We thank our colleagues for helpful discussions. We also thank Ilme Schlichting and Karl-Peter Hopfner for providing protein complexes. We acknowledge funding from the Federal Ministry for Education and Research (BMBF, ZIK programme, 03Z22HN22), the European Regional Development Funds (EFRE, ZS/2016/04/78115) and the MLU Halle-Wittenberg to C.S. and funding from the Wellcome Trust (109854/658 Z/15/Z) to A.P.

The authors have nothing to disclose.

Protocols are provided for mass spectrometry-based structural analysis of multi-subunit protein complexes. The two techniques, described in the protocol, mostly deliver complementary results and are well-suited to gain insights into the structural arrangements within protein (-ligand) complexes which are difficult to study by conventional structural techniques. Native mass spectrometry delivers insights into protein stoichiometries as well as protein interactions by analyzing subcomplexes and stable interaction modules. Cross-linking, on the other hand, yields information on direct contact sites. Depending on the cross-linker used, a certain flexibility can or should be included in the analysis.
The provided protocols are in general easy to perform and not time-consuming. The entire protocol can be executed within a week and can be applied to almost all protein complexes, although, a certain amount of the protein complex is required for successful analysis. Sample preparation is simple and does not require specifically purified protein complexes. However, one common pitfall is contamination of the sample during sample preparation for mass spectrometry-based protein identification. These contaminations in most cases include keratins which originate from dust, skin, or hair. Therefore, extra care such as wearing gloves and lab coats, filtrating aqueous buffers, and using high-purity solvents should be taken during sample preparation for mass spectrometry-based protein identification. Other contaminating proteins such as chaperones are usually introduced during protein purification, e.g., when using affinity tags. In these cases, protein purification should be improved, for instance by increasing washing steps. In any case, protein contaminations in the sample are easily identified during the database search by omitting the taxonomy filter (i.e., searching against proteins from all species). If only a few peptides are observed (i.e., a low protein coverage could be obtained), even though sufficient sample is available, it might be necessary to use a different proteinase during digestion. In general Trypsin yields a sufficient number of peptides; however, in some cases such as membrane proteins or membrane domains of proteins, the number of tryptic cleavage sites is reduced and other enzymes targeting hydrophobic amino acids are a better choice.
In terms of instrumentation, a particularly modified instrument is required for native mass spectrometry which maintains non-covalent interactions during transfer into the gas-phase. Several instrument types have been introduced, including Q-ToF and orbitrap&#160;instruments. While modified Q-ToF mass spectrometers are commercially available for native mass spectrometry since several years, the latter were only recently introduced and in most cases require specialized modification45. However, the application of high-resolution instruments allowed studying binding of multiple ligands and their quantification46,47 and is promising for future applications.
To identify cross-linked di-peptides by liquid chromatography-coupled mass spectrometry, standard procedures with few modifications can be applied. However, the database search is the limiting factor as specialized software can rarely deal with large databases, and reduced databases containing the protein subunits of the complexes are required. Recent studies used mass spectrometry-cleavable cross-linkers targeting protein interactions in entire cell lysates48,49. The use of chemical cross-linkers that fragment in tandem mass spectrometry experiments mostly yields linear peptides (modified by the cross-linker), which can be identified by further fragmentation and database searching of linear peptides, and this reduces search time and computational search space. However, to perform these experiments, an ion trap mass analyzer or a hybrid mass spectrometer with an ion trap is required. In general, as false-positives are an important issue, mass spectra of cross-linked peptides are often validated manually by the quality of their fragment spectra which extends data analysis time enormously. Developing robust scoring systems that can be applied without further validation steps are therefore potential future applications. One way to improve data analysis and to reduce the number of false-positives was the introduction of false discovery rate calculations and their application to cross-linking data sets50.
In general, the techniques described here can be complemented with further mass spectrometry techniques (e.g., covalent labeling) to increase the output from the analysis. Other modifications and improvements of the protocols can be easily implemented. As such comparative cross-linking34 unravels conformational changes in the protein assembly. Further developments in native mass spectrometry nowadays allow the analysis of membrane proteins51,52 and their interactions with lipids28,52,53,54. New developments of high-resolution mass spectrometers for native mass spectrometry have extended the application and ligand binding, e.g., binding of lipids to membrane proteins, can now be included in the analysis45,46. In combination with computational modeling approaches, these techniques can deliver structural models of varying resolution55. If no crystal structures are available for the intact complex or single subunits, mass spectrometry can deliver first insights into protein interactions and the topology of the unknown complex. Depending on the techniques used and the results obtained, low-resolution models of the unknown complex can be obtained56,57,58. If crystal structures or homology models are available, the structural information received from mass spectrometry can yield even near-native models59.
In comparison with other structural techniques, mass spectrometry has the advantage that it requires low sample amounts, it can deal with heterogeneous samples and is applicable to protein complexes of unlimited size. In addition, mass spectrometry allows the investigation of dynamic protein systems. Different populations of the protein or protein complex that exist in solution are usually analyzed together and therefore, unlike with other structural techniques which require selection of certain populations, all conformations are maintained during analysis and are assessable in one experiment. Quantitative cross-linking approaches were recently introduced34,60,61 and are promising for future applications describing conformational changes under different conditions.

The structural investigation of protein assemblies has become particularly important for the understanding of cellular processes. Consequently, many, techniques have been developed and improved in structural biology1. However, these techniques are sometimes limited in their application due to size, flexibility, or heterogeneity of the protein complexes under investigation. Mass spectrometry can deal with these challenges and, therefore, emerged as a powerful tool in structural biology2,3,4,5. The greatest advantage of mass spectrometry, however, is the ability to unambiguously identify proteins even in complex and heterogeneous mixtures6. To this end, proteins are usually digested with endoproteinases and the resulting peptide mixture is separated by liquid chromatography and directly eluted into the mass spectrometer. Peptide masses are subsequently determined and precursor ions are selected for further fragmentation of the peptides. Proteins are then identified by searching peptide and corresponding fragment masses against a known database. This procedure not only allows the identification of peptides/proteins but also their post-translational modifications which cause a mass shift of the precursor peptides and the fragment ions carrying the modification7. Some of the many techniques in structural mass spectrometry are based on this principle4,8. For instance, labeling techniques such as hydrogen deuterium exchange9,10, chemical labeling strategies11,12, or hydroxyl radical foot printing13,14, give insights into the surface accessibility of the proteins under certain conditions.
Another technique is (chemical) cross-linking involving the covalent linkage of amino acids in close proximity through their functional groups. For this, chemical reagents, UV-activatable reagents, or amino acids are employed15,16. After cross-linking, the proteins are usually hydrolyzed with proteases and cross-linked di-peptides are analyzed by liquid chromatography-coupled mass spectrometry. The identification of cross-linking products by database searching, however, requires the use of specialized software which concatenate peptide sequences of various proteins and disparate regions17,18,19. The use of chemical cross-linkers has the advantage that it can be employed to almost every protein complex of interest and does not require incorporation of UV-activatable amino acids, which can only be achieved when expressing the protein of interest in host cells. As such, cross-linking is a versatile tool and was successfully employed in many structural studies of even large protein assemblies20.
Mass spectrometry of intact protein complexes (sometimes called &#39;native&#39; mass spectrometry), on the other hand, involves the analysis of intact proteins and protein complexes without hydrolysis into peptides. It reveals composition, heterogeneity, stoichiometry, topology, and subunit interactions of protein complexes21,22. In combination with ion mobility, native mass spectrometry further allows determination of their conformation23,24. This makes it a powerful tool for the structural investigation of protein complexes that are difficult to assess by conventional structural techniques. However, native mass spectrometry requires analysis buffers which maintain non-covalent protein interactions during electrospray ionization. This is usually achieved by using aqueous, volatile buffers such as ammonium acetate25. In addition, instrument modifications are necessary to prevent dissociation during transmission into the gas phase of the mass spectrometer26. Applied in this way, many (large) protein complexes were analyzed. Impressive examples are the studies of intact ribosomes27, ATP synthases28, or viruses29.
The combination of native mass spectrometry and cross-linking proved particularly successful in previous studies. For instance, the stoichiometries of chaperone complexes, including an unexpected Hsp70 dimer, could be obtained from mass spectrometry experiments of the intact protein complexes, while chemical cross-linking revealed the arrangements of the proteins in the assemblies30,31. In a different study, the effects of post-translational modifications on an intact ATP synthase complex were studied. Native mass spectrometry provided insights into the protein complex stability in the presence or absence of phosphorylation or acetylation32,33. A comparative cross-linking strategy34 then revealed conformational changes of the protein complex under the different conditions.
Here, we provide the protocols for protein identification by mass spectrometry, (chemical) cross-linking, and native mass spectrometry, including data analysis and interpretation (Figure 1). The combination of complementary results obtained from these methods with the help of two well-characterized protein complexes is demonstrated35. Our protocol can be applied to any protein assembly which can be purified at certain purity and concentration. The approach is in some cases limited by data analysis of cross-linking data, i.e., the size of the database employed, which determines the required search space and time. In addition, manual validation of identified cross-links is often required and further reduces the output. Native mass spectrometry is mostly limited by the sample quality, e.g., buffers and adducts used during purification and the possibility to exchange them by aqueous and volatile buffers. However, protein complexes purified for structural analysis usually have the quality required for successful analysis with our protocols.

<table><tbody><tr><td>&lt;strong&gt;Chemicals, Reagents, Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES)</td><td>Sigma Aldrich</td><td>H4034</td><td>buffer</td></tr><tr><td>Acetic acid</td><td>Sigma Aldrich</td><td>695092</td><td>pH</td></tr><tr><td>Acetonitrile Optima LC/MS</td><td>Fisher Chemicals</td><td>A955-1</td><td>solvent</td></tr><tr><td>Amicon Ultra centrifugal devices (different MWCO)</td><td>Millipore</td><td>i.e. UFC500396</td><td>buffer exchange, concentration</td></tr><tr><td>Ammonium acetate solution</td><td>Sigma Aldrich</td><td>A2706</td><td>native MS</td></tr><tr><td>Ammonium bicarbonate</td><td>Sigma Aldrich</td><td>9830</td><td>in-gel digestion</td></tr><tr><td>Ammonium solution</td><td>Sigma Aldrich</td><td>9859</td><td>pH</td></tr><tr><td>Bis(Sulfosuccinimidyl)suberate-d0 (BS3-d0)</td><td>Thermo Scientific</td><td>21590</td><td>cross-linker</td></tr><tr><td>Bis(Sulfosuccinimidyl)suberate-d4 (BS3-d4)</td><td>Thermo Scientific</td><td>21595</td><td>cross-linker</td></tr><tr><td>Caesium iodide</td><td>Sigma Aldrich</td><td>203033</td><td>calibration</td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>C5670</td><td>in-gel digestion</td></tr><tr><td>Capillaries (1.0 OD &amp;times; 0.78 ID &amp;times; 100 L mm)</td><td>Harvard Apparatus</td><td>30-0038</td><td>native MS</td></tr><tr><td>Disuccinimidyl suberate (DSS)</td><td>Thermo Scientific</td><td>21655</td><td>cross-linker</td></tr><tr><td>DL-Dithiothreitol</td><td>Sigma Aldrich</td><td>D5545</td><td>in-gel digestion</td></tr><tr><td>DMSO (dimethyl sulfoxide)</td><td>Sigma Aldrich</td><td>D8418</td><td>solvent</td></tr><tr><td>Ethanol</td><td>Fisher Chemicals</td><td>BP2818</td><td>solvent</td></tr><tr><td>Formic acid Optima LC/MS</td><td>Fisher Chemicals</td><td>A117-50</td><td>solvent supplement</td></tr><tr><td>Instant Blue Coomassie staining solution</td><td>expedeon</td><td>ISB1L</td><td>staining solution for gel electrophoresis</td></tr><tr><td>Invitrogen NuPAGE 4-12% Bis-Tris gels (1 mm, 10 well)</td><td>life technologies</td><td>NP0323BOX</td><td>gel electrophoresis</td></tr><tr><td>Iodoacetamide</td><td>Sigma Aldrich</td><td>I1149</td><td>In-gel digestion</td></tr><tr><td>Isopropyl alcohol, HPLC grade</td><td>Fisher Chemicals</td><td>P750717</td><td>solvent</td></tr><tr><td>Methanol</td><td>Fisher Chemicals</td><td>A456-212</td><td>solvent</td></tr><tr><td>Micro BioSpin 6 columns</td><td>BioRad</td><td>732-6222</td><td>buffer exchange</td></tr><tr><td>NuPAGE Antioxidant</td><td>invitrogen</td><td>NP0005</td><td>running buffer, gel electrophoresis</td></tr><tr><td>NuPAGE LDS Sample Buffer (4 &amp;times;)</td><td>invitrogen</td><td>NP0007</td><td>sample loading buffer (non-reducing) for gel electrophoresis</td></tr><tr><td>NuPAGE MES SDS Running buffer</td><td>life technologies</td><td>NP0001</td><td>gel electrophoresis</td></tr><tr><td>NuPAGE MOPS SDS Running buffer</td><td>life technologies</td><td>NP0001</td><td>gel electrophoresis</td></tr><tr><td>NuPAGE Sample Reducing Agent (10 &amp;times;)</td><td>invitrogen</td><td>NP0004</td><td>reducing Agent for gel electrophoresis</td></tr><tr><td>PBS - phosphate buffered saline</td><td>Sigma Aldrich</td><td>P4417</td><td>buffer</td></tr><tr><td>SeeBlue Plus2 Prestained Standard Protein Marker</td><td>invitrogen</td><td>LC5925</td><td>prestained Protein Marker for gel electrophoresis</td></tr><tr><td>Sodium acetate</td><td>Sigma Aldrich</td><td>S2889</td><td>ethanol precipitation</td></tr><tr><td>Tris(hydroxymethyl)aminomethane (Tris)</td><td>Sigma Aldrich</td><td>252859</td><td>buffer</td></tr><tr><td>Trypsin</td><td>Sigma Aldrich</td><td>TRYPSEQ-RO (11418475001)</td><td>in-gel digestion</td></tr><tr><td>Vivaspin centrifugal devices (different MWCO)</td><td>Sartorius</td><td>i.e. VS0101</td><td>buffer exchange, concentration</td></tr><tr><td>Water for HPLC</td><td>Sigma Aldrich</td><td>270733-2.5L-M</td><td>solvent</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge Heraeus Fresco 21, bench-top centrifuge</td><td>Thermo Scientific</td><td>75002425</td><td></td></tr><tr><td>Flaming / Brown Micropipette puller Model P-1000</td><td>Sutter Instruments</td><td>P-1000</td><td></td></tr><tr><td>Gelelectrophoresis chamber Xcell SureLock MiniCell</td><td>Invitrogen</td><td>EI0001</td><td></td></tr><tr><td>Gold Coater Quorum Q150R S</td><td>Quorum Technologies Ltd.</td><td>Q150RS</td><td></td></tr><tr><td>Horizontal gel shaker Rotamax 120 T</td><td>Heidolph Instruments</td><td>544-41200-00</td><td></td></tr><tr><td>Q-TOF Ultima mass spectrometer, MS Vision high mass upgrade</td><td>Waters (Micromass)</td><td>-</td><td></td></tr><tr><td>reversed-phase C18 analytical column Acclaim PepMap (C18, 75 mm I.D., 50 cm, 3 mm pore size)</td><td>Thermo Scientific</td><td>164570</td><td></td></tr><tr><td>reversed-phase C18 pre-column (C18, 150 mm I.D., 2 cm, 5 mm pore size)</td><td>Thermo Scientific</td><td>164213</td><td></td></tr><tr><td>scalpel</td><td>Fisher Scientifc</td><td>10463989</td><td></td></tr><tr><td>SpeedVac SPD121P vacuum centrifuge</td><td>Thermo Scientific</td><td>SPD121DP-230</td><td></td></tr><tr><td>Thermomixer C</td><td>Eppendorf</td><td>5382000015</td><td></td></tr><tr><td>Tweezers AA</td><td>Sigma Aldrich</td><td>Z680184-1EA</td><td></td></tr><tr><td>Ultrasonic cleaner USC-TH, sonication bath</td><td>VWR</td><td>142-0084</td><td></td></tr><tr><td>UltiMate Dionex 3000 nano-LC system, coupled to Q-Exactive plus hybrid mass spectrometer (nano-ESI source)</td><td>Thermo Scientific</td><td>0726030+</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Software, Software Tools, Database search&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mascot</td><td>www.matrixscience.com</td><td></td><td></td></tr><tr><td>Massign</td><td>http://massign.chem.ox.ac.uk</td><td></td><td></td></tr><tr><td>MassLynx</td><td>Micromass</td><td></td><td></td></tr><tr><td>MassMatrix</td><td>Xu, H. J Proteome Res. 9 (2010)</td><td></td><td></td></tr><tr><td>MaxQuant</td><td>Cox, J. Nat. Biotechn. 26 (2008)</td><td></td><td></td></tr><tr><td>pLink</td><td>Yang, B. Nat Methods 9 (2012)</td><td></td><td></td></tr><tr><td>pXtract conversion tool</td><td>http://pfind.ict.ac.cn/downloads.html</td><td></td><td></td></tr><tr><td>UniDec</td><td>Marty, M.T. Anal. Chem. 87 (2015)</td><td></td><td></td></tr><tr><td>XCalibur</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>XiNET</td><td>http://crosslinkviewer.org</td><td></td><td></td></tr><tr><td>XLinkX</td><td>Liu, F. Curr Op Struct Biol 35 (2015)</td><td></td><td></td></tr><tr><td>xQuest</td><td>Rinner, O. Nat Methods 5 (2008)</td><td></td><td></td></tr><tr><td>Xvis</td><td>https://xvis.genzentrum.lmu.de</td><td></td><td></td></tr></tbody></table>

combining chemical cross-linking and mass spectrometry of intact protein complexes to study the architecture of multi-subunit protein assemblies

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is therefore fundamental for the understanding of cellular processes. However, many protein complexes are dynamic assemblies and are not accessible by conventional structural techniques. Mass spectrometry contributes to the structural investigation of these assemblies, and particularly the combination of various mass spectrometric techniques delivers valuable insights into their structural arrangement.
In this article, we describe the application and combination of two complementary mass spectrometric techniques, namely chemical cross-linking coupled with mass spectrometry and native mass spectrometry. Chemical cross-linking involves the covalent linkage of amino acids in close proximity by using chemical reagents. After digestion with proteases, cross-linked di-peptides are identified by mass spectrometry and protein interactions sites are uncovered. Native mass spectrometry on the other hand is the analysis of intact protein assemblies in the gas phase of a mass spectrometer. It reveals protein stoichiometries as well as protein and ligand interactions. Both techniques therefore deliver complementary information on the structure of protein-ligand assemblies and their combination proved powerful in previous studies.

The structural analysis of proteins and the complexes they form is fundamental for understanding their function. Mass spectrometry considerably contributes to the structural investigation in that it can be applied to almost every complex of interest irrespective of size or sample heterogeneity. We exemplify the protocol by using two well-characterized protein complexes; first, the hetero-dodecamer RvB1/B2 from C. thermophilum and second, the hetero-octameric CPS complex from E. coli.
First, we identified the protein components of the two complexes. For this, the proteins were separated by SDS-PAGE (Figure 2) and gel bands were cut from the gel. After in-gel digestion of the proteins, the peptide mixture was analyzed by liquid chromatography-coupled mass spectrometry and peptide and fragment masses were subjected to database searching. Following this workflow, we identified all protein subunits of the two complexes with high confidence, i.e., a high number of peptides with reasonable peptide scores was observed yielding high sequence coverage for all protein subunits (Table 1).
We then analyzed the intact RvB1/B2 complex by native mass spectrometry (Figure 3A). The mass spectrum revealed two species, one at approximately 8,000 m/z and another species at approximately 11,000-12,000 m/z. The calculated masses for these species correspond to the hexameric (RvB1)3(RvB2)3 ring (approximately 310 kDa) and the dodecameric double-ring (RvB1)6(RvB2)6 (approximately 620 kDa). Both peak series show two populations; these originate from a mixture of His-tagged and untagged RvB2 subunits in the complexes. A crystal structure for the RvB1/B2 complex was previously obtained36 and shows the arrangement of the double-ring (Figure 3B). The native mass spectrum therefore confirms the stoichiometry of the intact dodecamer and furthermore reveals a stable sub-complex. In addition, co-existing populations are identified.
To identify protein interaction sites in the RvB1/B2 complex, we chemically cross-linked the purified complex with BS3 cross-linker. We first titrated the amount of BS3 during the cross-linking reaction to determine the optimal concentration. BS3 is amine-specific and covalently links lysine side chains as well as the N-termini of proteins. The cross-linking reaction was followed by SDS-PAGE (Figure 2A). The non-cross-linked complex showed both RvB1 and RvB2 subunits. Adding BS3 to the reaction mixture caused covalent linkage of the proteins resulting in protein bands at higher molecular weight. The SDS gel shows that increasing amounts of BS3 yield higher amounts of cross-linked species while non-cross-linked protein subunits are reduced. We then cut the protein bands from the gel and followed the protocol provided above to identify protein interaction sites. An example spectrum of a cross-linked di-peptide is shown (Figure 3C). The spectrum shows y-ion series of both peptides confirming this protein interaction. In total, we obtained 14 protein interactions, including four cross-links between subunits RvB1 and RvB2 and two cross-links between two copies of RvB2 (Table 2). The results from BS3 cross-linking are visualized in an interaction network (Figure 3D) showing intra-molecular interactions as well as interactions between different subunits. Intra-molecular cross-links suggest that both RvB1 and RvB2 subunits fold in a way that N- and C-terminal domains are in close proximity. Note, that intra-molecular interactions cannot be distinguished from inter-molecular interactions of the same subunit in this case. Inter-molecular cross-links between the two subunits were also observed. Of these, two inter-molecular cross-links between RvB1 and RvB2 could be visualized in the structure validating the cross-linking approach. The other inter-molecular cross-links are located in flexible loops which are not included in the crystal structure. We also identified two cross-links in RvB2 containing the same peptide sequences. These cross-links can unambiguously be classified as inter-molecular as they must originate from two copies of the same protein (Figure 3D). Our cross-linking experiments reveal protein interaction sites within the complex but also within the protein subunits providing insights into their structural arrangement that could also be confirmed by the existing crystal structure (Figure 3B).
The second protein complex that we studied was CPS. The native mass spectrum (Figure 4A) revealed three protein complexes between 6,000 and 12,000 m/z. The largest complex of 640 kDa corresponds to the intact hetero-octamer. The smaller complexes represent two sub-complexes; the dimer of the small and large CPS subunits (160 kDa) and a hetero-tetramer containing two copies of each subunit (320 kDa). These sub-complexes deliver first insights into the protein assembly; i.e., the large and small subunits are in direct contact (as revealed by the hetero-dimer) and the tetramer might be a product of two dimers. To gain more information on the structural arrangement in the intact CPS complex, we performed tandem mass spectrometry (MS/MS) of the hetero-octamer and the hetero-tetramer. In both cases, the small subunit dissociated from the precursor suggesting that the small subunit is located in the periphery of the assembly (Figure 4B). Indeed, the small subunit is peripheral in the available crystal structure (Figure 4C)44.
Chemical cross-linking using the BS3 cross-linker was also performed. Using increasing amounts, the covalent linkage of the CPS subunits was enhanced. After digestion of the proteins and analysis of the peptides as described above, many protein interactions within the large subunit and one cross-link in the small subunit were obtained (Table 2). In addition, similar to the RvB1/B2 complex, we found two inter-molecular cross-links between two copies of the large CPS subunit. These cross-links place the two large subunits facing each other at their C-terminal sides. In a previous study, combining structural mass spectrometry and computational modeling35, we identified three additional interactions in the large subunit which most likely originate from the interface of two copies of the large subunit validated by the crystal structure and the obtained model (Figure 4C and Table 2). These interactions allow the arrangement of the CPS core complex consisting of four large subunits. However, no inter-subunit cross-links between the small and the large subunits were observed. By inspecting the available crystal structure (Figure 4C), it becomes apparent that the interaction surface between the tetrameric core of the complex, consisting of the large subunit, and the peripheral small subunits is very small, which might explain the absence of inter-subunit interactions. This is confirmed by native mass spectrometry which showed that the small subunit readily dissociates from the intact complexes most likely due to a small binding interface. Nonetheless, protein interactions in the CPS complex combined from chemical cross-linking and native mass spectrometry allow deducing their structural arrangement (Figure 4D).
Taken together, the combination of native mass spectrometry and chemical cross-linking coupled with mass spectrometric identification of cross-linked peptides, allows reconstitution of the structural arrangement of both example complexes. While chemical cross-linking revealed arrangement of the protein subunits, for instance the interactions between RvB1 and RvB2 or within the tetrameric core of CPS, native mass spectrometry delivered protein stoichiometries of the intact complexes and prevalent subcomplexes. In the case of CPS, for which no inter-molecular interactions between the two subunits could be observed by chemical cross-linking, native mass spectrometry suggests that each large subunit interacts with one small subunit (Figure 4D). Tandem mass spectrometry proposed the peripheral location of the small subunit in the complex and a small interface between both subunits.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56747/56747fig1.jpg" /> 
Figure 1: Workflow of native mass spectrometry and cross-linking. Both techniques deliver complementary results. While native mass spectrometry reveals stoichiometries and interaction modules, cross-linking gives insights into the protein interaction sites within the complexes. Note that chemical cross-linking only reveals binary interactions. (A) The first step in native mass spectrometry is buffer exchange to a volatile and aqueous buffer using filter units or gel filtration columns. Mass spectrometry of the intact protein complexes then reveals their stoichiometry. In tandem mass spectrometry experiments, peripheral subunits are dissociated. (B) For chemical cross-linking, the protein complex is incubated with a cross-linking reagent. The cross-linked proteins are then digested into peptides which are subsequently analyzed by liquid chromatography-coupled mass spectrometry. <a href="https://cloudflare.jove.com/files/ftp_upload/56747/56747fig1large.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/56747/56747fig2.jpg" /> 
Figure 2: SDS-PAGE of cross-linked RvB1/B2 (A) and CPS (B) complexes. (A) 2.5 &#181;M RvB1/B2 were loaded per gel lane. The concentration of BS3 was varied. Non-cross-linked RvB1/B2 shows the two protein subunits at approximately 50 kDa. Addition of BS3 caused covalent linkage of the protein subunits resulting in protein bands at higher molecular weight. The amount of cross-linked species is increased with higher BS3 concentrations. Optimal cross-linking conditions are highlighted (red). (B) 10 &#181;M CPS were loaded per gel lane. The large (90 kDa) and small (40 kDa) CPS subunits are obtained. Addition of BS3 caused covalent linkage of the protein subunits resulting in protein bands at higher molecular weight. Optimal cross-linking conditions are highlighted (red). <a href="https://cloudflare.jove.com/files/ftp_upload/56747/56747fig2large.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/56747/56747fig3.jpg" /> 
Figure 3: Native mass spectrometry and chemical cross-linking of RvB1/B2 complex. (A) The native mass spectrum reveals two species of RvB1/B2; the intact dodecamer (i.e., (RvB1)6(RvB2)6) at approximately 11,000 to 12,000 m/z and the hexameric ring (RvB1)3(RvB2)3 at approximately 8,000 m/z. Both species show two populations resulting from His-tagged and untagged RvB2. The spectrum has been modified from35. (B) The crystal structure of RvB1/B2 is shown (PDB ID 4WVY). Alternating RvB1 and RvB2 subunits form two hexameric rings. (C) Fragmentation spectrum of a cross-linked di-peptide. The N-terminus of RvB1 was cross-linked with K23 of RvB1. y-ion series were obtained for both peptides (red and cyan). (D) Intra- and inter-protein interactions obtained in the RvB1/B2 complex. Intra-cross-links are shown in red, inter-cross-links are shown in blue. The insert shows two inter-molecular cross-links between RvB1 and RvB2 subunits which could be visualized in the crystal structure (green, insert). Interactions that originate from two RvB2 copies are shown as blue dotted lines. <a href="https://cloudflare.jove.com/files/ftp_upload/56747/56747fig3large.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/56747/56747fig4.jpg" /> 
Figure 4: Native mass spectrometry and chemical cross-linking of CPS. (A) The native mass spectrum of CPS shows three complexes. The hetero-dimer (160 kDa), hetero-tetramer (320 kDa) and the hetero-octamer (640 kDa). The spectrum has been modified from35. (B) Tandem mass spectrometry of the tetrameric and octameric CPS complex revealed dissociation of the small CPS subunit. (C) The crystal structure of CPS is shown (PDB ID 1BXR). The large subunits form a tetrameric core and the small subunits are located in the periphery of the complex. Inter-molecular cross-links between two copies of the large subunit are shown (green). (D) Interactions of the large and small CPS subunits. Native mass spectrometry revealed subcomplexes and suggests a peripheral location of the small subunit. Chemical cross-links indicate arrangements in the tetrameric core of CPS. <a href="https://cloudflare.jove.com/files/ftp_upload/56747/56747fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Database search results. The proteins were identified by liquid chromatography-coupled mass spectrometry and database searching. The protein names, accession number, and description as well as protein mass are given. The protein score, number of observed spectra per protein, and the number of observed peptide sequences are listed. The five peptides with highest Mascot peptides scores are listed for each protein subunit. <a href="http://cloudflare.jove.com/files/ftp_upload/56747/Table1.xlsx">Please click here to download this file.</a>
Table 2: Cross-links observed in RvB1/B2 and CPS. The subunits of the complexes and the cross-linked residues are given. The type of cross-link (intra- or inter-molecular) was revealed from overlapping peptide sequences or a previous study35. <a href="http://cloudflare.jove.com/files/ftp_upload/56747/Table2.xlsx">Please click here to download this file.</a>

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Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

The architecture of protein complexes is essential for their function. Combining various mass spectrometric techniques proved powerful to study their assembly. We provide protocols for chemical cross-linking and native mass spectrometry and show how these complementary techniques help to elucidate the architecture of multi-subunit protein assemblies.

Proteins interact with their ligands to form active and dynamic assemblies which carry out various cellular functions. Elucidating these interactions is ...

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Protein Structure

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19.6K Views.  Martin Luther University Halle-Wittenberg. De architectuur van eiwitcomplexen is essentieel voor hun functie. Diverse massaspectrometrische technieken combineren bewezen krachtige te bestuderen van hun vergadering. Wij bieden protocollen voor chemische dwarsbinding en inheemse massaspectrometrie en laten zien hoe deze complementaire technieken helpen ophelderen van de architectuur van multi subeenheid eiwit assemblages.

Video: Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Biochemistry

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this work, the pre-endosomal anthrax toxin is assembled and transitioned into the endosomal complex. First, the N-terminal domain of a cysteine mutant lethal factor (LFN) is attached to a biolayer interferometry (BLI) biosensor through disulfide coupling in an optimal orientation, allowing protective antigen (PA) prepore to bind (Kd 1 nM). The optimally oriented LFN-PAprepore complex then binds to soluble capillary morphogenic gene-2 (CMG2) cell surface receptor (Kd 170 pM), resulting in a representative anthrax pre-endosomal complex, stable at pH 7.5. This assembled complex is then subjected to acidification (pH 5.0) representative of the late endosome environment to transition the PAprepore into the membrane inserted pore state. This PApore state results in a weakened binding between the CMG2 receptor and the LFN-PApore and a substantial dissociation of CMG2 from the transition pore. The thio-attachment of LFN to the biosensor surface is easily reversed by dithiothreitol. Reduction on the BLI biosensor surface releases the LFN-PAprepore-CMG2 ternary complex or the acid transitioned LFN-PApore complexes into microliter volumes. Released complexes are then visualized and identified using electron microscopy and mass spectrometry. These experiments demonstrate how to monitor the kinetic assembly/disassembly of specific protein complexes using label-free BLI methodologies and evaluate the structure and identity of these BLI assembled complexes by electron microscopy and mass spectrometry, respectively, using easy-to-replicate sequential procedures.

Here we present a protocol to monitor the assembly and disassembly of the anthrax toxin using biolayer interferometry (BLI). Following assembly/disassembly on the biosensor surface, the large protein complexes are released from the surface for visualization and identification of components of the complexes using electron microscopy and mass spectrometry, respectively.

In vivo, proteins are often part of large macromolecular complexes where binding specificity and dynamics ultimately dictate functional outputs. In this ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Quaternary Structure Modeling Through Chemical Cross-Linking Mass Spectrometry: Extending TX-MS Jupyter Reports

Protein-protein interactions can be challenging to study yet provide insights into how biological systems function. Targeted cross-linking mass spectrometry (TX-MS), a method combining quaternary protein structure modeling and chemical cross-linking mass spectrometry, creates high-accuracy structure models using data obtained from complex, unfractionated samples. This removes one of the major obstacles to protein complex structure analysis because the proteins of interest no longer need to be purified in large quantities. Cheetah-MS web server was developed to make the simplified version of the protocol more accessible to the community. Considering the tandem MS/MS data, Cheetah-MS generates a Jupyter Notebook, a graphical report summarizing the most important analysis results. Extending the Jupyter Notebook can yield more in-depth insights and better understand the model and the mass spectrometry data supporting it. The technical protocol presented here demonstrates some of the most common extensions and explains what information can be obtained. It contains blocks to help analyze tandem MS/MS acquisition data and the overall impact of the detected XLs on the reported quaternary models. The result of such analyses can be applied to structural models that are embedded in the notebook using NGLView.

Targeted cross-linking mass spectrometry creates quaternary protein structure models using mass spectrometry data acquired using up to three different acquisition protocols. When executed as a simplified workflow on the Cheetah-MS web server, the results are reported in a Jupyter Notebook. Here, we demonstrate the technical aspects of how the Jupyter Notebook can be extended for a more in-depth analysis.

Protein-protein interactions can be challenging to study yet provide insights into how biological systems function. Targeted cross-linking mass ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

Chemistry

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Immunology and Infection

Covalent Labeling with Diethylpyrocarbonate for Studying Protein Higher-Order Structure by Mass Spectrometry

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool for this purpose, especially for protein systems that are difficult to study by traditional methods. To study a protein&#39;s structure by MS, specific chemical reactions are performed in solution that encode a protein&#39;s structural information into its mass. One particularly effective approach is to use reagents that covalently modify solvent accessible amino acid side chains. These reactions lead to mass increases that can be localized with residue-level resolution when combined with proteolytic digestion and tandem mass spectrometry. Here, we describe the protocols associated with use of diethylpyrocarbonate (DEPC) as a covalent labeling reagent together with MS detection. DEPC is a highly electrophilic molecule capable of labeling up to 30% of the residues in the average protein, thereby providing excellent structural resolution. DEPC has been successfully used together with MS to obtain structural information for small single-domain proteins, such as &#946;2-microglobulin, to large multi-domain proteins, such as monoclonal antibodies.

The experimental procedures for performing diethylpyrocarbonate-based covalent labeling with mass spectrometric detection are described. Diethylpyrocarbonate is simply mixed with the protein or protein complex of interest, leading to the modification of solvent accessible amino acid residues. The modified residues can be identified after proteolytic digestion and liquid chromatography/mass spectrometry analysis.

Characterizing a protein&#39;s higher-order structure is essential for understanding its function. Mass spectrometry (MS) has emerged as a powerful tool ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Combining Chemical Cross-linking and Mass Spectrometry of Intact Protein Complexes to Study the Architecture of Multi-subunit Protein Assemblies

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