This work was supported by the following grants from the National Institutes of Health R35GM136255, R01GM086688, R01HL144778, R01GM097112, and S10RR025107.
DATA AVAILABILITY
The datasets generated during this study are available at XLinkDB - http://xlinkdb.gs.washington.edu/xlinkdb/HeLa_BDP_JoVE_2020_Bruce.php. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE&#160;42&#160;partner repository with the dataset identifier PXD023560.

NOTE: All materials, equipment, and software used here are described in Table of Materials.
1. Synthesis of BDP (Figure 3)
NOTE: The BDP-NHP cross-linker was synthetized using a CEM Liberty Lite peptide synthesizer, following the manufacturer&#39;s instructions. This protocol is based on a previous publication7.
<ol>
	<li>Prepare the following reagents.
	<ol>
		<li>Weigh out 0.63 g of Rink Amide resin (100-200 ...

<ol>
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J., Rappsilber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-linking+mass+spectrometry:+methods+and+applications+in+structural,+molecular+and+systems+biology.">Cross-linking mass spectrometry: methods and applications in structural, molecular and systems biology.</a> Nature Structural &amp; Molecular Biology. 25 (11), 1000-1008 (2018).</li><li>Tang, X., Munske, G. R., Siems, W. F., Bruce, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+Spectrometry+Identifiable+Cross-Linking+Strategy+for+Studying+Protein&#8722;Protein+Interactions.">Mass Spectrometry Identifiable Cross-Linking Strategy for Studying Protein&#8722;Protein Interactions.</a> Analytical Chemistry. 77 (1), 311-318 (2005).</li><li>Chavez, J. D., 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=Systems+structural+biology+measurements+by+in+vivo+cross-linking+with+mass+spectrometry.">Systems structural biology measurements by in vivo cross-linking with mass spectrometry.</a> Nature Protocols. 14 (8), 2318-2343 (2019).</li><li>Schweppe, D. K., 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=Mitochondrial+protein+interactome+elucidated+by+chemical+cross-linking+mass+spectrometry.">Mitochondrial protein interactome elucidated by chemical cross-linking mass spectrometry.</a> Proceedings of the National Academy of Sciences. 114 (7), 1732-1737 (2017).</li><li>Weisbrod, C. R., Chavez, J. D., Eng, J. K., Yang, L., Zheng, C., Bruce, J. 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XL-MS can provide information on protein structure and conformation (intra-links), protein-protein interaction and complex assembly (inter-links), and protein quantitation and solvent exposure (dead-ends). One limitation of the technique when used in vivo for complex samples is the low-resolution structural results on protein conformation and interactome. Thus, protein structures present in the PDB are useful for the modelling of structures and for the visualization of cross-links, which is automatically done by...

Biological processes are driven by multiple and complex mechanisms, with different molecules - nucleic acids, proteins, carbohydrates, lipids, etc. - playing key roles in each step. When studying proteins, several approaches can be used, but the ultimate objective is to understand how proteins with different domains and regulatory regions are structurally organized and functioning in a crowded cellular environment1,2. Besides structural information, assessing protein interactors and complexes is essential for the understanding of cellular mechanisms.
Chemical cross-linking of proteins with mass spectrometry (XL-MS), associated with other structural methods (e.g., X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy), has become a robust approach for protein structure and interactomics studies3,4,5. This technique is based on the reactivity of cross-linker groups to specific side chains of proteins, usually primary amines. The distance between these reactive groups (the length of the spacer) provides constraint information useful for modeling protein structures and protein complex topologies5.
Here, we describe a workflow for in-house synthesis and in vivo application of a protein interaction reporter (PIR) cross-linker6, the biotin aspartate proline-N-hydroxyphthalamide (BDP-NHP)7 (Figure 1). This cross-linker presents optimized features for interactome studies in vivo, such as membrane permeability, that enables the cross-linking of living cells, tissues, and isolated organelles (e.g., nuclei, vesicles, and mitochondria8), providing information on protein structure and complex assembly as close as possible to how it occurs in native and physiological conditions. Other features include a biotin tag for enrichment of cross-linked peptides - commonly found with low abundancy in complex samples, and MS-cleavable bonds, which ultimately facilitate the identification of cross-linked peptides6. This is achieved by relying on a predictable mass relationship between the released reporter ion and the cross-linked peptides after the collision induced dissociation (CID)-induced cleavage of chemical bonds. Detection of this mass relationship can be done (i) &#34;on-the-fly&#34; during the MS analysis, with Real-time Analysis for Cross-linked peptide Technology (ReACT)9, or (ii) post-acquisition using Mango10 for the in silico check of the mass relationship.
A quantitative XL-MS approach for interactomics studies can provide information on changes in protein conformation and complex composition when comparing different biological conditions. Our group has developed different cross-linkers and pipelines for quantitative XL-MS, including by MS1-based label-free quantitation11, targeted parallel reaction monitoring12, the stable isotope labeling of amino acids in cell culture (SILAC)-based quantitation of PIR cross-linked peptides11,12, and an isotope-labeled PIR cross-linker technology13. These quantitative XL-MS methods have been utilized to provide information on interactome changes in drug resistant cancer cells14, study protein conformational and interaction changes induced by mitotic inhibitors15 and heat shock protein 90 (Hsp90)16, and investigate the effects of phosphomimetic mutations and nucleotide binding on the interaction between Hsp90 and its cochaperone Aha117.
The in vivo cross-linking pipeline described here was performed with HeLa cells as a biological model (Figure 2), but we note that this protocol is applicable to other living systems7. To help interpret and visualize XL-MS results, we used XLinkDB18,19,20, a cross-linking database with algorithms to automatically generate structural models and facilitate molecular docking of cross-linked proteins. Recent updates in the platform include tools for visualization of different protein topologies and protein networks20.
The cross-linking results shown here provide insight into the HeLa cell interactome, including protein-protein interactions (inter-links) and protein conformations (intra-links), which provides information on a structural level. Moreover, monolinks - or dead-ends - are generated when one end of the cross-linker is covalently bound to a peptide and the other end is hydrolyzed21. Dead-ends can be informative on relative protein expression levels and protein structure as it is reflected in residue solvent accessibility, that ultimately translates to protein structure22. Here, we discuss ReACT and Mango mass spectrometry methods for a PIR-based cross-linking pipeline and highlight representative cross-linking results involving Hsp90B as a model. We also indicate instrument methods and in silico analysis for the identification of dead-ends when using the PIR technology.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile&#160;</td>
			<td>Fisher Scientific</td>
			<td>A955</td>
			<td>LC&#8211;MS grade</td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>A6141</td>
			<td>NH4HCO3</td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>&#160;Sigma-Aldrich</td>
			<td>221228</td>
			<td>NH4OH, ACS reagent, 28.0&#8211;30.0% NH3 basis</td>
		</tr>
		<tr>
			<td>Calcium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>AC42352</td>
			<td>CaCl2</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>Fisher Scientific</td>
			<td>AC610300010</td>
			<td>DCM, 99,9% (wt/wt)</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>Fisher Scientific</td>
			<td>AC610300010</td>
			<td>DCM; 99.9% (wt/wt)</td>
		</tr>
		<tr>
			<td>Diethyl ether 99.5% (wt/wt)</td>
			<td>Fisher Scientific</td>
			<td>AC364330010</td>
			<td></td>
		</tr>
		<tr>
			<td>Diisopropylcarbodiimide&#160;</td>
			<td>&#160;Sigma-Aldrich</td>
			<td>D125407</td>
			<td>DIC</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>&#160;Sigma-Aldrich</td>
			<td>276855</td>
			<td>DMSO</td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td>Fisher Scientific</td>
			<td>D119</td>
			<td>DMF</td>
		</tr>
		<tr>
			<td>DMEM&#160;</td>
			<td>Fisher Scientific</td>
			<td>11-965-118</td>
			<td>Cell culture medium&#160;</td>
		</tr>
		<tr>
			<td>EDTA 99% (wt/wt)</td>
			<td>Fisher Scientific</td>
			<td>AC118432500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl cyano(hydroxyimino)acetate</td>
			<td>Sigma-Aldrich</td>
			<td>8510860100</td>
			<td>Oxyma</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Atlas Biologicals</td>
			<td>FP-0500-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Asp(OtBu)-OH&#160;</td>
			<td>Millipore Sigma</td>
			<td>8520370005</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Gly-Wang resin</td>
			<td>Bachem</td>
			<td>D-1745</td>
			<td>100&#8211;200 mesh</td>
		</tr>
		<tr>
			<td>Fmoc-L-Lys(biotin)-OH&#160;</td>
			<td>P3 BioSystems</td>
			<td>41084</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Lys(Fmoc)-OH&#160;</td>
			<td>Millipore Sigma</td>
			<td>8520410025</td>
			<td></td>
		</tr>
		<tr>
			<td>Fmoc-Pro-OH&#160;</td>
			<td>Millipore Sigma</td>
			<td>8520170025</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid&#160;</td>
			<td>Fisher Scientific</td>
			<td>A117</td>
			<td>FA, 99.5+% (vol/vol), LC&#8211;MS grade</td>
		</tr>
		<tr>
			<td>HeLa cells&#160;</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td>Epithelial from human cervix</td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I1149</td>
			<td>IAA</td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>&#160;M8266</td>
			<td>MgCl2</td>
		</tr>
		<tr>
			<td>Methanol&#160;</td>
			<td>Fisher Scientific</td>
			<td>A456</td>
			<td>LC&#8211;MS grade</td>
		</tr>
		<tr>
			<td>Monomeric Avidin UltraLink Resin</td>
			<td>Pierce Biotechnology</td>
			<td>53146</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxyphthalimide</td>
			<td>Sigma-Aldrich</td>
			<td>H53704</td>
			<td>NHP</td>
		</tr>
		<tr>
			<td>Nitrogen (g) (99.998%)</td>
			<td>Praxair</td>
			<td>NI 4.8-T</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>SH30256LS</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium&#160;</td>
			<td>Fisher Scientific</td>
			<td>SH30264FS</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin&#8211;streptomycin (10,000 U/mL)</td>
			<td>Fisher Scientific</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Piperidine</td>
			<td>Sigma-Aldrich</td>
			<td>411027</td>
			<td>99.5% (vol/vol)</td>
		</tr>
		<tr>
			<td>Poly-Prep chromatography columns</td>
			<td>Bio-Rad</td>
			<td>7311550</td>
			<td>Disposable polypropylene columns</td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>KCl</td>
		</tr>
		<tr>
			<td>Potassium phosphate, monobasic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyridine</td>
			<td>Millipore Sigma</td>
			<td>MPX20127</td>
			<td>DriSolv, anhydrous septum-sealed bottle</td>
		</tr>
		<tr>
			<td>ReproSil-Pur, 5 micron, 120 &#197;</td>
			<td>Dr. Maisch</td>
			<td>r15.8e</td>
			<td></td>
		</tr>
		<tr>
			<td>Sep-Pak Vac 3cc (500mg) C18 cartridges</td>
			<td>Waters</td>
			<td>186004619</td>
			<td>reversed-phase C18 desalting column</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S 9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate, dibasic&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>AC20651</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate, monobasic (NaH2PO4; Sigma-Aldrich, cat. no. S9638)</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr>
			<td>Succinic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>239690</td>
			<td></td>
		</tr>
		<tr>
			<td>TFA acid&#160;</td>
			<td>Fisher Scientific</td>
			<td>LS121</td>
			<td>LC&#8211;MS grade</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid&#160;</td>
			<td>Fisher Scientific</td>
			<td>A116</td>
			<td>TFA acid, Optima LC&#8211;MS grade</td>
		</tr>
		<tr>
			<td>Trifluoroacetic anhydride</td>
			<td>Sigma-Aldrich</td>
			<td>106232</td>
			<td>TFA anhydride</td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride&#160;</td>
			<td>Fisher Scientific</td>
			<td>20491</td>
			<td>TCEP-HCl</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Promega</td>
			<td>&#160;V5113</td>
			<td>sequencing grade, modified, frozen</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>Sigma-Aldrich</td>
			<td>U5378</td>
			<td></td>
		</tr>
		<tr>
			<td>Water&#160;</td>
			<td>Fisher Scientific</td>
			<td>W6</td>
			<td>LC&#8211;MS grade</td>
		</tr>
		<tr>
			<td>Water with 0.1% (vol/vol) TFA acid</td>
			<td>Fisher Scientific</td>
			<td>LS119</td>
			<td>LC&#8211;MS grade</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5-mL LC autosampler vial</td>
			<td>Thermo Scientific</td>
			<td>MSCERT4000-39TR</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical/preparative HPLC system&#160;</td>
			<td>Agilent Technologies</td>
			<td>G1311C</td>
			<td></td>
		</tr>
		<tr>
			<td>CEM Liberty Lite peptide synthesizer</td>
			<td>CEM</td>
			<td></td>
			<td>Automated Microwave Peptide Synthesizer</td>
		</tr>
		<tr>
			<td>Conical centrifuge tubes&#160;</td>
			<td>Fisher Scientific</td>
			<td>14-959-53A and 12-565-270</td>
			<td>15 and 50 mL</td>
		</tr>
		<tr>
			<td>Extraction manifold</td>
			<td>Waters</td>
			<td>WAT200608</td>
			<td>20 position, 16 &#215; 75-mm tubes</td>
		</tr>
		<tr>
			<td>LC autosampler vial caps</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-622-289</td>
			<td></td>
		</tr>
		<tr>
			<td>LC autosampler vials&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>03-377-299</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td>Eppendorf</td>
			<td>22620444</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>02-681-320</td>
			<td>1.5 mL</td>
		</tr>
		<tr>
			<td>pH paper</td>
			<td>Fisher Scientific&#160;</td>
			<td>13-640-5070</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-Prep chromatography columns&#160;</td>
			<td>Bio-Rad</td>
			<td>7311550</td>
			<td></td>
		</tr>
		<tr>
			<td>Q Exactive Plus</td>
			<td>Thermo Scientific</td>
			<td>IQLAAEGAAPFALGMBDK</td>
			<td>High resolution mass spectrometer&#160;</td>
		</tr>
		<tr>
			<td>SCX column</td>
			<td>Phenomenex</td>
			<td>00G-4398-N0</td>
			<td>Luna column</td>
		</tr>
		<tr>
			<td>Sep-Pak Vac C18 cartridge, 3cc/500mg&#160;</td>
			<td>Waters</td>
			<td>186004619</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker with 15- and 1.5-mL sample blocks</td>
			<td>Eppendorf</td>
			<td>5355</td>
			<td>ThermoMixer R</td>
		</tr>
		<tr>
			<td>Thermal mixer</td>
			<td>Sigma-Aldrich</td>
			<td>T1317</td>
			<td>Eppendorf ThermoMixer compact</td>
		</tr>
		<tr>
			<td>Ultimate 3000</td>
			<td>Thermo Scientific</td>
			<td>ULTIM3000RSLCNANO</td>
			<td>nano-LC system</td>
		</tr>
		<tr>
			<td>Ultrasonic processor</td>
			<td>Cole-Palmer</td>
			<td>EW-04714-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum centrifuge</td>
			<td>SP Scientific</td>
			<td>EZ-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">ReAdW (https://sourceforge.net/projects/sashimi/files/ReAdW%20%28Xcalibur%20converter%29/)</td>
		</tr>
		<tr>
			<td colspan="2">Mango (https://github.com/jpm369/mango)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Comet 2018.01 or later (http://comet-ms.sourceforge.net/)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">XLinkProphet (https://github.com/brucelab/xlinkprophet)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Perl v.5.24.0+ (https://www.perl.org/get.html)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">All required software can be run on a standard personal computer equipped with a Linux operating system and at least 4 GB of RAM.</td>
		</tr>
	</tbody>
</table>

in vivo cross-linking mass spectrometry for protein and complex structural analysis

Chemical cross-linking of proteins with mass spectrometry (XL-MS) has increasingly become a powerful technique when studying protein structures and complexes. This approach is based on the reactivity of cross-linkers to specific protein sites - usually primary amines, including side chains of lysine residues and protein N-termini which yields information on protein-protein interactions and protein conformations. Information provided by XL-MS is complementary to that from other structural methods, such as X-ray crystallography, nuclear magnetic resonance, and cryo-electron microscopy. Here, we describe a protocol for in-house synthesis and use of a peptide-based cross-linker with optimized features for interactome studies of complex biological samples. These features comprise the protein interaction reporter (PIR) technology, MS-cleavable bonds, and an affinity tag, which ultimately facilitate the identification of cross-linked peptide pairs. The membrane permeability enables the cross-linking of living cells, tissues, and isolated organelles (e.g., nuclei and mitochondria), providing valuable structural and interaction data on proteins as they exist in their native environment. Moreover, quantitative XL-MS can be utilized for comparative interactome studies, providing information on protein conformational and interaction changes between varying biological states.

In this study, we performed in vivo cross-linking of HeLa cells using the PIR cross-linker BDP-NHP. After SCX fractionation and biotin-avidin enrichment of cross-linked peptides, two different methods were applied for the MS analysis of the fractionated samples - Mango and ReACT, as described in section 10. We used two technical replicates from Mango and ReACT for the in silico analysis. PeptideProphet27 and ProteinProphet28 are embedded in XLinkProphet fo...

We describe an integrated workflow for chemical cross-linking of proteins with mass spectrometry to study biological complexes in vivo. The protein interaction reporter (PIR) cross-linker presents features that enable the cross-linking of living cells with no prior protein isolation needed, providing information on protein conformations and protein-protein interactions.

In vivo Cross-Linking Mass Spectrometry for Protein and Complex Structural Analysis

Chemical cross-linking of proteins with mass spectrometry (XL-MS) has increasingly become a powerful technique when studying protein structures and ...

<em>In vivo</em> Cross-Linking Mass Spectrometry for Protein and Complex Structural Analysis

Coming Soon!