The protein samples used here in the representative results section are BSA, quantitatively succinylated BSA, and mixtures thereof yielding BSA samples with defined succinylation occupancy at 0%, 1%, 10%, 50%, and 100%, respectively (each sample will be prepared in 3 replicates). This protocol has also been performed using protein lysates from Escherichia coli and mouse liver⁴. The protocol can also be applied to other cell or tissue lysates.
2. Reduce 100 µL of BSA sample (100 µg) with 8 µL of 250 mM dithiothreitol (DTT) to reach a final concentration of 20 mM DTT and incubate the sample at 37 °C for 30 min with agitation at 1,400 rpm.
3. Alkylate the reduced BSA sample with 21.6 µL of 200 mM iodoacetamide to reach a final concentration of 40 mM iodoacetamide and incubate the sample in the dark at room temperature for 30 min.
   NOTE: It is important to have the iodoacetamide concentration slightly more than 2x of the DTT concentration to ensure that all reduced protein thiols are alkylated.
4. Proceed with preparing either acetic anhydride-d₆ (step 1.4.1) or succinic anhydride-d₄ (step 1.4.2) needed for chemical (quantitative) acetylation or succinylation of the samples.
   1. Determine the molarity (M) of the 1 g aliquot of aqueous acetic anhydride-d₆ solution from the molecular weight (108.13 g/mol) and density (1.143 g/mL at 25 °C).
      NOTE: The 1 g package contains 9,248 µmol of acetic anhydride-d₆ in 875 µL volume, yielding a 10.57 M solution used for per-acetylation of the samples.
   2. Prepare a 5 M solution of succinic anhydride-d₄ by dissolving the powder in anhydrous DMSO immediately prior to the reaction to avoid hydrolysis of the reagent. Dissolve 0.24 g of succinic anhydride-d₄ in 461 µL of anhydrous DMSO to obtain a 5 M solution.
5. Perform quantitative chemical acetylation or succinylation by adding 60 µmol of acetic anhydride-d₆ (6 µL of the 10.57 M solution) or succinic anhydride-d₄ (12 µL of the 5 M solution) respectively, and incubate the sample at 4 °C for 20 min on a vortex mixer.
   NOTE: Due to the acidity of anhydrides, proteins may precipitate. Take note and adjust as described in step 1.6.
6. Add 10 µL of 7.25 M NaOH after the incubation to increase the pH to ~8 to eliminate any O-acylation side-products. Vortex briefly and check the sample pH by spotting 1 µL on pH paper.
   NOTE: It may be necessary to add more than 10 µL of 7.25 M NaOH to reach pH 8. In addition, potentially precipitated proteins should re-dissolve.
7. Repeat the per-acylation and pH adjustment steps 1.5 and 1.6 for a total of three times. Check the pH values and note the volume of basic solution added per repetition. Work swiftly during the above chemical acylation steps because the anhydrides will hydrolyze and lose reactivity.
8. After the final labeling reaction and pH adjustment, add 10 µL of 50% hydroxylamine solution to revert O-acylation side reactions.

2. Digest Reacted Protein Samples Using Glu-C Endoproteinase

1. Dilute the urea concentration to 0.8 M using 50 mM TEAB, pH 8 and normalize the sample volumes.
2. Digest the protein samples by adding endoproteinase Glu-C at a 1:50 protease to substrate protein ratio (w/w) and incubate the samples overnight at 37 °C with agitation at 1,400 rpm.
3. Acidify and quench the protein digestion samples by adding formic acid to 1% by volume.
4. Desalt the samples using hydrophilic-lipophilic balance solid-phase extraction cartridges. Use one 30 mg sorbent cartridge per sample, maximum 5 mg material per cartridge^4,18.
   NOTE: It is important to keep the sorbent inside the cartridge wet throughout this desalting process. When necessary, turn off the vacuum pump to slow down the passage of solvent or sample through the cartridge.
   1. Insert the cartridge(s) into the port(s) on the 24-port glass block vacuum manifold and turn on the vacuum pump. Leave unused port(s) capped. Leave one or two ports un-capped to maintain lower pressure on the vacuum gauge if necessary.
   2. Wet each cartridge two times with 800 µL of 80% acetonitrile (ACN)/0.2% formic acid/19.8% water. Ensure solvent level remains 2–3 mm above the sorbent level. Ensure the vacuum gauge on the manifold reads 16.9–67.7 kPa (5–20 in Hg).
   3. Equilibrate each cartridge three times with 800 µL of 0.2% formic acid in water. Ensure the vacuum gauge on the manifold reads 16.9–67.7 kPa (5–20 in Hg).
   4. Load the peptide samples on to the cartridge. Ensure the vacuum gauge on the manifold reads 6.77–8.47 kPa (2–2.5 in Hg), and flow rate does not exceed 1 mL/min.
   5. Wash each cartridge three times with 800 µL 0.2% formic acid in water. Ensure the vacuum gauge on the manifold reads 16.9–67.7 kPa (5–20 in Hg).
   6. Remove the cartridge from the vacuum manifold and settle into a 1.5 mL microtube.
   7. Elute peptides one time with 800 µL of 80% ACN/0.2% formic acid/19.8% water and allow the solvent to incubate with sorbent for 2–3 min.
   8. Use a pipette to push solvent through the sorbent layer inside the cartridge. Repeat for the second elution of 400 µL 80% ACN/0.2% formic acid/19.8% water into the same 1.5 mL microtube.
5. Dry the eluate by vacuum centrifugation (fixed centrifugation rate at 268 x g), typically for 3 h.
   NOTE: If needed, dried eluate samples can be stored frozen at -20 °C until ready to proceed with fractionation.

3. Fractionate Peptides Using Offline Basic-pH Reversed Phase HPLC

1. Re-suspend the per-acylated and Glu-C digested samples (prepared as described in Sections 1 and 2) in 200 µL of Buffer A (10 mM ammonium formate in water, pH 10), mix the sample for 10 min on a vortex mixer at 4 °C, and centrifuge at 17,500 x g for 10 min at room temperature.
   NOTE: The resulting peptide sample is ready to be offline fractionated by high pH separation^19,20.
2. Program the offline HPLC fractionation method depending on the instrument and software used. The following method is specific for a binary HPLC pump system including a dual wavelength UV detector, an autosampler, a fraction collector, and a C18 column (4.6 mm x 250 mm, 5 µm particle size) that tolerates pH 10.
3. Collect 40 fractions each for 2.1 min per fraction and combine every fourth fraction, resulting in four final pooled fractions⁴. A greater number of pooled fractions can be used to further reduce sample complexity at the expense of mass spectrometry data collection time.
4. Dry the pooled fractions in a lyophilizer, re-suspend the dried peptide samples in 1 mL of 50% ACN and 1% formic acid in water, and transfer to a smaller sample container. Dry the transferred sample via vacuum centrifugation (fixed centrifugation rate at 268 x g), typically for 1 h.
   NOTE: The low vapor pressure of ammonium formate can make complete drying difficult. If significant amounts of ammonium formate salt remain as salt precipitate after drying, repeat the solid phase extraction.
5. Re-suspend the samples in 20 µL of 0.2% formic acid in water with indexed retention time (iRT) peptide standard mixture.
   NOTE: Samples are now ready for analysis by mass spectrometry.

4. DIA Analysis of Fractionated Peptide Samples

1. Analyze the samples using a DIA LC-MS/MS method, which can be adjusted according to the mass spectrometric instruments available. The analysis was performed using a nano-LC 2D HPLC system online connected to an orthogonal quadrupole time-of-flight (QqTOF) mass spectrometer.
   1. Program the instrument software so that peptide mixtures are transferred onto a C18 pre-column chip (200 µm x 6 mm C18-CL chip, 3 µm, 300 Å) and wash at 2 µL/min for 10 min with the loading solvent (H₂O/0.1% formic acid) for desalting.
   2. Separate the peptides chromatographically on a 75 µm x 15 cm C18-CL chip (3 µm, 300 Å) at a flow rate of 300 nL/min with a 2 h gradient using typical (and LC-system specific) aqueous and ACN solvent buffers⁴.
   3. Generate a variable window DIA method, where the mass range windows of variable width (5 to 90 m/z) are passed from the Q1 quadrupole into the collision cell in incremental steps over the full mass range (m/z 400-1,250).
      NOTE: The cycle time of 3.2 s includes a 250 ms precursor ion scan followed by 45 ms accumulation time for each of the 64 SWATH segments^21,22.
2. Identify peptides using a parallel analysis of samples by DDA MS and building of spectral libraries, or alternatively, peptides can be identified directly from DIA data using PIQED software²³, which handles all technical steps of data processing, identification, and import into Skyline for subsequent quantification.

5. Example Data Analysis Tutorial

1. Download the Skyline software (http://proteome.gs.washington.edu/software/skyline) and create a PanoramaWeb account (https://panoramaweb.org).
   NOTE: For detailed tutorials describing use of Skyline with data, please see (https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorials). Other software algorithms may be used instead to extract light and heavy fragment ion peak areas from the DIA acquisitions, such as Open SWATH²⁴, SWATH 2.0 plugin into PeakView²⁵, and Spectronaut²⁶.
   1. Download the Skyline data file for the succinylated BSA representative results (Figure 3) from Panorama Public: https://panoramaweb.org/labkey/JoVEsuccBSA.url. On the Panorama webpage, go to the "Targeted MS Runs" table, and download the sky.zip file choosing the DOWNLOAD link on the right side. Extract the downloaded .zip folder and double click on the skyline.sky file to open it in Skyline. Check the peptide fragment ion target tree (left panel) and the four chromatograms from defined percentages of light succinylation (right panels).
   2. Select the "View" menu, navigate to "Peak Areas | Replicate Comparison". The "Peak Areas – Replicate Comparison" panel containing bar graphs will appear on the right side of the window. In the Peak Areas panel, right-click and select "Transitions | Single", which shows the fragment ion peak area for the selected fragment ion. Finally, right-click the "Peak Areas" panel and select "Order | Document".
   3. In the target tree panel on the left side of the window, right-click on the peptide "E.LCKVASLRE.T | Ratios To |Oneheavy", which calculates the ratio of light ion signal over heavy ion signal, Light/Heavy.
      NOTE: This is not the same as site occupancy stoichiometry ratio of Light/(Heavy+Light).
   4. Notice that the target tree now reports the ratios L/H to the right of the light fragment ions.
2. Determine the "differentiation fragment ions" that contain the acylation site of interest.
   NOTE: As shown in Figure 3, featuring this exact example, the differentiating ions were determined as y₇ (highest ranked), y₈, b₃, and b₄. Notice that the non-differentiating fragment ions show a ratio of 1 in the target tree.
   1. In the left panel target tree, under the 588.30++ sub node of peptide E.LCKVASLRE.T, click on the y7 differentiating fragment ion
      K [y₇ ] – 902.49+ (rank1) [5]
      Notice the increasing peak height and area.
   2. In the left panel target tree, under the 588.30++ sub node of peptide E.LCKVASLRE.T, click on the y₅ non-differentiating fragment ion A[y₅ ] – 575.31+ (rank 6) [1]. Observe that the y₅ fragment ion chromatograms and peak areas remain in the same range for all samples.
   3. In the target tree panel, browse through other differentiating (ratio other than 1) and non-differentiating (ratio of 1) fragment ions and notice the changes and patterns in the peak area bar graph and chromatograms.
   4. Determine the exact peak areas for the light and heavy pairs of the differentiating ions in order to calculate the stoichiometry (for additional details also see Meyer et al.⁴). Click on the "View | Document Grid" and the document grid will pop up. In the top left of the document grid, click on "Views | Transition Results" to see a multi-column table with fragment ion information, such as Peptide Sequence, Precursor mz, Replicate Name, etc.
   5. Change the report format to "pivot" by again clicking on "Views" in the document grid and selecting "Edit View" to open the Customize View window. At the bottom left of the "Customize View" window, check the "Pivot Replicate Name" and select "Ok" to close the Customize View window. Observe that now the "Document Grid: Transition Results" table window has re-arranged to group the Replicate Name, Retention Time, Area, Background, Peak Rank columns by Replicate (1%, 10%, 50%, 100% succinylation).
   6. For the peptide "E.LCKVASLRE.T" in the "Document Grid: Transition Result" table, find the "Precursor Mz" column, the "Fragment Ion" column, and the 1pct_light_sw1 Area (1% succinylation) column. Observe that there are two precursor ions for this peptide, light (Precursor Mz of 588.30 in the first 8 rows), and heavy (Precursor Mz of 590.32 in the last 8 rows).
   7. In the "Fragment Ion" column, find the two rows for the y8 fragment ion corresponding to the light and the heavy precursor ions. From the same rows in the 1pct_light_sw1 Area column, record the y8 areas for the light (15,820), and the heavy (1,426,461).
3. Using these peak area values, compute the succinylation stoichiometry, or the proportion modified, according to the formula below and obtain a stoichiometry ratio of 0.01, or 1% succinylation, which matches the proportion of light succinylation in this defined mixture:
   
4. Calculate the stoichiometry ratio for 10% succinylation using the y8 differentiating fragment ion peak area values from the 10pct_light_sw1 Area column, 144,953 (light) and 1,188,041 (heavy), to obtain a ratio of 0.10, or 10% succinylation.
5. Calculate the stoichiometry ratio for 100% succinylation using the y8 differentiating fragment ion peak area values from the 100pct_light_sw1 Area column, 954,513 (light) and 16,407 (heavy), to obtain a ratio of 0.98, or 98% succinylation.
6. Similarly, calculate the stoichiometry ratio for 1% succinylation using the b3 differentiating fragment ion peak area values from the 1pct_light_sw1 Area column, 55,697 (light) and 3,149,119 (heavy), to obtain a ratio of 0.01, or 1% succinylation.
7. Continue the stoichiometry ratio calculations using the formula for all the differentiating fragment ions, both y and b ions, to obtain the lysine succinylation stoichiometry values for 1, 10, 50, and 100% succinylation mixtures.