This work was supported by the NIH National Institute of Diabetes and Digestive and Kidney Diseases NIH-NIDDK grant R24 DK085610 (PI: E. Verdin). JGM was supported by a NIH T32 fellowship (T32 AG000266, PI: J. Campisi). The authors acknowledge support from the NIH shared instrumentation grant for the TripleTOF 6600 system at the Buck Institute (1S10 OD016281, PI: B.W. Gibson).

1. Quantitatively Acetylate and/or Succinylate Proteins Using Isotope-labeled Acetic or Succinic Anhydride
<ol>
	<li>Prepare 3 replicates of 100 &#181;g of bovine serum albumin (BSA) protein sample in parallel, at a concentration of 1 &#181;g/&#181;L (total of 100 &#181;L), 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.</li>
	<li>Reduce 100 &#181;L of BSA sample (100 &#181;g) with 8 &#181;L of 250 mM dithiothreitol (DTT) to reach a final concentration of 20 mM DTT and incubate the sample at 37 &#176;C for 30 min with agitation at 1,400 rpm.</li>
	<li>Alkylate the reduced BSA sample with 21.6 &#181;L 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.</li>
	<li>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.
	<ol>
		<li>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 &#176;C). 
		NOTE: The 1 g package contains 9,248 &#181;mol of acetic anhydride-d6 in 875 &#181;L volume, yielding a 10.57 M solution used for per-acetylation of the samples.</li>
		<li>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 &#181;L of anhydrous DMSO to obtain a 5 M solution.</li>
	</ol>
	</li>
	<li>Perform quantitative chemical acetylation or succinylation by adding 60 &#181;mol of acetic anhydride-d6 (6 &#181;L of the 10.57 M solution) or succinic anhydride-d4 (12 &#181;L of the 5 M solution) respectively, and incubate the sample at 4 &#176;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.</li>
	<li>Add 10 &#181;L 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 &#181;L on pH paper. 
	NOTE: It may be necessary to add more than 10 &#181;L of 7.25 M NaOH to reach pH 8. In addition, potentially precipitated proteins should re-dissolve.</li>
	<li>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.</li>
	<li>After the final labeling reaction and pH adjustment, add 10 &#181;L of 50% hydroxylamine solution to revert O-acylation side reactions.</li>
</ol>
2. Digest Reacted Protein Samples Using Glu-C Endoproteinase
<ol>
	<li>Dilute the urea concentration to 0.8 M using 50 mM TEAB, pH 8 and normalize the sample volumes.</li>
	<li>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 &#176;C with agitation at 1,400 rpm.</li>
	<li>Acidify and quench the protein digestion samples by adding formic acid to 1% by volume.</li>
	<li>Desalt the samples using hydrophilic-lipophilic balance solid-phase extraction cartridges. Use one 30 mg sorbent cartridge per sample, maximum 5 mg material per cartridge4,18. 
	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.
	<ol>
		<li>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.</li>
		<li>Wet each cartridge two times with 800 &#181;L of 80% acetonitrile (ACN)/0.2% formic acid/19.8% water. Ensure solvent level remains 2&#8211;3 mm above the sorbent level. Ensure the vacuum gauge on the manifold reads 16.9&#8211;67.7 kPa (5&#8211;20 in Hg).</li>
		<li>Equilibrate each cartridge three times with 800 &#181;L of 0.2% formic acid in water. Ensure the vacuum gauge on the manifold reads 16.9&#8211;67.7 kPa (5&#8211;20 in Hg).</li>
		<li>Load the peptide samples on to the cartridge. Ensure the vacuum gauge on the manifold reads 6.77&#8211;8.47 kPa (2&#8211;2.5 in Hg), and flow rate does not exceed 1 mL/min.</li>
		<li>Wash each cartridge three times with 800 &#181;L 0.2% formic acid in water. Ensure the vacuum gauge on the manifold reads 16.9&#8211;67.7 kPa (5&#8211;20 in Hg).</li>
		<li>Remove the cartridge from the vacuum manifold and settle into a 1.5 mL microtube.</li>
		<li>Elute peptides one time with 800 &#181;L of 80% ACN/0.2% formic acid/19.8% water and allow the solvent to incubate with sorbent for 2&#8211;3 min.</li>
		<li>Use a pipette to push solvent through the sorbent layer inside the cartridge. Repeat for the second elution of 400 &#181;L 80% ACN/0.2% formic acid/19.8% water into the same 1.5 mL microtube.</li>
	</ol>
	</li>
	<li>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 &#176;C until ready to proceed with fractionation.</li>
</ol>
3. Fractionate Peptides Using Offline Basic-pH Reversed Phase HPLC
<ol>
	<li>Re-suspend the per-acylated and Glu-C digested samples (prepared as described in Sections 1 and 2) in 200 &#181;L of Buffer A (10 mM ammonium formate in water, pH 10), mix the sample for 10 min on a vortex mixer at 4 &#176;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 separation19,20.</li>
	<li>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 &#181;m particle size) that tolerates pH 10.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Re-suspend the samples in 20 &#181;L 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.</li>
</ol>
4. DIA Analysis of Fractionated Peptide Samples
<ol>
	<li>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.
	<ol>
		<li>Program the instrument software so that peptide mixtures are transferred onto a C18 pre-column chip (200 &#181;m x 6 mm C18-CL chip, 3 &#181;m, 300 &#197;) and wash at 2 &#181;L/min for 10 min with the loading solvent (H2O/0.1% formic acid) for desalting.</li>
		<li>Separate the peptides chromatographically on a 75 &#181;m x 15 cm C18-CL chip (3 &#181;m, 300 &#197;) at a flow rate of 300 nL/min with a 2 h gradient using typical (and LC-system specific) aqueous and ACN solvent buffers4.</li>
		<li>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 segments21,22.</li>
	</ol>
	</li>
	<li>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.</li>
</ol>
5. Example Data Analysis Tutorial
<ol>
	<li>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.
	<ol>
		<li>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 &#34;Targeted MS Runs&#34; 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).</li>
		<li>Select the &#34;View&#34; menu, navigate to &#34;Peak Areas | Replicate Comparison&#34;. The &#34;Peak Areas &#8211; Replicate Comparison&#34; panel containing bar graphs will appear on the right side of the window. In the Peak Areas panel, right-click and select &#34;Transitions | Single&#34;, which shows the fragment ion peak area for the selected fragment ion. Finally, right-click the &#34;Peak Areas&#34; panel and select &#34;Order | Document&#34;.</li>
		<li>In the target tree panel on the left side of the window, right-click on the peptide &#34;E.LCKVASLRE.T | Ratios To |Oneheavy&#34;, 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).</li>
		<li>Notice that the target tree now reports the ratios L/H to the right of the light fragment ions.</li>
	</ol>
	</li>
	<li>Determine the &#34;differentiation fragment ions&#34; 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.
	<ol>
		<li>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 ] &#8211; 902.49+ (rank1) [5] 
		Notice the increasing peak height and area.</li>
		<li>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 ] &#8211; 575.31+ (rank 6) [1]. Observe that the y5 fragment ion chromatograms and peak areas remain in the same range for all samples.</li>
		<li>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.</li>
		<li>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 &#34;View | Document Grid&#34; and the document grid will pop up. In the top left of the document grid, click on &#34;Views | Transition Results&#34; to see a multi-column table with fragment ion information, such as Peptide Sequence, Precursor mz, Replicate Name, etc.</li>
		<li>Change the report format to &#34;pivot&#34; by again clicking on &#34;Views&#34; in the document grid and selecting &#34;Edit View&#34; to open the Customize View window. At the bottom left of the &#34;Customize View&#34; window, check the &#34;Pivot Replicate Name&#34; and select &#34;Ok&#34; to close the Customize View window. Observe that now the &#34;Document Grid: Transition Results&#34; table window has re-arranged to group the Replicate Name, Retention Time, Area, Background, Peak Rank columns by Replicate (1%, 10%, 50%, 100% succinylation).</li>
		<li>For the peptide &#34;E.LCKVASLRE.T&#34; in the &#34;Document Grid: Transition Result&#34; table, find the &#34;Precursor Mz&#34; column, the &#34;Fragment Ion&#34; 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).</li>
		<li>In the &#34;Fragment Ion&#34; 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).</li>
	</ol>
	</li>
	<li>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: 
	<img alt="Equation" src="/files/ftp_upload/57209/57209eq1.jpg" /></li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+differential+interactomes+by+affinity+purification+coupled+with+data-independent+mass+spectrometry+acquisition.">Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition.</a> Nat Methods. 10 (12), 1239-1245 (2013).</li><li>Bruderer, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extending+the+limits+of+quantitative+proteome+profiling+with+data-independent+acquisition+and+application+to+acetaminophen-treated+three-dimensional+liver+microtissues.">Extending the limits of quantitative proteome profiling with data-independent acquisition and application to acetaminophen-treated three-dimensional liver microtissues.</a> Mol Cell Proteomics. 14 (5), 1400-1410 (2015).</li><li>Weinert, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+Quantification+of+Site-specific+Acetylation+Stoichiometry+Reveals+the+Impact+of+Sirtuin+Deacetylase+CobB+on+the+E.+coli+Acetylome.">Accurate Quantification of Site-specific Acetylation Stoichiometry Reveals the Impact of Sirtuin Deacetylase CobB on the E. coli Acetylome.</a> Mol Cell Proteomics. 16 (5), 759-769 (2017).</li></ol>

The authors have nothing to disclose.

This protocol provides a novel and accurate method to quantify site-specific lysine acetylation and succinylation occupancy that can be applied to an entire proteome. This method relies on measurement of endogenous light peptides and exogenous heavy peptides, the latter of which are generated in vitro using quantitative chemical acylation of proteins with deuterated acetic anhydride-d6 or succinic anhydride-d4 (Figure 1). Similar methods have used stable isotopic labeling of native unmodified lysine residues and performed site occupancy quantification based on either precursor ion-only12 or fragment ions from DDA acquisitions13,14. This protocol applies several steps during sample preparation to improve acylation efficiency and extends the chemical labeling to succinylation. Data collection using DIA acquisitions obtains both precursor ion and MS2 fragment ion intensities over the entire detectable mass range, thus reducing the fragment ion interferences. Analysis and quantification via Skyline and custom scripts developed in-house allow the site occupancy calculation for peptides containing more than one lysine residue and more than one acylation at one site.
There are several critical steps during the sample preparation stage of this protocol that should be followed closely. Since the entire protocol relies on efficient chemical modification of all lysine residues, this step is of utmost importance. Anhydrides react with free amines, so amine-containing buffer or contaminant molecules in the protein lysate must be avoided. Also during the chemical per-acylation step, ensure that the pH of the reaction mixture is adjusted back to pH 8 after each of the three incubations with anhydride reagent as this reaction acidifies the mixture, and O-acylation side-reactions may form. Furthermore, dilution of the 8 M urea-containing reaction mixture to around 0.8 M urea prior to digestion and checking that the pH is within the optimal range is important for the optimum activity of the endoproteinase Glu-C. Another key component of our protocol is the data collection step and the introduction of a DIA workflow, which overcomes any DDA data sampling inconsistencies and which allows for quantification at the MS2 fragment ion level. One main advantage of the DIA methodology is the decrease of interferences which are typically much more prone and problematic when quantifying the MS1 precursor ion signal (as some of the previous publications have suggested).
Several modifications to the workflow can be made when preparing highly complex samples. The number of fractions pooled after the offline basic-pH reversed-phase HPLC fractionation can be decreased to lower the complexity of the pooled samples that will be acquired by LC/MS. Additionally, longer chromatographic gradients can also be used during acquisition to allow for better peptide separation. Alternatively, multiple proteases may be used to digest samples to produce a larger variety of cleaved peptides and increase coverage of protein lysine residues. Trial runs and optimization can be conducted using commercial BSA containing specific percentages of acetylation or succinylation.
One limitation to this protocol is potentially slight site occupancy overestimation due to unaccounted other lysine modifications, such as methylation or ubiquitination, etc., at the same site4. Calculated from the peak areas of light and heavy acyl modifications, L/(L+H), the site occupancy is based on the assumption that the total level of modification at a lysine site consists of only endogenous &#39;light&#39; acylation (L) and chemically labeled &#39;heavy&#39; acylation (H). However, the actual total acylation occupancy at a site in vivo may include other modifications beyond acetylation and/or succinylation. Additionally, the reported isotopic purity of the purchased reagents acetic anhydride-d6 (99%) and succinic anhydride-d4 (&#62;98%) may contribute to a small overestimation of up to 2% (succinyl) or 1% (acetyl) of the endogenous acylation level4. Together, these slight overestimations add to the difficulty in quantifying sites with less than 1% occupancy. Large abundance differences between the complete chemically acylated peptides and the native acylated peptides also contribute to potential site occupancy quantification errors, especially for low endogenous acylated peptides27. A recent study by Weinert et al.27 found that decreased abundance differences between complete per-acetylated peptides can reduce the quantification error27.
Stoichiometry quantifications gathered using this protocol can pinpoint important acylation hotspots in a particular protein and form hypotheses for biological follow-up experiments, such as site-directed mutagenesis of certain sites of interest. This protocol could also reveal combined effects of low acylation stoichiometry sites that could exert indirect or subtle influences on protein structural stability and localized cellular environments. Additionally, implementation of this protocol to measure acetylation and succinylation and other possible lysine acylation modifications beyond acetylation would uniquely offer insights into the dynamic acylation effects on proteins under different biological conditions.

N&#400;-acylation of protein lysine residues is an important regulator of protein function. Lysine acetylation and other acylations, such as succinylation, malonylation, and glutarylation, are thought to regulate metabolism, cell signaling, and other cellular processes1,2,3,4, and have implications in metabolic disorders5. Numerous studies have found that lysine acyl modifications undergo large fold-changes under different conditions in mammalian tissues and cell lines5,6,7,8 as well as bacteria9,10,11, however, these relative fold changes do not provide insights into the proportion of the total protein modified. Studies reporting measurements of acylation site occupancy are scarce12,13,14, despite the relevance and need for such studies as they provide more detail than relative fold change. For example, a 10-fold change could represent a site occupancy increase from 0.01 to 0.1%, 1 to 10%, or even 10 to 100%. Accurate stoichiometry measurements are required to interpret biological significance of acylation and to predict impact regarding the magnitude of protein structural, and possibly, functional changes.
One previous method to quantify site occupancy utilizes heavy stable-isotope chemical labeling of unmodified lysine residues followed by mass spectrometry to measure the ratio between endogenous &#34;light&#34; acetylation in comparison to the exogenous, chemically-labeled &#34;heavy&#34; acetyl-lysine using precursor ion intensities12. Another recent study by Zhou et al.13 described a similar approach to assess lysine acetylation stoichiometry that also employed a complete chemical acetylation of all unmodified lysine residues in proteins with stable isotope labeling, but used fragment ion intensities as measured by DDA. Nakayasu et al.14 used a similar DDA approach, but instead used the ratio of light and heavy acetyl-lysine immonium ions for quantification. Quantification based on fragment ions, immonium or sequence ions (MS2), in most cases results in less signal interferences compared to processing intact peptide precursor ions (MS1). However, quantification of fragment ions from DDA-generated MS/MS spectra can suffer from stochastic sampling deficiencies, where the high-abundance precursor ions are more likely to be selected for MS/MS and thus, lead to a biased and incomplete sampling of precursor ions.
This novel workflow4 (Figure 1) uses conceptually a stable isotope chemical labeling approach originally developed by Baeza et al.12, however the workflow is subsequently coupled with DIA to collect both precursor and multiple fragment ion abundances over the detectable mass range15,16 providing accurate stoichiometry calculations.
Peak areas from fragment ions that contain the acylation site of interest, or &#39;differentiating fragment ions&#39;, are used to quantify acylation site occupancy (Figure 2). Fragment ions that do not contain the modification have identical light and heavy m/z values, and are used for peptide identification but not for quantification. Skyline software17 is used to extract precursor and fragment peak areas. The presence of multiple fragment ions containing a given acylation site provides flexibility if interferences are detected in some of the fragment ions. Data visualization in Skyline allows for critical inspection of light-to-heavy fragment ion ratios. In addition to manual data analysis, an open-source R package written in-house, StiochiolyzeR (https://github.com/GibsonLab/StoichiolyzeR), was developed, which uses the precursor ion and fragment ion data collected through DIA and quantifies site-specific acylation stoichiometry from peptides containing multiple lysine residues, a feature not possible with precursor ion-only intensity measurements4.
This protocol also demonstrates the use of endoproteinase Glu-C, which is specific for C-terminal cleavage at glutamic and aspartic acid residues, instead of using trypsin or Arg-C protease, as the latter often generate very large and potentially multiply acylated proteolytic peptides resulting from blocked trypsin cleavage at acylated lysine residues. The chemical labeling procedure was also extended to include use of succinic anhydride (Figure 1), thereby enabling the quantification of succinylation stoichiometry succinylation. In addition to improvements to the sample preparation, the implementation of peptide fractionation by offline, basic pH, reversed-phase (bRP) separation of peptides further decreased quantification interferences, allowing better de-convolution of acylation stoichiometry in whole proteome samples. Together, this method features and highlights several advantages: (1) increased efficiency of chemical acylation; (2) measurement of protein succinylation stoichiometry in addition to acetylation; and (3) improved quantification accuracy. The improved quantification is due to decreased interferences in fragment ion signals from DIA compared to DDA precursor signal, as well as the implementation of off-line peptide pre-fractionation by bRP.

<table><tbody><tr><td>aqueous acetic anhydride-&lt;em&gt;d&lt;sub&gt;6&lt;/sub&gt;&lt;/em&gt;</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>175641-1G</td><td>1% isotopic purity</td></tr><tr><td>succinic anhydride-&lt;em&gt;d&lt;sub&gt;4&lt;/sub&gt;&lt;/em&gt;</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>293741</td><td>2% isotopic purity</td></tr><tr><td>sequencing grade endoproteinase Glu-C</td><td>Roche, Indianapolis, IN, USA (through Sigma)</td><td>10791156001</td><td>1:50 protease to substrate protein ratio (wt:wt)</td></tr><tr><td>Oasis HLB Solid-Phase Extraction (SPE) cartridges</td><td>Waters Corp., Milford, MA, USA</td><td>WAT 094225</td><td></td></tr><tr><td>HPLC acetonitrile</td><td>Burdick and Jackson, Muskegon, MI, USA</td><td>BJAH015-4</td><td></td></tr><tr><td>HPLC grade water</td><td>Burdick and Jackson, Muskegon, MI, USA</td><td>BJAH365-4</td><td></td></tr><tr><td>iodoacetamide</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>I1149-25G</td><td></td></tr><tr><td>dithiothreitol (DTT)</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>D9779-5G</td><td></td></tr><tr><td>FLUKA formic acid (FA) for mass spec</td><td>Thomas Scientific, Swedesboro, NJ, USA</td><td>C988C27</td><td>~98%</td></tr><tr><td>urea</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>PI29700</td><td></td></tr><tr><td>bovine serum albumin (BSA)</td><td>Pierce, Rockford, IL, USA</td><td>88341</td><td></td></tr><tr><td>triethylammonium bicarbonate (TEAB)</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>T7408-100ML</td><td></td></tr><tr><td>anhydrous dimethyl sulfoxide (DMSO)</td><td>Alfa Aesar, Tewksbury, MA, USA</td><td>43998</td><td></td></tr><tr><td>sodium hydroxide (NaOH)</td><td>EMDMillipore, Burlington, MA, USA</td><td>SX0590</td><td></td></tr><tr><td>50% hydroxylamine solution in water</td><td>Sigma Aldrich, St. Louis, MO, USA</td><td>438227-50ML</td><td></td></tr><tr><td>LC-MS grade water</td><td>Burdick and Jackson, Muskegon, MI, USA</td><td>BJLC365-4</td><td></td></tr><tr><td>LC-MS acetonitrile</td><td>Burdick and Jackson, Muskegon, MI, USA</td><td>BJLC015-4</td><td></td></tr><tr><td>Eppendorf Themomixer Compact</td><td>Eppendorf AG, Hamburg, Germany</td><td>T1317-1EA</td><td></td></tr><tr><td>Thermo Scientific Savant SPD131DDA SpeedVac Concentrator</td><td>Thermo Fisher Scientific, Waltham, MA, USA</td><td>SPD131DDA-115</td><td></td></tr><tr><td>Waters 1525 binary HPLC pump system</td><td>Waters Corp., Milford, MA, USA</td><td>WAT022939</td><td></td></tr><tr><td>Waters 2487 Dual Wavelength UV detector</td><td>Waters Corp., Milford, MA, USA</td><td>WAT081110</td><td></td></tr><tr><td>Waters 717plus Autosampler</td><td>Waters Corp., Milford, MA, USA</td><td>WAT022939</td><td></td></tr><tr><td>Waters Fraction Collector III</td><td>Waters Corp., Milford, MA, USA</td><td>186001878</td><td></td></tr><tr><td>Agilent Zorbax 300Extend C18 column</td><td>Agilent Technologies, Inc., Santa Clara, CA, USA</td><td>770995-902</td><td></td></tr><tr><td>Labconco Freeze Dry System Freezone 4.5 Lyophilizer</td><td>Labconco, Kansas City MO, USA</td><td>7751000</td><td></td></tr><tr><td>indexed retention time (iRT) normalization peptide standard</td><td>Biognosys AG, Schlieren, Zurich, Switzerland</td><td>Ki-3002-2</td><td></td></tr><tr><td>Ultra Plus nano-LC 2D HPLC system</td><td>Sciex LLC, Eksigent Division, Framingham, MA, USA</td><td>model# 845</td><td></td></tr><tr><td>orthogonal quadrupole time-of-flight (QqTOF) TripleTOF 5600 mass spectrometer</td><td>Sciex LLC, Framingham, MA, USA</td><td>per quote</td><td></td></tr><tr><td>orthogonal quadrupole time-of-flight (QqTOF) TripleTOF 6600 mass spectrometer</td><td>Sciex LLC, Framingham, MA, USA</td><td>per quote</td><td></td></tr><tr><td>C18 pre-column chip (200 &amp;mu;m &amp;times; 6 mm ChromXP C18-CL chip, 3 &amp;mu;m, 300 &amp;Aring;)</td><td>Sciex LLC, Framingham, MA, USA</td><td>804-00006</td><td></td></tr><tr><td>SWATH 2.0 plugin into PeakView 2.2</td><td>Sciex LLC, Framingham, MA, USA</td><td>software download Sciex</td><td></td></tr><tr><td>Spectronaut</td><td>Biognosys AG, Schlieren, Zurich, Switzerland</td><td>Sw-3001</td><td></td></tr><tr><td>C18-CL chip (75 &amp;micro;m x 15 cm ChromXP, 3 &amp;micro;m, 300 &amp;Aring;)</td><td>Sciex LLC, Framingham, MA, USA</td><td>804-00001</td><td></td></tr></tbody></table>

quantification of site-specific protein lysine acetylation and succinylation stoichiometry using data-independent acquisition mass spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

After data acquisition, differentiating MS2 fragment ions were determined from the proteolytic acylated peptides, subsequently extracted ion chromatograms (XIC) were processed in Skyline, and corresponding light and heavy peak areas were exported which were finally used to calculate site occupancy. Figure 2A shows a conceptual illustration of how the precursor-ion XIC may appear featuring both light and heavy peptide signals, and Figure 2B presents an example of the corresponding fragment ion XICs with color coding (red = light; blue = heavy) for differentiating fragment ions containing the lysine acylation modifications.
Figure 3 shows data resulting from an occupancy experiment, such as described above analyzing pre-defined ratios of light/heavy succinylated BSA generated in-house at 1, 10, 50, or 100%, and the determination of succinylation occupancy thereof. Importing DIA occupancy data into Skyline enables easy visualization of the target tree, displaying peptide sequences, and fragment ions both for the light and heavy precursor ions (Figure 3A). Data from DIA-MS of each defined succinylated BSA ratio revealed immanently the relative differences in ratio of light-to-heavy y7 ion, the highest-ranked differentiating fragment ion from the identified peptide-spectra match (indicated in red, Figure 3B). Figure 4 shows an overview of the processed results from the MS2 occupancy calculation that confirmed the succinylation percentages occupancy of input protein, here consisting of the pre-succinylated BSA. Measured lysine succinylation occupancy for 20 proteolytic succinylated peptides obtained from commercial pre-succinylated BSA, succinylated at defined percentages (e.g., at 0, 1, 10, 50, and 100%) are shown in Table 2. For each of the 20 proteolytic BSA peptides, the highest ranked, differentiating MS2 fragment ion was used to calculate lysine succinylation (Ksucc) occupancy, as L/(L+H) in %. Overall, the MS2-based quantification showed very good accuracy in determining acylation occupancy even at low stoichiometries.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57209/57209fig1.jpg" /> 
Figure 1: Stoichiometry workflow. (A) Proteins are incubated three times with acetic anhydride-d6 to acetylate unmodified lysine residues. Next, acetylated proteins are digested with endoproteinase Glu-C, followed by HPLC fractionation of the proteolytic peptides using basic-pH reversed-phase chromatography. Finally, peptides are analyzed by LC-MS using DIA with variable precursor window widths. (B) The same workflow is used to determine the succinylation stoichiometry as described in (A), except the heavy acylation reagent is changed to succinic anhydride-d4. Figure adapted from Meyer et al.4 (J. Am. Soc. Mass Spectrom., Open Choice). <a href="https://cloudflare.jove.com/files/ftp_upload/57209/57209fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57209/57209fig2.jpg" /> 
Figure 2: Example of possible data obtained and stoichiometry calculations. (A) Light (L) and heavy (H) MS1 precursor ions containing one acetylated lysine differ by 3 mass units. XICs are generated for both light and heavy isotopic envelopes indicated in red and blue, respectively. (B) Quantification from the MS2 fragment ion XICs from DIA acquisitions can be performed using &#39;differentiating&#39; light and heavy fragment ions that contain the acetylation site indicated in red and blue. Common fragment ions are displayed in dotted black and do not contain the site of modification. Figure adapted from Meyer et al.4 (J. Am. Soc. Mass Spectrom., Open Choice). <a href="https://cloudflare.jove.com/files/ftp_upload/57209/57209fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57209/57209fig3.jpg" /> 
Figure 3: Skyline visualization of succinylated proteolytic BSA peptide at different occupancy levels. (A) Skyline Target Tree showing the proteolytic peptide LCKsuccVASLRE and its resulting fragment ions. (B) Skyline extracted fragment ion chromatograms at varying levels of succinylation occupancy. The highest ranked differentiating ion, y7, increases in peak area 1, 10, 50, and 100% correlating to the input percentage of pre-succinylated BSA (generated in-house). <a href="https://cloudflare.jove.com/files/ftp_upload/57209/57209fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57209/57209fig4.jpg" /> 
Figure 4: Assessing BSA acylation stoichiometry of pre-succinylated BSA. Five different BSA samples at defined percentages of heavy modification (e.g., at 0, 1, 10, 50, and 100%) were subjected to MS2 occupancy determination. Site occupancy for 20 succinylated BSA peptides were determined from the highest ranked differentiating fragment ions for five different BSA samples at defined percentages of light modification (e.g., at 0, 1, 10, 50, and 100%). The Box Whisker plot displays the distribution of the 20 succinyl peptides, values from 50% of peptides are in the &#39;box&#39; (25% percentile to 75% percentile), the upper whisker indicates the values from 75% percentile to maximum (100%), and the lower whisker indicates the values from 25% percentile to minimum (0% percentile). (J. Am. Soc. Mass Spectrom., Open Choice). Quantification of measurements can be found in Table 2. <a href="https://cloudflare.jove.com/files/ftp_upload/57209/57209fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time (minute)</td>
			<td>% A</td>
			<td>% B</td>
		</tr>
		<tr>
			<td>0.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>7.27</td>
			<td>92</td>
			<td>8</td>
		</tr>
		<tr>
			<td>45.27</td>
			<td>73</td>
			<td>27</td>
		</tr>
		<tr>
			<td>49.27</td>
			<td>69</td>
			<td>31</td>
		</tr>
		<tr>
			<td>65.27</td>
			<td>61</td>
			<td>39</td>
		</tr>
		<tr>
			<td>72.27</td>
			<td>40</td>
			<td>60</td>
		</tr>
		<tr>
			<td>80.00</td>
			<td>10</td>
			<td>90</td>
		</tr>
		<tr>
			<td>85.00</td>
			<td>10</td>
			<td>90</td>
		</tr>
		<tr>
			<td>86.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td>120.00</td>
			<td>100</td>
			<td>0</td>
		</tr>
		<tr>
			<td colspan="2">Flow rate: 0.7 mL/min</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="3">Buffer A: 10 mM ammonium formate in water, pH 10</td>
		</tr>
		<tr>
			<td colspan="3">Buffer B: 10 mM ammonium formate in 90% ACN and 10% water, pH 10</td>
		</tr>
		<tr>
			<td colspan="3">Note: The pH of both mobile phases adjusted to 10 with neat ammonia&#160;</td>
		</tr>
	</tbody>
</table>
Table 1: Gradient for offline basic-pH reversed-phase HPLC fractionation. Gradient length: 120 min. Buffer A: 10 mM ammonium formate in water, pH 10. Buffer B: 10 mM ammonium formate in 90% ACN and 10% water, pH 10.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>L/L+H in %</td>
			<td>L/L+H in %</td>
			<td>L/L+H in %</td>
			<td>L/L+H in %</td>
			<td>L/L+H in %</td>
		</tr>
		<tr>
			<td>&#160;Ksucc 0%</td>
			<td>Ksucc 1%</td>
			<td>Ksucc 10%&#160;</td>
			<td>Ksucc 50%&#160;</td>
			<td>Ksucc 100%&#160;</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>1.7</td>
			<td>11.1</td>
			<td>50.9</td>
			<td>99.2</td>
		</tr>
		<tr>
			<td>1.2</td>
			<td>2.3</td>
			<td>12.3</td>
			<td>49.4</td>
			<td>97.6</td>
		</tr>
		<tr>
			<td>2.9</td>
			<td>3.8</td>
			<td>14</td>
			<td>48.6</td>
			<td>99.5</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>1.8</td>
			<td>11.7</td>
			<td>50.8</td>
			<td>99.5</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>1.7</td>
			<td>11.1</td>
			<td>48.3</td>
			<td>98.2</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>1.2</td>
			<td>11</td>
			<td>47.5</td>
			<td>96.1</td>
		</tr>
		<tr>
			<td>0.3</td>
			<td>1.6</td>
			<td>12.8</td>
			<td>51</td>
			<td>99.2</td>
		</tr>
		<tr>
			<td>3.7</td>
			<td>5.2</td>
			<td>14.7</td>
			<td>51.9</td>
			<td>89.5</td>
		</tr>
		<tr>
			<td>1.5</td>
			<td>1.8</td>
			<td>12.5</td>
			<td>47.2</td>
			<td>91.1</td>
		</tr>
		<tr>
			<td>0.8</td>
			<td>1.5</td>
			<td>11.3</td>
			<td>48.4</td>
			<td>96.8</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>1.6</td>
			<td>13.9</td>
			<td>49.7</td>
			<td>98.9</td>
		</tr>
		<tr>
			<td>0.1</td>
			<td>1.1</td>
			<td>10.7</td>
			<td>48.2</td>
			<td>98.6</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>0.9</td>
			<td>10.3</td>
			<td>49.4</td>
			<td>99.4</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>2.5</td>
			<td>17.2</td>
			<td>52.3</td>
			<td>97.5</td>
		</tr>
		<tr>
			<td>0.1</td>
			<td>3.5</td>
			<td>20.8</td>
			<td>51</td>
			<td>99</td>
		</tr>
		<tr>
			<td>0.3</td>
			<td>1.9</td>
			<td>10.7</td>
			<td>49.6</td>
			<td>98.2</td>
		</tr>
		<tr>
			<td>2</td>
			<td>1.7</td>
			<td>10.9</td>
			<td>47.7</td>
			<td>96.3</td>
		</tr>
		<tr>
			<td>0.3</td>
			<td>1.2</td>
			<td>10</td>
			<td>53</td>
			<td>98</td>
		</tr>
		<tr>
			<td>1.1</td>
			<td>1.8</td>
			<td>12.9</td>
			<td>57.9</td>
			<td>94.1</td>
		</tr>
		<tr>
			<td>0.2</td>
			<td>1.4</td>
			<td>11.6</td>
			<td>48.6</td>
			<td>98.8</td>
		</tr>
	</tbody>
</table>
Table 2: Quantification of measured BSA lysine succinylation occupancy for 20 proteolytic succinylated peptides. Succinylated peptides obtained from commercial pre-succinylated BSA, succinylated at defined percentages (e.g., at 0, 1, 10, 50, and 100%). For each of the 20 proteolytic BSA peptides, the highest ranked, differentiating MS2 fragment ion was used to calculate lysine succinylation (Ksucc) occupancy, as L/(L+H) in %.

Watch this Scientific Journal Video about Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry at JoVE.com

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

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

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Amines

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11.5K Views.  Buck Institute for Research on Aging. Hier presenteren wij onbevooroordeelde kwantificering van site-specific eiwit acetylation en/of succinylation bezetting (stoichiometrie) van een volledige Proteoom door middel van een analyse van de ratiometric van endogene aangebrachte wijzigingen na kwantitatieve chemische acylering met behulp van stabiele isotoop-geëtiketteerden anhydriden. In combinatie met de Spectrometrie van de massa van de gevoelige gegevens-onafhankelijke acquisitie, worden nauwkeurige site beze...

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

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

Chemistry

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

Biochemistry

Quantitative Analysis of Chromatin Proteomes in Disease

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due to the high percentage of binucleated cells,1 the predominantly heterochromatic state of the DNA, and the non-dividing nature of the cardiomyocyte which renders adult nuclei in a permanent state of interphase.2 Transcriptional regulation during development and disease have been well studied in this organ,3-5 but what remains relatively unexplored is the role played by the nuclear proteins responsible for DNA packaging and expression, and how these proteins control changes in transcriptional programs that occur during disease.6 In the developed world, heart disease is the number one cause of mortality for both men and women.7 Insight on how nuclear proteins cooperate to regulate the progression of this disease is critical for advancing the current treatment options.
Mass spectrometry is the ideal tool for addressing these questions as it allows for an unbiased annotation of the nuclear proteome and relative quantification for how the abundance of these proteins changes with disease. While there have been several proteomic studies for mammalian nuclear protein complexes,8-13 until recently14 there has been only one study examining the cardiac nuclear proteome, and it considered the entire nucleus, rather than exploring the proteome at the level of nuclear sub compartments.15 In large part, this shortage of work is due to the difficulty of isolating cardiac nuclei. Cardiac nuclei occur within a rigid and dense actin-myosin apparatus to which they are connected via multiple extensions from the endoplasmic reticulum, to the extent that myocyte contraction alters their overall shape.16 Additionally, cardiomyocytes are 40% mitochondria by volume17 which necessitates enrichment of the nucleus apart from the other organelles. Here we describe a protocol for cardiac nuclear enrichment and further fractionation into biologically-relevant compartments. Furthermore, we detail methods for label-free quantitative mass spectrometric dissection of these fractions-techniques amenable to in vivo experimentation in various animal models and organ systems where metabolic labeling is not feasible.

Advances in mass spectrometry have allowed the high throughput analysis of protein expression and modification in a host of tissues. Combined with subcellular fractionation and disease models, quantitative mass spectrometry and bioinformatics can reveal new properties in biological systems. The method described herein analyzes chromatin-associated proteins in the setting of heart disease and is readily applicable to other in vivo models of human disease.

In the nucleus reside the proteomes whose functions are most intimately linked with gene regulation. Adult mammalian cardiomyocyte nuclei are unique due ...

Medicine

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Detection of Protein S-Acylation using Acyl-Resin Assisted Capture

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty acids via a labile thioester bond. S-acylation, which is emerging as a widespread regulatory mechanism, can modulate almost all aspects of the biological activity of proteins, from complex formation to protein trafficking and protein stability. The recent progress in understanding of the biological function of protein S-acylation was achieved largely due to the development of novel biochemical tools allowing robust and sensitive detection of protein S-acylation in a variety of biological samples. Here, we describe acyl resin-assisted capture (Acyl-RAC), a recently developed method based on selective capture of endogenously S-acylated proteins by thiol-reactive Sepharose beads. Compared to existing approaches, Acyl-RAC requires fewer steps and can yield more reliable results when coupled with mass spectrometry for identification of novel S-acylation targets. A major limitation in this technique is the lack of ability to discriminate between fatty acid species attached to cysteines via the same thioester bond.

Acyl-RAC (Acyl-Resin Assisted Capture) is a highly sensitive, reliable and easy to perform method to detect reversible lipid modification of cysteine residues (S-acylation) in a variety of biological samples.

Protein S-acylation, also referred to as S-palmitoylation, is a reversible post-translational modification of cysteine residues with long-chain fatty ...

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli

Genetic code expansion serves as a powerful tool to study a wide range of biological processes, including protein acetylation. Here we demonstrate a facile protocol to exploit this technique for generating homogeneously acetylated proteins at specific sites in Escherichia coli cells.

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

Immunology and Infection

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small molecule studies and drug development 4. Many epigenetic regulators have only recently been discovered and are still in the process of being classified. Among these enzymes are lysine demethylases which remove methyl groups from lysines on histones and other proteins. Due to the novel nature of this class of enzymes, few assays have been developed to study their activity. This has been a road block to both the classification and high throughput study of histone demethylases. Currently, very few demethylase assays exist. Those that do exist tend to be qualitative in nature and cannot simultaneously discern between the different lysine methylation states (un-, mono-, di- and tri-). Mass spectrometry is commonly used to determine demethylase activity but current mass spectrometric assays do not address whether differentially methylated peptides ionize differently. Differential ionization of methylated peptides makes comparing methylation states difficult and certainly not quantitative (Figure 1A). Thus available assays are not optimized for the comprehensive analysis of demethylase activity.
Here we describe a method called MassSQUIRM (mass spectrometric quantitation using isotopic reductive methylation) that is based on reductive methylation of amine groups with deuterated formaldehyde to force all lysines to be di-methylated, thus making them essentially the same chemical species and therefore ionize the same (Figure 1B). The only chemical difference following the reductive methylation is hydrogen and deuterium, which does not affect MALDI ionization efficiencies. The MassSQUIRM assay is specific for demethylase reaction products with un-, mono- or di-methylated lysines. The assay is also applicable to lysine methyltransferases giving the same reaction products. Here, we use a combination of reductive methylation chemistry and MALDI mass spectrometry to measure the activity of LSD1, a lysine demethylase capable of removing di- and mono-methyl groups, on a synthetic peptide substrate 5. This assay is simple and easily amenable to any lab with access to a MALDI mass spectrometer in lab or through a proteomics facility. The assay has ~8-fold dynamic range and is readily scalable to plate format 5.

We present a method for using MALDI mass spectrometry and reductive methylation chemistry to quantify changes in lysine methylation.

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...

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

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