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

Summary

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.

Abstract

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.

Introduction

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 environment^1,2. 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 studies^3,4,5. 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) "on-the-fly" 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 peptides^11,12, 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 XLinkDB^18,19,20, 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.

Protocol

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's instructions. This protocol is based on a previous publication⁷.

1. Prepare the following reagents.
   1. Weigh out 0.63 g of Rink Amide resin (100-200 mesh) for 0.05 mmol.
   2. Weigh out 1.73 g of aspartate (Fmoc-Asp(OtBu)-OH) and dissolve it in 21 mL of DMF.
   3. Weigh out 23 g of succinic anhydride and dissolve in 23 mL of DMF.
   4. Weigh out 1 g of biotinylated lysine (Fmoc-Lys(biotin)-OH) and dissolve it in 32 mL of DMF.
      NOTE: Increase the solubility of biotinylated lysine in DMF by heating the solution up to 70 °C, with constant mixing.
   5. Weigh out 1.42 g of proline (Fmoc-Pro-OH) and dissolve in 21 mL of DMF.
   6. Weigh out 1.3 g of lysine (Fmoc-Lys(Fmoc)-OH) and dissolve it in 11 mL of DMF.
2. Fill the wash bottle with 400 mL of DMF.
3. Fill the deprotection bottle with 60 mL of the deprotection solution [20% (vol/vol) piperidine in DMF].
4. Fill the activator bottle with 40 mL of activator solution (0.5 M DIC in DMF).
5. Fill the activator base bottle with 20 mL of 1 M Oxyma in DMF.
6. Run the peptide synthesizer with the following coupling steps:
   Resin: 0.5-mmol resin swelling
   Fmoc-Lys(biotin)-OH: 0.5-mmol triple coupling
   ​NOTE: Divide the resin-lysine coupling in 3 separate steps due to the large volume of the lysine solution (32 mL).
   Fmoc-Lys(Fmoc)-OH: 0.5-mmol single coupling
   Fmoc-Pro-OH: 0.5-mmol double coupling
   Fmoc-Asp(OtBu)-OH: 0.5-mmol double coupling
   Succinic anhydride: 0.5-mmol succinyl coupling
7. Transfer the BDP-containing resin to a 50 mL tube.

2. Synthesis of TFA-NHP

1. Weigh out 5.86 g of N-hydroxyphthalamide (NHP, 163.139 g/mol) and place in 50 mL round bottom flask.
2. Add a four-fold molar excess of trifluoroacetic acid anhydride (210.03 g/mol).
3. Allow the reaction to proceed for 1.5 h in dry nitrogen gas atmosphere, with constant mixing.
4. Evaporate excess TFA anhydride under vacuum. The product should appear as dry, white, crystalline solid.
5. Transfer the content to 2 mL microcentrifuge tubes.
   ​NOTE: The protocol can be paused here. Store the TFA-NHP-containing tubes at -20 °C up to a year.

3. BDP-NHP cross-linker esterification and purification

1. Transfer the BDP-containing resin to a 50 mL tube.
2. Swell the BDP-containing resin with a minimal volume of 1 mL of DMF for 5 min.
3. Weigh out a 12-fold molar excess of TFA-NHP from 2.5 (approximately 300 mg for a 0.1 mmol batch).
4. Dissolve the amount of TFA-NHP in 10 mL of dry pyridine.
5. Transfer the TFA-NHP-pyridine solution to the tube containing the swelled resin and incubate for 20 min at room temperature, with constant mixing.
6. Transfer the resulting solution from 3.5 to an empty disposable polypropylene chromatography column.
7. Using a vacuum system, wash the resin three times with 20 mL of DMF.
8. Seal the column with the outlet and incubate the resin with 20 mL of DMF for 20 min.
9. Wash the resin three times with a 20 mL of DCM.
10. Seal the column with the outlet and incubate the resin with 10 mL of DCM for 20 min.
11. Cleave the BDP-NHP cross-linker from the resin by adding 5 mL of the cleavage solution [95% trifluoroacetic acid (TFA)/ 5% DCM] for 3 h at room temperature, with constant mixing.
12. Collect the flow-through in a 15 mL tube.
13. Add 5 mL of the cleavage solution to the column and incubate for 5 min at room temperature.
14. Collect the flow-through and add the volume to the 15 mL tube from 3.12.
15. Add 150 mL of cold diethyl ether separately to four 50 mL tubes.
16. To precipitate the cross-linker, slowly add 2.5 mL of the recovered final solution from 3.12 and 3.14 to each diethyl-ether-containing tube.
    NOTE: At this point, a white precipitate should be visible.
17. Centrifuge the tubes at 3,400 × g for 30 min at 4 °C to pellet the cross-linker.
18. Suspend each resulting pellet in 5-10 mL of cold diethyl ether, combine the volumes in one 50 mL tube, and repeat the centrifugation step 3.17.
19. Decant the diethyl-ether to waste and completely dry the cross-linker pellet under vacuum (for approximately 20 min).
20. Weigh the dry pellet and suspend it in DMSO to produce a stock solution with concentration of 100-300 mM.
21. Calculate the yield based on the measured mass of product - usually around 95%. Convert the measured mass of dried cross-linker into moles by dividing by the average molecular mass of BDP-NHP (1,414.45 g/mol) and divide by the theoretical maximum number of moles (i.e., 0.5 mmol) on the basis of the amount of resin used.
22. Check the purity of the cross-linker by direct infusion ESI-MS analysis of a 1:1,000 dilution of the concentrated stock solution in a solution of 49% (vol/vol) methanol, 49% (vol/vol) water, 2% (vol/vol) acetic acid. A purity range of 80-90% is expected based on the relative intensity of the quasi-molecular ion peak for the PIR cross-linker ([M+H]⁺ = 1,414.526 m/z) (Figure 4).
23. Measure the concentration of BDP-NHP by UV-Vis absorbance. The concentration of BDP-NHP can be measured by UV-Vis absorbance.
    1. Dilute 1 µL of the concentrated stock solution of cross-linker in 0.1 M NH₄HCO₃, pH 8.0, to achieve a concentration of 0.5 mM and quench the crosslinker, releasing free NHP with yellow color.
    2. Measure the absorbance of NHP at 410 nm and compare to a calibration curve for free NHP in 0.1 M NH₄HCO₃, pH 8.0, preparing dilutions from 0.1 to 2 mM to calculate the concentration of released NHP. The concentration of BDP cross-linker in the stock solution is half the calculated concentration of NHP multiplied by the dilution factor.
24. The stock cross-linker solution can be stored at -80 °C for up to 1 year.
    ​NOTE: The protocol can be paused here.

4. Cell culture and harvest

1. Culture HeLa cells in 15 cm plates with 20 mL of DMEM, supplemented with 10% FBS (vol/vol) and 1% (vol/vol) penicillin-streptomycin.
2. Maintain the cells in a cell incubator at 37 °C with 5% CO₂.
3. When cells are 80-90% confluent (approximately 2.0 × 10⁷ cells), wash the culture twice with 5 mL of 1x PBS.
4. Incubate the cells with 5 mL of PBS 1 × with 20 mM EDTA, at 37 °C for 3-5 min.
5. Visualize the cells in a microscope to confirm detachment of the cells from the plate.
6. Transfer the cell content to a 15 mL tube, centrifuge at 300 × g for 3 min at 20 °C and discard the supernatant.
7. Suspend the remaining pellet in PBS with calcium and magnesium.
8. Centrifuge at 300 × g for 3 min at 20 °C and discard the supernatant.
9. Suspend the cell pellet in 10 mL of 1x PBS.
10. Centrifuge at 300 × g for 3 min at 20 °C and discard the supernatant.
11. Repeat 4.9 and 4.10 two additional times.

5. In vivo cross-linking

1. Suspend the cell pellet from 4.11 in one-pellet volume of 170 mM Na₂HPO₄ (approximately 500 µL).
2. Add 10 mM of BDP-NHP cross-linker.
   NOTE: Pipette the corresponding volume of cross-linker in the tube without touching the pipet tip to the solution - it can precipitate in the tip - and immediately vortex the sample on a gentle setting to not disrupt the integrity of the cells.
3. Incubate for 30 min at room temperature, with constant mixing.
   NOTE: The solution will turn yellow as the BDP-NHP cross-linking reaction happens.
4. Add one pellet volume of 0.1 M NH₄HCO₃ to quench the cross-linking reaction.
5. Pellet the cells by centrifugation at 300 × g for 3 min at 20 °C and discard the supernatant.
6. Suspend the cell pellet in 1 mL of 0.1 M NH₄HCO₃ and repeat centrifugation as in 5.5.
7. Repeat 5.6 3 times or until no yellow color remains in the supernatant.

6. Protein extraction, reduction, alkylation, and digestion

1. Prepare the cell lysis buffer with 8 M urea in 0.1 M NH₄HCO₃.
2. Suspend the cell pellet from 5.7 in 1 mL of the cell lysis buffer.
3. Sonicate the cells in lysis buffer using an ultrasonic processor, applying five pulses at amplitude 40 for 5 s.
4. Add 5 mM of TCEP-HCl to the sample to disrupt disulfide bonds. Incubate at room temperature for 30 min, with constant mixing.
5. Add 10 mM of IAA to alkylate reduced thiol groups. Incubate for 30 min at room temperature, with constant mixing.
   NOTE: IAA is light sensitive. Protect the sample from light during the reaction.
6. Add 0.1 M NH₄HCO₃ to dilute the sample and reduce the urea concentration to < 1 M.
7. Quantify the protein content using the Bradford assay - or other protein quantitation method of choice.
8. Add 1 µg of sequencing-grade modified trypsin for every 200 µg of protein (1:200 ratio) in the sample and digest for 16-18 h at 37 °C, with constant shaking at 650 rpm.
9. Acidify the sample with 1% TFA (vol/vol) to a pH < 3.
10. Centrifuge sample at 16,000 × g for 15 min at room temperature.

7. Sample desalting

1. Prepare the reversed-phase C18 desalting column with the suitable protein capacity and wash it with one-column volume of methanol.
2. Add one-column volume of 0.1% (vol/vol) TFA acid in acetonitrile to condition the column.
3. Pass the solution through the column using the vacuum manifold.
4. Repeat steps 7.2 and 7.3 two additional times.
5. Add one-column volume of 0.1% (vol/vol) TFA acid in water to equilibrate the column.
6. Pass the solution through the column using the vacuum manifold.
7. Repeat steps 7.5 and 7.6 two additional times.
8. Add the supernatant from step 6.10 to the Sep-Pak column, considering the maximum volume allowed by the cartridge.
9. Pass the solution through the column using the vacuum manifold.
10. Repeat steps 7.8 and 7.9 until the entire volume from 6.10 has passed through the column.
11. Repeat steps 7.5 and 7.6 to wash the columns and eliminate remaining salts.
12. Place an open 1.5 mL microcentrifuge tube under each column outlet.
13. Add one-column volume of 80% (vol/vol) acetonitrile/ 0.1% (vol/vol) TFA acid to elute the peptides.
14. Collect the flow-through using the extraction manifold vacuum.
15. Dry out the sample by vacuum centrifugation, then reconstitute the peptides with ~10 µL of 30% ACN, 0.1% TFA.
    ​NOTE: The protocol can be paused here. Store samples at -80 °C.

8. Strong cation exchange (SCX) chromatography for sample fractionation

1. Add 500 µL of SCX solvent A [7 mM KH₂PO₄, pH 2.6, 30% (vol/vol) acetonitrile] to the sample from step 7.15.
2. Centrifuge the sample at 16,000 × g for 15 min at room temperature.
3. Transfer the supernatant to a 1.5 mL LC autosampler vial.
4. Inject the sample into a liquid chromatography system equipped with an SCX column (Table of Materials).
5. Fractionate the sample applying a 1.5 mL/min flow rate on a 97.5-min gradient with increasing concentration of SCX solvent B [7 mM KH₂PO₄, pH 2.6, 350 mM KCl, 30% (vol/vol) acetonitrile], as follows: 0% B at 0 min, 5% B at 7.5 min, 60% B at 47.5 min, 100% B at 67.5 min, 100% B at 77.5 min, 0% B at 77.51 min, and 0% B at 97.5 min.
6. Collect the 14 fractions per experiment with 500 mL each and pool the fractions 1-5, 6-7, and 11-14.
7. Dry out each fraction (6-7, 8, 9, 10, 11-14) by vacuum centrifugation, then reconstitute the peptides with 2-3 mL of 100 mM NH₄HCO₃.
   NOTE: Fractions 1-5 are usually not analyzed because most cross-linked peptides begin eluting in fraction 6. However, these fractions are useful for the identification of dead-ends.
8. Adjust the pH to 8.0 using 1 M NaOH.

9. Avidin-biotin enrichment of cross-linked peptides

1. Add 200 µL of monomeric avidin resin to each resulting fraction from section 8.
2. Incubate the fractions for 30 min at room temperature, with constant mixing.
3. Use a disposable poly-propylene column for enrichment of BDP-cross-linked peptides from each fraction. Place the columns in the extraction manifold.
4. Transfer avidin-bound peptides from each fraction to the corresponding disposable poly-propylene column.
5. Add 2 mL of 0.1 M NH₄HCO₃ pH 8.0 to each column.
6. Remove the liquid using the extraction manifold vacuum. Discard the flow-through.
7. Repeat steps 9.5 and 9.6 five additional times.
8. Incubate the avidin resin with 1 mL of 70% (vol/vol) acetonitrile and 1% (vol/vol) formic acid (FA) for 5 min.
9. Place a 1.5 mL microcentrifuge tube under each cartridge. Apply vacuum and collect the flow-through.
10. Concentrate the fractions (6-7, 8, 9, 10, and 11-14) to a final volume of approximately 10 µL in a vacuum centrifuge.
    ​NOTE: The experiment can be paused here. Store the samples at -80 °C.

10. LC-MS analysis

NOTE: This protocol used a high-resolution LC-MS pipeline on a Q-Exactive Plus for a Mango-oriented data analysis¹⁰, and also LC-MS on a hybrid ion trap LTQ Velos FT-ICR instrument²³ for the ReACT method⁹. Both methods apply DDA with selection of ions with charge state of +4 to +8. Briefly, Mango relies on tandem mass spectra (MS2) for the mass-relationship check and for the identification of peptides, while ReACT uses MS2 for the "on-the-fly" mass relationship check (mass of precursor = mass reporter + mass peptide 1 + mass peptide 2), and then MS3 events for the fragmentation of cross-linked peptides and further peptide search/identification. For the BDP-NHP, the mass of the reporter ion is 693.405431 Da.

1. Suspend pooled fractions from section 9 in 30 µL of 0.1% (vol/vol) FA.
2. Centrifuge sample fractions at 16,000 × g for 10 min at room temperature.
3. Transfer the supernatant of each sample to LC autosampler vials.
4. Inject 1-5 µL of sample (approximately 1 µg) from each pooled fraction into the nano-LC system.
   NOTE: Analyze each fraction 2-3 times for technical replicates of the experiment.
5. Load each peptide fraction onto a trap column [3 cm × 100-µm i.d., stationary phase ReproSil-Pur C8 (5-µm diameter and 120-Å-pore-size particles)] with a flow rate of 2 µL/min of mobile phase: 98% (vol/vol) LC-MS solvent A [0.1% (vol/vol) FA in water] and 2% (vol/vol) LC-MS solvent B [0.1% (vol/vol) FA in acetonitrile].
6. Chromatographically separate the peptides using an analytical column [60 cm × 75-µm i.d., stationary phase ReproSil-Pur C8 (5-µm diameter and 120-Å-pore-size particles)] and apply a 240-min linear gradient: from 95% LC-MS solvent A, 5% LC-MS solvent B to 60% LC-MS solvent A, 40% LC-MS solvent B, at a flow rate of 300 nL/min.
7. Apply a voltage of 2.5 kV to the spray tip for the ESI-based ionization of peptides.
8. For Mango, set the high-resolution mass spectrometer (e.g., Q-Exactive Plus) to data-dependent acquisition (DDA) of the 5 most intense ions with charge state of +4 to +8, with a dynamic exclusion of 30 s and isolation window of 3 Da. Each MS1 scan (70,000 resolving power at 200 m/z, automated gain control (AGC) of 1 × 10⁶, scan range 400 to 2,000 m/z, dynamic exclusion of 30 s) is followed by 5 MS2 scans (70,000 resolving power at 200 m/z, AGC of 5 × 10⁴, normalized collision energy of 30).
9. For ReACT, set the mass spectrometer, in this case a hybrid ion trap LQT Velos FT-ICR instrument, to data-dependent acquisition (DDA) of the 6 most intense ions with charge state of +4 to +8, with dynamic exclusion of 45 s and isolation window of 3 Da. Each MS1 scan (50,000 resolving power at 200 m/z, mass range 500 to 2,000 m/z) was followed by 1 MS2 acquisition for the "on-the-fly" mass relationship check - mass precursor = mass reporter + mass peptides - (12,500 resolving power at 200 m/z, CID activation, normalized collision energy of 25), and 4 MS3 acquisitions of the selected ions with a 20 ppm tolerance for mass error after the mass relationship check, and with 1+ and 2+ charge states (CID activation, normalized collision energy of 35).
   NOTE: ReACT can be performed in any instrument with a high resolution MS2 and low resolution MS3 capacity⁹.
10. For an LC-MS analysis of dead-ends, use an MS method similar to the one described in section 10.8, but with charge state of 1+ to 8+, MS1 events with 70,000 resolving power at 200 m/z, and MS2 scans with 17,500 resolving power at 200 m/z.
    NOTE: Besides fractions 6 to 14, include pooled fractions 1 to 5 for this specific LC-MS analysis. Most cross-linked peptides begin eluting in fraction 6, with charge state of 4+ to 8+, but dead-ends will elute early during the SCX chromatography, with charge state 1+ to 8+.

11. Data analysis

NOTE: All required software run on a Linux operating system.

1. Mango data analysis
   1. Use ReAdW to convert the instrument files, e.g., .raw files, to the .mzXML format. ReAdW is available for download at https://sourceforge.net/projects/sashimi/files/ReAdW%20%28Xcalibur%20converter%29/.
   2. Use the Mango tool, available at https://github.com/jpm369/mango, to generate .ms2 files containing individual precursor masses of released peptides for each spectrum and .peaks file containing all relationships within a 20 ppm tolerance.
   3. Use the following command to write the Mango parameters
      mango.exe -p
   4. Use the following command to run Mango on .mzXML files:
      mango.exe *.mzXML
      NOTE: Hardklör ²⁴ is embedded in Mango and is used for charge deconvolution and deisotoping of complex MS2 spectra.
   5. Run the Comet²⁵ search engine (http://comet-ms.sourceforge.net/) on the .ms2 files resulted from 11.1.2.
   6. Use the following command to write the comet parameters:
      ​comet -p
   7. Use the following command to run Comet on .ms2 files:
      runCometQ *.ms2
   8. Proceed to step 11.3.
2. ReACT data analysis
   1. Use ReAdW to convert the instrument files, e.g., .raw files, to the .mzXML format.
   2. Use the following command to write the comet parameters:
      comet -p
   3. Run the Comet search engine (http://comet-ms.sourceforge.net/) on the .mzXML files resulted from 11.2.1.
      runCometQ *.mzXML
   4. Run react2csv to map ReACT2 results to the sequences in the database.
      react2csv *.pep.xml
   5. Proceed to step 11.3.
3. XLinkProphet²⁶ for validation of results
   1. Run XLinkProphet (https://github.com/brucelab/xlinkprophet) on the pep.xml files resulted from the Comet search to apply probability values and validate results.
   2. Use the following command to run XLinkProphet parameters:
      ​runXLinkProphet.pl WRITEPARAMS
   3. Add the PIR cross-linker reporter mass to the command. In the case of the BDP-NHP cross-linker, the reporter mass is 693.405431, thus use the following command:
      runXLinkProphet.pl '*.pep.xml' REPORTERMASS=693.405431
4. Upload the results from XLinkProphet in the .pep.xml format to XLinkDB¹⁹, available in http://xlinkdb.gs.washington.edu/xlinkdb/, following the platform instructions.

Results

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...

Discussion

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...

Materials

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