We acknowledge the support from the NIH shared instrumentation grant for the TripleTOF system at the Buck Institute (1S10 OD016281). This work was also supported by the National Institute of Allergy and Infectious Disease (R01 AI108255 to B.S.) and the National Institute of Diabetes and Digestive and Kidney Diseases (R24 DK085610 to Eric Verdin; R01 DK090242 to Eric Goetzman). X.X. was supported by a grant from the National Institutes of Health (NIH grant T32GM8806, to Judith Campisi and Lisa Ellerby), N.B. was supported by a postdoctoral fellowship from the Glenn Foundation for Medical Research.

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P., Lamoliatte, F., Thibault, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cross+talk+between+SUMOylation+and+ubiquitylation+using+a+sequential+peptide+immunopurification+approach.">Identification of cross talk between SUMOylation and ubiquitylation using a sequential peptide immunopurification approach.</a> Nature Protocols. 12 (11), 2342-2358 (2017).</li><li>Venne, A. S., Kollipara, L., Zahedi, R. 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J., 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=Label-free+quantitative+proteomics+of+the+lysine+acetylome+in+mitochondria+identifies+substrates+of+SIRT3+in+metabolic+pathways.">Label-free quantitative proteomics of the lysine acetylome in mitochondria identifies substrates of SIRT3 in metabolic pathways.</a> Proceedings of the National Academy of Science U. S. A. 110 (16), 6601-6606 (2013).</li><li>Rardin, M. J., 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=SIRT5+regulates+the+mitochondrial+lysine+succinylome+and+metabolic+networks.">SIRT5 regulates the mitochondrial lysine succinylome and metabolic networks.</a> Cell Metabolism. 18 (6), 920-933 (2013).</li><li>Carrico, C., Meyer, J. G., He, W., Gibson, B. 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The authors have nothing to disclose.

This protocol describes a novel technique for simultaneous multiple PTM enrichment to more effectively understand PTM crosstalk. Alternative methods of reaching this objective tend to be prohibitively time-consuming and expensive, and they require large amounts of protein to be successful11,13. This protocol presents a multi-PTM enrichment workflow that involves incubation in antibody-conjugated beads for two PTMs at once to improve the overall efficiency of the experiment. This method also involves the use of DDA for spectral library generation and DIA MS acquisitions to detect and quantify the peptides present with reduced interference from fragment ions22,23. Software programs, such as MS database search engines are used to analyze and quantify data from DDA acquisitions, whereas Quantitative Proteomics Software24 and specific DIA Quantitative Analysis Software25 are necessary to interpret the complex spectra produced by DIA acquisitions.
There are several critical steps within this protocol that should be followed carefully. As the main goal of the protocol is to enrich for multiple PTMs simultaneously, the antibody-affinity enrichment step (section 2) is critical to success of the experiment. When performing washes on the beads, it is necessary to ensure none of the beads are aspirated accidentally. Ensuring the urea concentration has been diluted to 1 M prior to digestion with trypsin (step 1.8) is also necessary. Although 8 M urea is required earlier in the protocol for protein solubilization, urea concentrations above 1 M will inhibit trypsin&#39;s enzymatic activity. In addition, it is important to consistently check the pH of the sample throughout the protocol. This is especially important prior to digestion. If the pH of the sample and the trypsin solution are not neutralized appropriately prior to the incubation, it can result in an inefficient digestion wherein many cleavage sites may be missed, resulting in fewer peptide identifications.
A few modifications to the protocol may be helpful when preparing samples. For a protein lysate procured from 1 mg of starting material, a quarter of the antibody beads provided in each PTM scan tube can be used as a cost-effective alternative. A greater amount of starting material can be used for better results, as long as the amount of antibody beads used are increased proportionately. Another modification that can enhance the workflow is to digest samples with another protease in addition to trypsin. This modification would result in more variability in the peptides cleaved, providing increased coverage of protein residues. Although not necessary, it is recommended that trypsin be one of the enzymes used for PTM analysis due to its high cleavage specificity26.
A limitation of this protocol is that the PTMs being studied need to have similar chemistries in order to be enriched simultaneously14. The procedures for the different antibody-conjugated beads must be similar, utilizing similar solvents and solutions, including similar elution conditions, and preferably from the same vendor. For this reason, the method outlined here consistently uses acetylation and succinylation as an example, both of which use antibody-conjugated beads (Cell Signaling Technology, Inc). Although this method can theoretically be applied to any number of PTMs, additional studies would be needed to assess the exact limitation of the protocol in this respect. Furthermore, as this is an antibody-based enrichment method, the method can only provide a relative quantification of PTM sites.
In comparison with existing multi-PTM enrichment methods, this workflow is a more feasible and cost-effective alternative. From this experiment, it was observed that the efficacy of this method compares extremely well with alternative methods, such as individual or serial enrichments. Figure 2B shows that the median CV for modified peptide peak areas was actually decreased in the one-pot method in comparison to single-PTM enrichments and serial-PTM enrichments13. We further analyzed the experimental results assessing site-level quantifications for acetylation or succinylation. Additionally, Spearman correlation analysis (Figure 2C,D) demonstrated that the one-pot PTM enrichment performed similarly to the single-PTM enrichment workflows. This was also true for the peptide- and fragment-level correlations. The same observation held for all three correlations when comparing one-pot with serial-PTM enrichment.
This protocol allows researchers to make fascinating biological insights into PTM crosstalk in a quick and cost-effective manner. The DIA component of the workflow allows researchers to understand more about PTMs, as it provides information about site localization and overcomes challenges such as low site occupancy of PTMs. Precursor ions tend to be excluded with DDA, which is particularly important when studying PTMs, as site occupancy is often low to the point where these peptides do not get selected for MS/MS but still contain crucial information. Follow-up experiments could be performed to assess the upper limit of how many PTMs can be enriched simultaneously using this method. A future improvement of this workflow may include development of more advanced software platforms to further automate the analysis of site localization and PTM site occupancy.

A myriad of post-translational modifications dynamically regulate proteins and pathways through effects on the activity1, signaling2, and turnover3,4. For example, protein kinases are activated or deactivated by the addition of phosphate groups5, and histone acetylation and other modifications provide a mechanism to change the chromatin structure and serve as transcriptional regulatory mechanisms6,7. In recent years, evidence has mounted that multiple PTMs work in concert or compete to regulate protein function or activity8,9,10,11. Therefore, understanding PTM crosstalk is an emerging need in PTM research. However, most available proteomic workflows to identify and quantify PTM sites focus on single modifications, rather than the interplay of multiple modifications. The described workflow correlates specific protein modification &#34;hot-spots&#34; and lysine residues that are modified by multiple different PTMs.
There is a growing need in the scientific community for feasible methods to study multiple PTMs simultaneously12. Most methods to globally identify and quantify the sites of multiple types of PTMs are challenging due to the high costs and amount of tissue required12,13. Not only are multi-PTM enrichment experiments time-consuming in terms of sample preparation, data acquisition, and data analysis, but these studies typically require large and often prohibitive amounts of protein11. Described here is a protocol for simultaneous enrichment and analysis of multiple PTMs, which also addresses several of these barriers and enables large-scale PTM profiling and assessing crosstalk between various PTMs14. This one-pot workflow outlines a practical way for biomedical researchers to globally profile multiple PTMs, identify co-modified peptides, and study PTM crosstalk in an efficient and cost-effective way14,15.
Here, this method is showcased by examining mitochondrial protein acylation, which was first studied over 50 years ago16. It specifically concentrates on lysine acetylation17 and succinylation18, including the co-occurrence of these modifications on proteins and even co-modification at the peptide level. Since the study uses a sirtuin 5 (SIRT5) knockout mouse model, it was chosen to focus on enrichment of acetylation and succinylation sites. This decision was made because succinylation sites are targets of SIRT5 desuccinylase and are thus expected to show significant upregulation in KO mice, making them the most relevant PTMs in this case. Both PTMs are biologically relevant as recently summarized by Carrico et al.19. In general, acetylation shows important effects on gene expression and metabolism, and succinylation has been reported to regulate heart metabolism and function20.
The described protocol can be performed with a low amount of protein input material (e.g., 1 mg of protein lysate) and reduces the total duration of the experiment by reducing the time spent for sample processing, MS acquisition, and data analysis. A workflow schematic is provided in Figure 1. We have also used even lower amounts of starting material (down to 100 &#181;g of protein lysate, scaling down the amounts of beads used accordingly), which as expected, reduces the overall yield of identified acylated peptides; however, it still provides highly valuable results and quantifiable acylated peptides.
While so-called top-down or middle-down workflows typically do not use proteolytic digestion approaches (and thus maintain the connectivity of multiple PTMs within one protein), this protocol focuses on a peptide-based affinity enrichment approach to gain extra depth and sensitivity for PTM identification and quantification (Figure 1). In addition, this peptide-centric workflow utilizes modern mass spectrometry methods, including 1) a combination of data-dependent acquisition (DDA) to generate spectral libraries, and 2) data-independent acquisition (DIA) for accurate PTM quantification in a label-free workflow.
DIA workflows overcome sampling stochasticity of typical DDA scan schemes by comprehensively fragmenting all peptide signals within the sampled m/z range21. This feature is also extremely beneficial in terms of site localization, because it is easier to gain information regarding which specific fragment ions are modified within the peptide. In addition, DIA workflows allow identification and quantification of minor PTM-peptide isoforms with identical precursor ions. DIA methods can also determine a specific PTM site localization within a peptide based on specific corresponding fragment ions that are comprehensively measured at all times. However, DDA approaches often utilize &#34;dynamic exclusion&#34; features that exclude multiple sampling of MS/MS for the same precursor ion, thus missing minor PTM isoforms.
The simultaneous enrichment strategy described here is ideally suited for studies that will benefit from the global profiling and quantification of multiple PTMs, examining PTM crosstalk, and understanding the dynamic interactions of post-translational modifications. Identification of multiple enriched PTMs in one combined workflow have been described by: global, serial or parallel enrichment of PTM containing proteolytic peptides, or alternatively by the analysis of intact proteins. In direct comparison with serial enrichment of acetylation and succinylation, the efficiency of the one-pot methodology was established as being very similar14. These alternative protocols require significant amounts of starting material and time and can be prohibitively expensive. In contrast, the one-pot protocol provides an inexpensive and efficient method for enrichment of more than one PTM with subsequent analysis and identification.
Mouse liver tissues are obtained from SIRT5 knockout mice and are used here as starting material. This protocol can also be performed for protein lysates from different tissues or cell culture experiments. This protocol can be applied to protein lysates obtained from tissues or cell culture pellets.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 M Triethylammonium biocarbonate buffer (TEAB)</td>
			<td>Sigma Aldrich, St. Louis, MO, USA</td>
			<td>T7408</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, Burdick and Jackson LC-MS grade</td>
			<td>Burdick and Jackson, Muskegon, MI, USA</td>
			<td>36XL66</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioruptor sonicator</td>
			<td>Diagenode, Denville, NJ, USA</td>
			<td>B01020001</td>
			<td></td>
		</tr>
		<tr>
			<td>C18 pre-column chip (200 &#181;m x 6 mm ChromXP C18-CL chip, 3 um, 120 A)</td>
			<td>SCIEX, Framingham, MA, USA</td>
			<td>5015841</td>
			<td></td>
		</tr>
		<tr>
			<td>C18-CL chip (75 &#181;m x 15 cm ChromXP, 3 &#181;m, 300 &#197;)</td>
			<td>SCIEX, Framingham, MA, USA</td>
			<td>804-00001</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>Eppendorf Thermomixer Compact</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>T1317-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tube (2.0 mL Safelock)</td>
			<td>Eppendorf AG, Hamburg, Germany</td>
			<td>22363352</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma Aldrich, St. Louis, MO, USA</td>
			<td>F0507-500ML</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>Iodoacetamide (IAA)</td>
			<td>Sigma Aldrich, St. Louis, MO, USA</td>
			<td>I1149-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>mapDIA</td>
			<td></td>
			<td>web link</td>
			<td>software for interference removal of DIA datasets</td>
		</tr>
		<tr>
			<td>Methanol, Burdick and Jackson LC-MS grade</td>
			<td>Burdick and Jackson, Muskegon, MI, USA</td>
			<td>BJLC230-4</td>
			<td></td>
		</tr>
		<tr>
			<td>PURELAB flex 1 ultrapure water dispenser</td>
			<td>VWR International, Radnor, PA, USA</td>
			<td>89204-088</td>
			<td></td>
		</tr>
		<tr>
			<td>mProphet in Skyline</td>
			<td></td>
			<td>incorporated in Skyline</td>
			<td>integrated statistical algorithms for FDR assessments</td>
		</tr>
		<tr>
			<td>Oasis HLB SPE cartridges</td>
			<td>Waters Corp., Milford, MA, USA</td>
			<td>WAT094225</td>
			<td>cartridges for desalting protein lysates, up to 50 mg material</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline solution</td>
			<td>Life Technologies</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Assay</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>ProteinPilot 5.0 - &#39;MS database search engine&#39;</td>
			<td>SCIEX, Framingham, MA, USA</td>
			<td>software download SCIEX</td>
			<td>MS database search engine</td>
		</tr>
		<tr>
			<td>PTMScan Succinyl-Lysine Motif [Succ-K] Kit #13764</td>
			<td>Cell Signaling Technology</td>
			<td>13764</td>
			<td>antibody beads for affinity enrichment</td>
		</tr>
		<tr>
			<td>PTMScan Acetyl-Lysine Motif [Ac-K] Kit #13416</td>
			<td>Cell Signaling Technology</td>
			<td>13416</td>
			<td>antibody beads for affinity enrichment</td>
		</tr>
		<tr>
			<td>Sequencing-grade lyophilized trypsin</td>
			<td>Life Technologies</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Skyline - &#39;Quantitative Proteomics Software&#39;</td>
			<td>MacCoss lab (academic)</td>
			<td>open source software</td>
			<td>Quantitative Proteomics Software (academic)</td>
		</tr>
		<tr>
			<td>Spectronaut - &#39;DIA Quantitative Analysis Software&#39;</td>
			<td>Biognosys AG, Schlieren, Zurich, Switzerland</td>
			<td>Sw-3001</td>
			<td>DIA Quantitative Analysis Software / PTM site localization</td>
		</tr>
		<tr>
			<td>Thermo Scientific Savant SPD131DDA Speedvac Concentrator</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>SPD131DDA-115</td>
			<td>instrument to concentrate liquid volume of samples</td>
		</tr>
		<tr>
			<td>TissueLyser II</td>
			<td>Qiagen, Hilden, Germany</td>
			<td>85300</td>
			<td>instrument for efficient lysis of tissue</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid (TFA)</td>
			<td>Sigma Aldrich, St. Louis, MO, USA</td>
			<td>T6508-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>TripleTOF 6600: orthoganol quadrupole time-of-flight (QqTOF)mass spectrometer</td>
			<td>SCIEX, Framingham, MA, USA</td>
			<td>Per quote</td>
			<td>high resolution mass spectrometer</td>
		</tr>
		<tr>
			<td>Ultra Plus nano-LC 2D HPLC system</td>
			<td>SCIEX, Eksigent Division, Framingham, MA, USA</td>
			<td>Model #845</td>
			<td>chromatographic separation system</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Thermo Fisher Scientific, Waltham, MA, USA</td>
			<td>PI29700</td>
			<td></td>
		</tr>
		<tr>
			<td>Water, Burdick and Jackson LC-MS</td>
			<td>Burdick and Jackson, Muskegon, MI, USA</td>
			<td>600-30-76</td>
			<td></td>
		</tr>
		<tr>
			<td>ZipTip C18 Pipette Tips, P10</td>
			<td>Merck Millipore Ltd, Tullagreen, Carrigtwohill, Co. Cork, IRL</td>
			<td>ZTC18S096</td>
			<td>C-18 resin loaded tips for desalting of peptide mixtures</td>
		</tr>
	</tbody>
</table>

simultaneous affinity enrichment of two post-translational modifications for quantification and site localization

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into protein function. Despite the importance of multi-PTM enrichment studies, few studies investigate more than one PTM at a time, due partially to the expenses, time, and large protein quantities required to perform multiple global proteomic analysis of PTMs. The &#34;one-pot&#34; affinity enrichment detailed in this protocol overcomes these barriers by permitting the simultaneous identification and quantification of peptides with lysine residues containing acetylation and succinylation PTMs with low amounts of sample input. The protocol involves preparation of protein lysate from mouse livers of SIRT5 knockout mice, performance of trypsin digestion, enrichment for PTMs, and performance of mass spectrometric analysis using a data-independent acquisition (DIA) workflow. Because this workflow allows for the enrichment of two PTMs from the same sample simultaneously, it provides a practical tool to study PTM crosstalk without requiring large amounts of samples, and it greatly reduces the time required for sample preparation, data acquisition, and analysis. The DIA component of the workflow provides comprehensive PTM-specific information. This is particularly important when studying PTM site localization, as DIA provides comprehensive sets of fragment ions that can be computationally deciphered to differentiate between different PTM localization isoforms.

Figure 1 shows a general diagram of the workflow, including harvesting the tissue from mouse livers, using 1 mg of protein for digesting the protein lysate with trypsin, incubating peptides with antibody-conjugated beads, acquiring the samples on the MS, and finally performing DIA/SWATH analysis of the data using various quantitative proteomics software packages (academic and commercial).
Figure 2A shows how the timeline of the workflow and the amounts of sample and protein required, compared to alternative methods currently being used for multi-PTM enrichment studies. The one-pot method can be performed in half as much time and with half the number of samples as these alternative methods. Compared to the two single-PTM enrichment method, the one-pot protocol also requires half the amount of protein.
This protocol has been shown to be a feasible and cost-effective alternative. Figure 2B shows that the median coefficient of variation (CV) for modified peptide areas was lower in the one-pot method than in the single-PTM and serial-PTM enrichments. Figure 2C,D shows that, while comparing the one-pot PTM and single-PTM enrichment methods, no noteworthy differences were apparent between the correlations of site-level quantifications for the two modifications. This was also true for the peptide-level and fragment-level correlations. The same observation held for all three correlations when comparing the one-pot and serial-PTM enrichments. All underlying MS raw data and processed Excel results sheets associated with a recent report by Basisty et al.14 are available and can be downloaded from MassIVE (MSV00081906) and ProteomeXchange (PXD008640).
In general, while antibody enrichment strategies may show certain limitations, such as potential epitope occlusion or limited specificity, the antibodies used in this study are mixtures of independently generated clones and thus provide wider ranges of specificities.
Experimental results document the possibility of detecting and assessing PTM crosstalk. Figure 3 displays data from a successful enrichment and illustrates an example for a peptide containing multiple and different acyl modifications visualizing PTM crosstalk. Figure 3A shows a peptide that is acetylated on one lysine residue and succinylated on the other, and Figure 3B shows the same peptide succinylated at both lysines. This demonstrates that the same lysine residue can be modified with both acylation groups, and there is a possibility of crosstalk occurring at that site. Figure 4 displays the number of lysine residues that were identified as described in sections 7.1-7.3 to carry both modifications, also pointing towards possible PTM crosstalk.
As Figure 5 demonstrates, processing DIA PTM datasets with quantitative proteomics software allows us to pinpoint which specific lysine residues are modified. This is a concept known as site localization, which is an essential step for any analysis for determination of possible PTM crosstalk. Figure 5 displays two potential isoforms along with the confirming and refuting ions for each that could be visualized and assessed as described in sections 8.1-8.3 (specifically steps 8.3.2 and 8.3.3). Based on this information, we were able to confidently identify which of the two isoforms was present in the original sample. The MS/MS spectrum of the confirmed isoform KQYGEAFEKacR demonstrates clearly that the y ions (y2-y5) containing the acetylated lysine residue, which shifted by 42 m/z (the increment mass of an acetyl group), confirmed the specific lysine residue in the peptide that was modified.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60780/60780fig1.jpg" /> 
Figure 1: Typical workflow for one-pot enrichment of PTMs. Tissue (here, livers) are harvested from SIRT5 KO and wild-type (WT) mice, and proteins are lysed, trypsin-digested into peptides, and desalted. Peptides are then enriched by immunoaffinity with combinations of succinyl- and acetyl-antibody beads. Parallel MS workflows measure both 1) small aliquots of whole lysate protein expression changes (for protein normalization) and 2) enriched acyl-containing peptides for acylation site identification (DDA-MS) and site localization, followed by quantification (DIA-MS). <a href="https://www.jove.com/files/ftp_upload/60780/60780fig1large.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/60780/60780fig2.jpg" /> 
Figure 2: Comparison of one-pot workflow with alternative methods. (A) Comparison of time, costs, and materials required for the one-pot workflow, serial-PTM enrichment, and two single-PTM enrichments. (B) Comparison of CVs between the one-pot workflow, single acetyl-lysine PTM enrichment, and single succinyl-lysine PTM enrichment. Spearman correlation analysis comparing the acyl peptide peak areas obtained from the one-pot workflow, and the single-PTM enrichments: corresponding plots of the log2 peak area results for (C) acetylation sites and (D) succinylation sites. Regression slopes and correlation factors are indicated in the individual panels14. Two independent biological replicates were processed for each of the conditions. This figure has been modified from Basisty et al.14. <a href="https://www.jove.com/files/ftp_upload/60780/60780fig2alarge.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/60780/60780fig3.jpg" /> 
Figure 3: Crosstalk between acetylation and succinylation modifications of lysine residues. MS/MS spectra of tryptic peptides from mitochondrial 3-ketoacyl-CoA thiolase that show the same amino acid sequence but have been modified at two lysine residues with different PTMs. (A) MS/MS of peptide AANEAGYFNEEMAPIEVKsuccTKacK and (B) MS/MS of peptide AANEAGYFNEEMAPIEVKsuccTKsuccK. This figure has been modified from Basisty et al.14. <a href="https://www.jove.com/files/ftp_upload/60780/60780fig3large.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/60780/60780fig4a.jpg" /> 
Figure 4: Overlap and crosstalk between the acetylated and succinylated lysine residues&#8211;specific examples in protein complexes. (A) Venn diagram displaying overlap between 2,235 acetylation and 2,173 succinylation sites. Of these, 943 sites were both acetylated and succinylated. Liver from a SIRT5 (de-succinylase) knockout mouse was analyzed, and many succinylation sites were identified. In fact, they were more abundant than normally observed in mouse liver (modified peptides were filtered at a Q value of &#60;0.05). (B) Protein complexes showing the percentage of their subunits containing both acetylated and succinylated sites (bold red line represents the significance as determined by Fisher&#39;s exact test). (C) Diagram of ATP synthase complex: protein subunits in red depict the subunits that contain both acetylated and succinylated sites. This figure has been modified from Basisty et al.14. <a href="https://www.jove.com/files/ftp_upload/60780/60780fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60780/60780fig5.jpg" /> 
Figure 5: Quantitative proteomics software deciphers the peptide site localization of PTMs. Based on MS/MS fragmentation of the peptide, it is possible to provide information about the specific lysine residue the acetyl group is modifying. This showcases the ability of the software to offer valuable information on site localization of PTMs. (A) Two possibilities of lysine residue modification and PTM site localization: KQYGEAFEKacR (left) and KacQYGEAFEKR (right). &#34;Confirming&#34; and &#34;refuting&#34; fragment ions are shown for each of the potential site localization isoforms of the peptide. Based on this information, confirming scores and refuting scores are assigned, confirming the presence of isoform KQYGEAFEKacR in the sample. (B) MS/MS spectrum corresponding to the confirmed isoform KQYGEAFEKacR indicating that all y ions including the acetylated lysine residue (y2 and higher) carry an increment mass of +42 m/z, which corresponds to the acetyl modification. Observed b ions do not contain the modification. (C) Extracted ion chromatogram (XIC) with abundant peak areas resulting from y2 and y3 ions, both of which make up the acetylation site in the confirmed isoform KQYGEAFEKacR. <a href="https://www.jove.com/files/ftp_upload/60780/60780fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization at JoVE.com

Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Studying multiple post-translational modifications (PTMs) of proteins is a crucial step to understand PTM crosstalk and gain more holistic insights into ...

simultaneous-affinity-enrichment-two-post-translational-modifications

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JoVE Journal

Biology

6.7K Views.  Buck Institute for Research on Aging. This workflow describes the performance of time- and cost-efficient enrichment of multiple protein post-translational modifications (PTMs) simultaneously for quantitative global proteomic analysis. The protocol utilizes peptide-level PTM enrichment with multiple conjugated antibodies, followed by data-independent acquisition mass spectrometry analysis to gain biological insights into PTM crosstalk.

Video: Simultaneous Affinity Enrichment of Two Post-Translational Modifications for Quantification and Site Localization

Principles of Site-Specific Recombinase (SSR) Technology

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular biology. Site-specific recombinases are proteins that bind to distinct DNA target sequences. The Cre/lox system was first described in bacteriophages during the 1980's. Cre recombinase is a Type I topoisomerase that catalyzes site-specific recombination of DNA between two loxP (locus of X-over P1) sites. The Cre/lox system does not require any cofactors. LoxP sequences contain distinct binding sites for Cre recombinases that surround a directional core sequence where recombination and rearrangement takes place. When cells contain loxP sites and express the Cre recombinase, a recombination event occurs. Double-stranded DNA is cut at both loxP sites by the Cre recombinase, rearranged, and ligated ("scissors and glue"). Products of the recombination event depend on the relative orientation of the asymmetric sequences.
SSR technology is frequently used as a tool to explore gene function. Here the gene of interest is flanked with Cre target sites loxP ("floxed"). Animals are then crossed with animals expressing the Cre recombinase under the control of a tissue-specific promoter. In tissues that express the Cre recombinase it binds to target sequences and excises the floxed gene. Controlled gene deletion allows the investigation of gene function in specific tissues and at distinct time points. Analysis of gene function employing SSR technology --- conditional mutagenesis -- has significant advantages over traditional knock-outs where gene deletion is frequently lethal.

Principles of Site-Specific Recombinase SSR Technology

The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and has proven itself as a valuable tool for assessing gene function in transgenic animals. This interview discusses the mechanism of site specific recombination by Cyclization recombinase (Cre) and how the use of this enzyme has led to the development of conditional mutagenesis, which has significant advantages over traditional knock out strategies.

Site-specific recombinase (SSR) technology allows the manipulation of gene structure to explore gene function and has become an integral tool of molecular ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

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

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.

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 ...

Worm-align and Worm_CP, Two Open-Source Pipelines for Straightening and Quantification of Fluorescence Image Data Obtained from Caenorhabditis elegans

An issue often encountered when acquiring image data from fixed or anesthetized C. elegans is that worms cross and cluster with their neighbors. This problem is aggravated with increasing density of worms and creates challenges for imaging and quantification. We developed a FIJI-based workflow, Worm-align, that can be used to generate single- or multi-channel montages of user-selected, straightened and aligned worms from raw image data of C. elegans. Worm-align is a simple and user-friendly workflow that does not require prior training of either the user or the analysis algorithm. Montages generated with Worm-align can aid the visual inspection of worms, their classification and representation. In addition, the output of Worm-align can be used for subsequent quantification of fluorescence intensity in single worms, either in FIJI directly, or in other image analysis software platforms. We demonstrate this by importing the Worm-align output into Worm_CP, a pipeline that uses the open-source CellProfiler software. CellProfiler&#8217;s flexibility enables the incorporation of additional modules for high-content screening.&#160;As a practical example, we have used the pipeline on two datasets: the first dataset are images of heat shock reporter worms that express green fluorescent protein (GFP) under the control of the promoter of a heat shock inducible gene hsp-70, and the second dataset are images obtained from fixed worms, stained for fat-stores with a fluorescent dye.

Worm-align and Worm_CP, Two Open-Source Pipelines for Straightening and Quantification of Fluorescence Image Data Obtained from Caenorhabditis elegans

Worm-align/Worm_CP is a simple FIJI/CellProfiler workflow that can be used to straighten and align Caenorhabditis elegans samples and to score whole-worm image-based assays without the need for prior training steps. We have applied Worm-align/Worm_CP to the quantification of&#160;heat-shock induced expression in live animals or lipid droplets&#160;in fixed samples.

An issue often encountered when acquiring image data from fixed or anesthetized C. elegans is that worms cross and cluster with their neighbors. This ...