eXL-MS: An Enhanced Cross-Linking Mass Spectrometry Workflow To

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eXL-MS: An enhanced Cross-Linking Mass Spectrometry Workflow to Study Protein Complexes Martial Rey, Mathieu Dupré, Isabel Lopez-Neira, Magalie Duchateau, and Julia Chamot-Rooke Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00737 • Publication Date (Web): 20 Aug 2018 Downloaded from http://pubs.acs.org on August 20, 2018

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Analytical Chemistry

eXL-MS: An enhanced Cross-Linking Mass Spectrometry Workflow to Study Protein Complexes. Martial Rey, Mathieu Dupré, Isabel Lopez-Neira, Magalie Duchateau, Julia Chamot-Rooke* Mass Spectrometry for Biology Unit, CNRS USR 2000, Institut Pasteur, Paris, France 75015. ABSTRACT: The analysis of proteins and protein complexes by cross-linking mass spectrometry (XL-MS) has expanded in the last decade. However, mostly used approaches suffer important limitations in term of efficiency and sensitivity. We describe here a new workflow based on the advanced use of the trifunctional cross-linker NNP9. NNP9 carries an azido group allowing the quantitative and selective introduction of a biotin molecule into cross-linked proteins. The incorporation is performed by click-chemistry using an adapted version of the enhanced filter-aided sample preparation (eFASP) protocol. This protocol, based on the use of a molecular filter, allows a very high recovery of peptides after enzymatic digestion and complete removal of contaminants. This in turn offers the possibility to analyze very large membrane proteins solubilized in detergent. After trypsin digestion, biotinylated peptides can be easily enriched on monoavidin beads and analyzed by LC-MS/MS. The whole workflow was developed on creatine kinase in presence of detergent. It led to a drastic improvement in the number of identified cross-linked peptides (407 vs 81), compared to the conventional approach using a gel-based separation. One great advantage of our eXL-MS workflow is its high efficiency, allowing the analysis of very low amount of material (15 micrograms). We also demonstrate that Higher-Energy Collision Dissociation (HCD) outperforms Electron-Transfer/Higher-Energy Collision Dissociation (EThcD) in term of number of cross-linked peptides identified, but EThcD leads to better sequence coverage than HCD and thus easier localization of cross-linking sites.

Introduction In past years, chemical cross-linking (XL) coupled to mass spectrometry (MS) has proved to be a powerful approach to probe proteins and protein complexes structures. When realized under controlled conditions, the cross-linking reaction links together residues close in space within the range of the cross-linking agent.1 Mass spectrometry is then used to unambiguously identify cross-linked peptides and cross-linking sites with high sensitivity and accuracy. Combined together chemical cross-linking and mass spectrometry provide structural data of high quality and can be used to validate or help building protein structural models.2 Although most of the applications concerned purified complexes, some reagents have also been proved to be useful for large-scale in vivo cross-linking.3– 7

In a XL-MS experiment, the efficiency of the cross-linking reaction and the detection/identification of the reaction products after enzymatic digestion are two key issues. Many different cross-linkers have been synthesized in the past decade with various types of reactive groups or chemical functions allowing either an easier identification of cross-linked peptides from MS (isotopically labeled cross-linkers)8–11 or MS/MS data (gas phase cleavable cross-linkers).12–16 In the past five years many dedicated software solutions have also been released, providing automated identification of cross-linked peptides such as xQuest,17 Stavrox,18 pLink,19 CrossWork,20 XlinkX,21 Mass Spec Studio,22 with some statistical analysis and graphical outputs. However, a major issue in XL-MS experiments remains the complexity of the peptide mixture obtained after the enzymatic digestion. Indeed, a mix of unmodified, singly modified peptides (called dead-end or monolink), and both intra- and intermolecular cross-linked peptides is generated.23 Moreover, if proper conditions are used to avoid non-native, unspecific intermolecular interactions, only a few cross-linked peptides are generated, which largely complicates their identification. Several groups have developed new strategies to tackle this

important issue. One of the most useful approaches is the enrichment of cross-linked species using strong cation exchange (SCX).9,24–27 Another is to perform size exclusion chromatography (SEC) before the MS analysis.28,29 These developments allowed for the characterization of several very large protein complexes like RNA polymerase II subunit,24 26S proteasome,30 protein kinase 2D network,31 mammalian mitochondrial ribosome32 and the nuclear pore complex in combination with other structural techniques like X-ray crystallography.33 Another promising approach was to introduce an affinity tag (biotin) on the cross-linked peptides via click chemistry to enrich them by avidin capture.34,35 Using this method, Sohn et al. successfully identified cross-linked ubiquitin peptides diluted in yeast lysate, thus demonstrating the benefit of an affinity enrichment of cross-linked peptides.35 Despite recent progress, the overall XL-MS process remains fastidious, and there is room for improvement at various stages of the protocol. First, the cross-linking reaction yield is quite low due to the fast hydrolysis of the NHS-ester groups present in most cross-linking reagents in aqueous solution. Second, the classical SDS-PAGE step often used to remove the excess of cross-linker and other contaminants, and to separate the different cross-linked species one from the others, makes the extraction of the large and branched cross-linked peptides from the gel difficult. This step participates greatly in lowering the signal of the peptides of interest. Third, large amount of proteins are required for non-specific enrichment like SEC or SCX. For instance, in their paper on CK and other proteins,28 Leitner and his coworkers started from 200 µg of material to further enrich their cross-linked peptides by SEC. In their study on BSA, Schmidt and Sinz indicated that at least 50 µg of tryptic peptides were required for an efficient fractionation using OASIS MCX (Waters).27 The requirement of such quantities of starting material can preclude the analysis of many real biological samples of interest. In addition, in the case of the use of isotopically labelled cross-linkers, data analysis can be complicated by the presence of the isotopic pairs and the signal intensity of the peptide of interest is reduced by a factor

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of 2, making the low abundant peaks corresponding to crosslinked peptides disappearing in the noise. Finally, despite efforts toward the development of automated software tools, confident cross-linked peptide identification still requires manual validation. In the process of optimizing XL-MS methods, we developed a few years ago a new trifunctional cross-linking agent, named ‘NNP9’, and used it to characterize with high sensitivity the interface between Hsc70 and α-Synuclein, without applying any specific enrichment strategy.36 This cross-linker is based on the reactivity of NHS-carbamate groups, which are more stable in aqueous solution than the classical NHS-esters, and therefore improves the cross-linking efficiency. NNP9 also carries an azido “clickable” group that allows further affinity enrichment of cross-linked peptides. The choice of having a “clickable” group instead of a real affinity tag was made to reduce the bulkiness of the cross-linker, increasing its accessibility to the amino acids. As pointed out by other groups, large cross-linkers can decrease their efficiency, impair their reactivity and their fragmentation in gas phase. 37–39 In the present paper, we describe an enhanced gel-free XLMS workflow based on the use of NNP9 and inspired by the enhanced Filter Aided Sample Preparation (eFASP) protocol developed recently to increase sample recovery and thus proteome coverage in quantitative proteomic experiments.40 Our gel-free workflow is entirely compatible with membrane proteins and is not limited by the size of the protein complex. Click-chemistry is used to introduce quantitatively a biotin group into cross-linked proteins, which allows further enrichment using avidin but also generates reporter ions in MS/MS spectra of cross-linked peptides, increasing our confidence in the identification. Using creatine kinase (CK) as a model protein, we show that the eXL-MS workflow leads to drastic improvement in the number of cross-linked peptides identified compared to all previous studies, with HCD outperforming EthCD. EThcD leads however to better sequence coverage than HCD and thus easier localization of cross-linking sites. The outstanding results achieved with the eXL-MS workflow could be obtained with a low amount of starting material (15 µg) showing its high efficiency and sensitivity. Methods Cross-linking reaction. Cross-linking of rabbit creatine kinase (10 µM) was carried out in PBS, pH 7.4, with freshly prepared NNP9 (10 mM in DMSO) using 20:1 crosslinker/protein molar ratio. Cross-linking with NPP9 was performed at 4°C for 30 min and stopped by the addition of ammonium bicarbonate (AB, final concentration 50 mM) for 15 min at 4°C. SDS-PAGE. After cross-linking, protein were separated by SDS-PAGE and digested as described in Nury et al.36 Filter-Aided Sample Preparation. After cross-linking, dodecyl maltoside (DDM) was added at 0.05 % (w:w) final concentration and samples were transferred into a passivated molecular filter device (Amicon ultra 0.5 mL with 30 kDa molecular cut-off ) as described in 40 and centrifuged at 10,000 g for 5 min. The concentrate was then washed 4 times by concentration-dilution cycle with AB 50 mM to remove the excess of cross-linker.

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Click-Chemistry and Enzymatic Digestion. The biotin tag was introduced into cross-linked proteins via click-chemistry. Biotin-PEG4-Alkyne (BP4A, 1 mM final concentration) was added in the molecular filter device containing the crosslinked proteins with 1 mM CuSO4, 10 mM Tris(3hydroxypropyltriazolylmethyl) amine (THPTA) in DMSO and 10 mM sodium ascorbate (freshly prepared). The clickchemistry reaction was performed for several times at various temperature. The cross-linked and clicked proteins were then washed 6 times by concentration/dilution cycle with 50 mM AB to remove the reactants. The labelled proteins were then digested overnight at 37°C by addition of trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega) at an enzyme:protein ratio of 1:50 (w:w) under 900 rpm shaking. The peptides were recovered through the molecular filter device by centrifugation (10 min at 10,000g). To improve the peptide recovery 50 µL of 50 mM AB were further added onto the filter and a second centrifugation step was performed. Enrichment of biotinylated peptides. The biotinylated tryptic peptides were enriched by affinity purification on monomeric avidin agarose beads (Pierce) or on streptavidin agarose beads (Pierce) according to the manufacturer conditions with minor modifications. Briefly, after the cross-linking and the click-chemistry reactions, 10 µg of tryptic peptides were added onto 1 µL of monomeric avidin or 1 µL of streptavidin agarose beads previously treated with biotin molecules as required by the manufacturer. After 30 min of gentle mixing at room temperature, the beads were pelleted by centrifugation 1 min at 5,000 g and the supernatant was removed. The monomeric avidin beads were resuspended with 10 µL of formic acid (FA) 0.1 % (v:v) and placed at 95 °C for 10 min to recover the biotinylated peptides. The streptavidin beads were washes 3 times with 50 mM AB, resuspended in 50 % formamide (v:v) and placed at 95 °C for 15 min. Each supernatant was then dried out on a Speedvac and the peptides were resuspended in 0.1 % FA (v:v) prior to mass spectrometry analysis. NanoLC-MS/MS Analysis. Tryptic digests (0.25 µg) were analyzed by nanoLC-MS/MS using an Ultimate 3000 Nano HPLC system (Dionex, Thermo-Scientific) coupled to the nanoelectrospray ion source of an Orbitrap Fusion Lumos mass spectrometer (Thermo-Scientific). Peptides were loaded on an Acclaim PepMap C-18 precolumn (C-18, 3 µm, 100 Å, 5 mm, Thermo Scientific) at a flow rate of 10 µL/min of solvent A (0.1 % FA (v:v) in water) for 5 minutes, and the separation was performed using an in-house packed 25 cm nanoHPLC column (75 µm inner diameter) with C-18 resin (1.7 µm particles, 100 Å pore size, Aeris PEPTIDE XB-C18, Phenomex). Peptides were separated at a flow rate of 250 nL/min using a gradient of 2 % to 30 % solvent B (80 % acetonitrile (v:v), 0.1 % FA (v:v) in water) for 60 min, followed by a 10 min at 60% solvent B. The column and precolumn were then washed 10 min at 100% solvent B and a reconditioned at 2 % B for 10 min. NanoLC-MS/MS experiments were conducted in Data-Dependent Acquisition mode with a top speed mode. After a MS survey scan at a resolution of 60,000 (at m/z 400 in the Orbitrap) the most intense ions, above an intensity threshold of 5,000 counts, were selected for fragmentation during 3 seconds using a resolution of 30,000. Different NCE values were tested for HCD (15, 30, 25, 30 and 35) and different reaction time for ETD (15, 25, 50 75 ms). For EThcD experiments, the following combination were tested: 15 ms

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with Supplemental Activation (SA) of 20 NCE, 25 ms with SA of 15, 20 or 25 NCE, 50 ms with SA of 15, 20 or 25 NCE and 75 ms with SA of 15 NCE. The FT automatic gain control (AGC) was set at 1*106 for MS and 5*104 for MS/MS experiments. Data Processing. NanoLC-MS/MS data were processed automatically using Mass Spec Studio v2.022 with methionine oxidation as a variable modification and lysine, serine, threonine, and tyrosine as residues targeted with NNP9. Mass modifications were set to 771.337 Da for intra- or intermolecular cross-linked peptides, 745.358 and 788.364 for dead-end modifications when NNP9 reacted respectively with water or ammonia. Indeed the dead-end reaction with water leads to the formation of a carboxylic acid that undergoes a rapid decarboxylation to leave a –NH2 group instead of a NH(CO)-NH2 in case of ammonia. The theoretical m/z of 5 reporter ions were added, i.e. 558.307, 501.249, 420.216, 270.127, 227.085. Cross-linked peptides were searched between 300 and 1,500 m/z with “Mass Pairing Generator” algorithm using the following parameters: charge states between 3 and 8, peptides size between 3 and 50 amino acids. The mass accuracy was set to 10 ppm for both MS and MS/MS, the elution width was set to 0.5 min and no thresholding of the data were selected. MS/MS spectra of all cross-linked candidates were further assessed using a FDR estimated at 0.1 % (using a decoy database) and further manually validated. The presence of at least 2 reporter ions masses was mandatory to confirm their identification. Data are available via ProteomeXchange with identifier PXD010422. Results and Discussion We introduce here a new XL-MS workflow, which aims at being more simple, efficient and sensitive than the current ones and compatible with very large protein complexes such as those found in membranes (Figure 1). This workflow is based on the use of (i) a trifunctional reagent (NNP9) for

improved cross-linking efficiency, (ii) a molecular filter for sample preparation (eFASP), (iii) enrichment of cross-linked peptides with affinity purification after the introduction of a biotin moiety into the cross-linked proteins by click-chemistry, (iv) an improved MS/MS method. CK was used as a model protein for this study, to compare with preliminary results obtained with different analytical strategies. Dodecyl maltoside (a detergent often used for membrane proteins) was added just after the XL quenching step to test our workflow in realistic conditions for membrane proteins. It was not introduced earlier to avoid any change in the native structure of CK, which is a soluble protein. We also analyzed a sample without detergent, with DMPG, triton X-100 or CHAPS and similar results were obtained in all cases. Use of a molecular filter (eFASP protocol). One limitation of the classical XL-MS approach is the use of a SDS-PAGE separation after the cross-linking step, which leads to a poor recovery of cross-linked peptides. We therefore decided to replace the gel-based separation by a passivated molecular filter for sample preparation as proposed in the eFASP approach developed for quantitative proteomic experiments.40,41 The first benefit of this approach is that the enzymatic digestion can be performed in solution, which induces a much better recovery of cross-linked peptides. The second is to allow extensive washes of cross-linked proteins, without sample loss, which leads to an efficient elimination of the excess of cross-linking and click-chemistry reagents, as well as detergent. Finally, it removes the size limitation imposed by the gel-based separation. Therefore both the click-chemistry step, for biotin incorporation into cross-linked proteins, and trypsin digestion were designed to take place in this unique filter device, which maximized the overall recovery of the sample: 83.4 % of the initial protein load could be retrieved through the filter as peptides (average value obtained for three biological replicates calculated by measuring the OD at 280 nm before and after filtration).

Figure 1: eXL-MS Protocol. After cross-linking with NNP9, proteins are transferred into a passivated filtering device. A biotin-PEG4alkyne (BP4A) molecule is then added to perform a click-chemistry reaction with the azido group of the cross-linker. After several washing cycles, cross-linked proteins are trypsin digested and peptides are recovered through the filter. Cross-linked peptides are enriched on monoavidin beads and analyzed by nanoLC-MS/MS. Data are processed with Mass Spec Studio and manually validated.



Optimization of the click-chemistry step for biotin incorporation into cross-linked proteins. This step was critical since it had to be quantitative to further enrich all cross-linked peptides. To optimize the click-chemistry conditions, we first checked the dependency of the reaction with temperature (Figure 2A). We followed by mass spectrometry the conversion of 13 intense peptides modified with NNP9 and thus carrying an azido group (cross-linked peptides and dead-ends) into biotinylated species with a fixed reaction time of 20 min. As expected for this type of chemical reaction, the biotin incorporation was found to be proportional to the temperature and maximum for the highest one (95°C). Unfortunately the filter device could not handle such temperature and got damaged twice out of three experiments. We therefore selected 75°C as a safe and efficient temperature for the clickchemistry reaction. At this temperature, we were able to reach a complete biotinylation of cross-linked peptides (Figure 2B) in 60 min. The sample was then digested with trypsin and all peptides were recovered through the molecular filter.

Figure 2: Effect of temperature (A) and time (B) on the efficiency of the click-chemistry reaction between NNP9 and cross-linked proteins. The efficiency of biotinylation is followed by monitoring the intensity ratio of 13 intense biotinylated peptides with their non-modified counterparts.

Optimization of the nanoLC-MS/MS analysis. Before testing our enrichment protocol, we first optimized the fragmentation conditions on the non-enriched sample to evaluate the best method between HCD (High energy Collision Energy), ETD (Electron Transfer Dissociation) and a combination of both (EThcD) (Table S1). We only targeted ions with 3 charges or more for fragmentation to preferentially sequence cross-linked peptides, which theoretically carry more than 2

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charges. We tested different normalized collision energies (NCE) and reaction times (ET) and followed two parameters given by Mass Spec Studio. The first one is the number of identifications with a score above 10 (ID10). The second one is the mean score of the top 50 identification (S50). For an easier comparison we compared the values obtained for ID10 x S50. For HCD, the best results were obtained using a normalized collision energy (NCE) of 30 leading to S50=22.8 and ID10=303 (ID10 x S50 = 6,908; Table S1). For ETD, we compared the fragmentation efficiency without and with supplemental activation (EThcD). The best results were then obtained using a reaction time of 25 ms with a supplemental activation of 20 NCE leading to S50=22.0 and an ID10=127 (ID10 x S50 = 2,794; Table S1). Applying a supplemental activation in ETD clearly improved the results. For the mean score of the top 50 identification (S50), HCD and EThcD methods led to comparable values (22.8 and 22.0 respectively). However, a drastic difference was observed for the number of identifications with a score above 10 (ID10): ID10 was found to be 127 for EThcD experiments and 308 for HCD. This means that HCD led to twice the number of identifications than EThcD. This result is not very surprising since EThcD is much slower than HCD and for very complex mixtures a fast fragmentation technique is required to fragment as many precursor ions as possible. Interestingly, we also noticed that the fragmentation of the biotin moiety generated intense reporter ions (Figure S1), spreading from m/z 227.085 to m/z 558.307 depending on the energy and on the fragmentation mode. Structures for these reporter ions are proposed in Figure S2. These reporter ions were of high interest to confirm the presence of the cross-linker. The total ion chromatogram (TIC) obtained for the nonenriched sample with HCD is shown in Figure 3A as an example. It is dominated by a few very intense peaks corresponding to non-modified peptides, which is typical for a cross-linking experiment without any enrichment. Data analysis indicated that cross-linked species represented only a very small fraction of the signal. HCD and EThcD fragmentations led respectively to the identification of 68 and 50 cross-linked peptides (Figure S3B, Table S2). Nevertheless, both methods were complementary, 41 cross-linked peptides were uniquely obtained by HCD and 23 by EThcD. Combining both types of experiments, the non-enriched sample allowed identifying 91 unique cross-linked peptides (Figure 4A). This is 10 times more than our previous study based on a gel-based separation, a LTQ-Velos Orbitrap and Stavrox/X-Quest for data analysis.36 This is also slightly better than a gel-based approach followed by Orbitrap Fusion Lumos and Mass Spec Studio analysis, which led to 81 cross-linked peptides (Table S2). Considering these results, we decided to use both HCD (NCE 30) and EThcD (25ms, NCE 20) for the analysis of the enriched sample. All experiments were performed in three replicates and we considered all non-redundant cross-linked peptides. Results are gathered in Table S2. Enrichment of cross-linked peptides with affinity purification. Typical affinity purification strategies involve trapping of the biotinylated material on agarose beads coated with streptavidin or avidin and therefore we decided to test both.


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The strong affinity of streptavidin for biotin (Kd 10-15 M) allowed extensive washes to be performed and thus many contaminants to be removed. Nevertheless, the release of the biotinylated molecules turned out to be complicated and led to an important loss of material (data not shown). To overcome this limitation, we decided to purify our biotinylated peptides using agarose beads coated with monomers of avidin, for which the biotin affinity is weaker (Kd 10-7 M). We therefore analyzed the monoavidin enriched sample by nanoLC-MS/MS (using HCD or EThcD) and compared the results with those obtained previously. The amount of material injected was adjusted to give comparable maximum signal intensity (~1.2E10 arbitrary units) between the non-enriched and en-

riched preparation (Figure 3). The TIC obtained for the monoavidin enriched fraction analyzed by HCD was found to be much denser than for the non-enriched sample (Figure 3B) and many new signals showed up in regions that were previously completely empty (such as between 38 and 48 minutes). For example, the signal of the cross-linked peptide HNNHMAKVLTPDLYK-PFGNTHNKYK (m/z 626.98; z=6), which was absent in the non-enriched sample was clearly visible in the enriched one, and turned out to be sufficiently intense to be selected for fragmentation (Figure 3, inserts). Note however that the major peaks observed in the nonenriched sample were still present here but in lower intensity.

A. 100 Intensity (%)

No Enrichment 50 No signal 0 627.32

m/z 626.98 (6+)

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627.48

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Figure 3: Comparison of the TIC obtained without (A) and with (B) monoavidin enrichment. The extracted ion chromatogram of the isotopic envelop of the +6 charge state of the HNNHMAKVLTPDLYK-PFGNTHNKYK cross-linked peptide is displayed in black. The insert represents the MS signal between m/z 626.5 and 628.5 at 41.5 min corresponding to this peptide.

After the enrichment, a total of 407 unique cross-linked peptides were identified combining all HCD and EThcD runs, corresponding to a 4-fold increase compared to the nonenriched fraction and a 5-fold increase compared to the gelbased approach (Figure 4B, Table S3). Among these peptides, 360 were found to be interlinks with 65.8% corresponding to K-K linkages, 30% to K-X (X=T,S or Y) linkage and only 4.2% do not contain K (Table S4). Therefore, as for NHS-ester cross-linkers, NHS-carbamates primarily target lysine residues. As observed for the non-enriched fraction, HCD led to more cross-linked peptide identification than EthcD with respectively 375 and 222 cross-linked peptides identified (Figure 4B). However, EThcD gives rise to more fragments than HCD (34.6 vs 22.3 on average, Table S5) and leaves the cross-linker attached to both peptides, which facilitates the exact localization of the cross-linking site (Table S5). For instance, as shown in Figure 5 respectively 62 and 23 fragments were obtained for the cross-linked peptide GGVHVKLAHLSK-VISMEKGGNMK. Moreover, 17 frag-

ment ions are found containing both peptide in EThcD (Figure 5B) instead of 3 in HCD (Figure 5A). Indeed, even if this site is clearly identified by HCD in the peptide β, this information is lost in the case of the peptide α.

Figure 4: Venn diagram representing the number of cross-linked peptides obtained in different conditions A. for monoavidin en-



richment (blue) or no enrichment (red) combining HCD and EThcD results. B. for monoavidin enrichment depending on the fragmentation mode (HCD in purple and EThcD in yellow).

It is worth noting that the whole workflow was done with 15 µg of starting material, which is much less then what is normally used when a chromatographic fractionation is performed. For instance in their study of CK, Leitner et al. used a SEC enrichment step (after digestion with 5 different enzymes) to increase the number of cross-linked peptides identified.28 The authors started from 200 µg CK to finally identify 19 cross-linked peptides in total. Even if in the present study a single protein was used, and not a mixture, the amount of sample remains much lower than in all other studies and thus we are confident that our workflow could easily be extended to more complex samples. Distance Constraint Analysis. In total, our new eXL-MS protocol allowed to identify 407 different cross-linked peptides in CK (Figure 6). This number is much higher than what was described in earlier studies, thus we decided to check the validity of the cross-linked peptides by fitting them to the CK structure present in the PDB (1U6R). Since it not possible to make the distinction between intramolecular and intermolecular cross-linked peptides, cross-linked peptides were considered intermolecular when part of the sequence was shared between alpha and beta peptides. Note that only the dimer structure is available although CK is known to also form tetramers and higher-order structures.36,42 The distribution of the distance obtained for a random distribution of cross-linked peptides was calculated and compared to our experimental

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results (respectively defined as “random” and “measured” in Figure S4). The two distributions were found significantly different. We identified 194 cross-linked peptides to be within the 26 Å expected distance in the dimer (Table S6). This population is clearly visible on the distribution with an average distance of 12.5 Å (Figure S4) and corresponds to the crosslinked peptides of the known dimer structure (PDB code 1U6R). Among these cross-linked peptides, 8 were found to involve two different monomers and the rest to be intramolecular. Note that only one intermolecular cross-linked peptide was found in the previous study with NNP9. An example of these intermolecular cross-linked peptides showing the interaction between the N-termini of two CK monomers is depicted in Figure S5. One hundred and thirty three other cross-linked peptides were found to be between 26 and 40 Å, and could be explained by dynamic events such as breathing motions or large domain movements in the protein complex, as already observed in other studies.36,42 Finally, we found 80 crosslinked peptides exceeding 40 Å. Looking in detail into these long cross-linked peptides, 46 were found to describe interactions occurring between a single patch constituted of 13 residues in close vicinity (K222, K241, K246, K297, S302, K303, K306, K318, K357, K364, K365, K368, S371) and the CK Nterminus (T5, K8, Y9, K10, K14, Y38, K39 and K40). One hypothesis is that these interactions arise from minor structures of CK that are different from the one present in the PDB but would be captured by our workflow, which operates in solution.


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Figure 5: HCD (A) and EThcD (B) fragmentation spectra of the [GGVHVKLAHLSK (α) + VISMEKGGNMK (β)] cross-linked peptide exported from Mass Spec Studio. The fragments identified are reported on the sequences. The fragments reported far from the sequence contain the biotin moiety and the linked peptide. The fragments in pink arise from the biotin moiety. ID10*S50 obtained for the automatic identification of cross-linked peptides; S2. List of cross-linked peptides identified; S3. Number of cross-linked peptides identified; S4. Type and percentages of residue linkages; S5. Average scores and number of fragments for MS/MS spectra; S6. List of minimum Cα-Cα distances on the CK dimer structure.

AUTHOR INFORMATION Corresponding Author * E-mail : [email protected]

Author Contributions Figure 6: Structure of the creatine kinase dimer (PDB 1U6R) with the 162 crosslinks < 26 Å indicated as red dashed line.

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes Conclusion This paper describes the development of an enhanced crosslinking mass spectrometry workflow named eXL-MS. This workflow comprises the following steps: cross-linking with a trifunctional reagent named NNP9, click-chemistry reaction for introducing a biotin moiety in all cross-linked proteins in eFASP conditions, trypsin digestion, monoavidin enrichment of biotinylated peptides, nanoLC-MS/MS analysis with HCD or EThCD on an Orbitrap Fusion Lumos and data analysis with MassSpecStudio. It relies on major modifications compared to the usual protocol that, in combination, led to a drastic improvement of efficiency and sensitivity. First, removing the SDS-PAGE step and introducing a filtration device allowed to slightly increase the sensitivity of the approach (91 cross-linked peptides were identified for the creatine kinase instead of 81 with the gel-based separation). Second, the introduction of a biotin molecule into cross-linked proteins using click-chemistry allowed a drastic enrichment of cross-linked peptides on monoavidin beads. This enrichment step greatly improved the number of cross-linked peptides identified, which was more than quadrupled (final number 407). Finally we showed that HCD and EThcD fragmentation gave complementary results, EThcD leading to better sequence coverage and thus better cross-linking site localization. The unique sensitivity of our workflow, performed with 15 µg of starting material, and the fact that it was designed to be compatible with detergent and large biological systems paves the way to the structural analysis of extra-large membrane protein complexes that could not be addressed by other means.

The authors declare no competing financial interest.

ACKNOWLEDGMENT Synthesis and characterization of NNP9 were performed by the COBRA lab (Mont-Saint-Aignan, University of Rouen). This work has been partially supported by Institut Pasteur and CNRS. Financial support from l’Agence Nationale de la Recherche (CLICKMASSLINK -ANR 09-PIRI-0006) and from the “Investissement d’Avenir” Bioinformatique program (grant BIP:BIP, ANR-10-BINF-03-13). The authors are grateful to Q. GiaiGianetto for the statistical analysis of cross-linked peptides distances and to Jonathan Dhenin for his help on Figure 6.

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ASSOCIATED CONTENT Supporting Information This information is available free of charge via the Internet at http://pubs.acs.org/ Figure S1. EThcD spectrum; S2. Proposed structures for the reporter ions; S3. Venn diagrams representing cross-linked peptides; S4. Distance distribution of cross-linked peptides; S5. First amino acids of the CK dimer structure. Table S1. ID10, S50 and

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