This work was supported by the Madison and Lila Self Graduate Fellowship (AJM), NIH 5T32GM008359 (PTO), KUMC Biomedical Research Training Program (PTO), NIH R01AI090085 (MTF), KU Bridging Funds and the Biomolecule Interaction Technologies Center (BITC) Grant (CT and MTF). The authors would like to thank Barbara Fegley at KUMC Electron Microscopy Core for assistance with TEM image acquisition.

1. Assembly of Defined Macromolecular Complexes on Biolayer Interferometry (BLI) Biosensor Surfaces
<ol>
	<li>Assembly of prepore anthrax toxin complex on a PDEA-modified amine reactive biosensor surface
	<ol>
		<li>Hydrate an amine reactive second generation (AR2G) BLI biosensor tip in 250 &#181;L of water for ten minutes.</li>
		<li>Program step times, listed in Table 1, on the software controlling the BLI unit (see Table of Materials).</li>
		<li>Start the BLI run by immersing the biosensor tip in 250 &#181;L water for 30 s to measure an initial baseline of biosensor thickness and density.</li>
		<li>Activate the biosensor by immersing the tip into 250 &#181;L 50 mM NHS (N-hydroxysuccinimide) and 200 mM EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)) for 7 minutes.</li>
		<li>Immerse the activated biosensor in 50 mM PDEA (2-(2-pyridinyldithio)ethanamine) dissolved in 0.1 M borate buffer (pH 8.5) for 5 minutes to generate an activated thiol-reactive surface.</li>
		<li>Immerse the activated thiol-reactive biosensor into 250 &#181;L of solution containing 100 nM E126C LFN in 10 mM sodium acetate pH 5.0, 100 mM NaCl, buffer for 5 minutes.</li>
		<li>Immerse the LFN tip in 50 mM L-cysteine, 1 M NaCl, 0.1 M sodium acetate, pH 5.0, for 4 minutes, to quench any remaining reactive free thiol-reactive groups.</li>
		<li>Immerse the quenched LFN tip in 50 mM Tris, 50 mM NaCl for 2 minutes to establish buffer baseline.</li>
		<li>Immerse the quenched LFN tip into 0.5 &#181;M protective antigen prepore (PAprepore), 50 mM Tris, 50mM NaCl for 5 minutes to create the LFN-PAprepore complex.</li>
		<li>Once PAprepore is associated, remove the tip from the PAprepore solution and immerse the tip into 50 mM Tris, 50 mM NaCl for 30 seconds to wash away any non-specifically bound PAprepore.</li>
		<li>Immerse the LFN-PAprepore complex into 0.5 &#181;M CMG2 receptor (without the transmembrane domain), 50 mM Tris, 50 mM NaCl, for 5 minutes.</li>
		<li>Immerse the LFN-PAprepore-CMG2 complex into 50 mM Tris, 50 mM NaCl for 30 seconds to wash away any unbound CMG2 to form pre-endosomal complex.</li>
		<li>For EM analysis, release the LFN-PAprepore-CMG2 complex from the biosensor tip by immersing the tip into 5 &#181;L of 50 mM DTT, 50 mM Tris, 50 mM NaCl, inside a PCR tube.</li>
		<li>For tandem MS analysis of the peptides from the complex, release the LFN-PAprepore-CMG2 complex from the biosensor tip by immersing the biosensor into 5 &#181;L 50 mM DTT, 6 M GuHCl (keratin-free), 25 mM ammonium bicarbonate pH 8.0 inside a PCR tube. This is performed on a different biosensor than the one used for EM analysis.</li>
	</ol>
	</li>
	<li>Assembly of pore anthrax toxin complex on PDEA-modified amine reactive biosensor surface
	<ol>
		<li>To view the complex after pH transition, perform steps 1.1.1-1.1.12 in the previous section to generate the LFN-PAprepore-CMG2 complex.</li>
		<li>Immerse the biosensor tip into a 10 mM acetate pH 5.0 for 5 minutes to initiate the transition of the PAprepore to PApore transition. This transition is indicated by increasing amplitude (approximately 0.2 nm) followed by a larger amplitude decline that is hypothesized to be substantial or complete receptor dissociation due to diminished binding affinity.</li>
		<li>Immerse LFN-PApore tip in 50 mM Tris, 50 mM NaCl, pH 8.0, for 30 seconds to wash off acidic buffer.</li>
		<li>For the EM analysis sample, immerse the biosensor tip into a solution containing 1.25 mM micelles (2.5 mM MSP1D1, 25 mM Na-cholate, 162.5 mM POPC) for 5 minutes to prevent aggregation in solution after disulfide release.</li>
		<li>For EM analysis, release the LFN-PApore-Micelle complex from the biosensor tip by immersing the tip into 5 &#181;L of 50 mM DTT, 50 mM Tris, 50 mM NaCl inside a PCR tube.</li>
		<li>For tandem MS analysis of the peptides from the complex, release the LFN-PApoore complex (no micelle) from the biosensor tip by immersing the biosensor into 5 &#181;L 50 mM DTT, 6 M GuHCl (keratin-free), 25 mM ammonium bicarbonate pH 8.0 inside a PCR tube. This is performed on a different biosensor than the one used for EM analysis.</li>
	</ol>
	</li>
</ol>
2. Visualizing and Validating Released Macromolecular Assemblies from BLI Biosensors by Negative Stain Electron Microscopy
<ol>
	<li>Glow discharge a carbon-coated Cu 300 grid. Typical glow discharge settings are 0.38 mBar stable atmosphere pressure, negative 15 mAmps, 20 seconds, then venting with air.</li>
	<li>Secure grid between a pair of clean tweezers.</li>
	<li>Pipette 4 &#181;L of released complex sample in PCR tube onto the grid and allow adsorption for 60s.</li>
	<li>Wick away remaining liquid with a filter paper wedge.</li>
	<li>Stain the grid by pipetting 5 &#181;L of 0.75% 0.02 micron filtered uranyl formate and wicking away excess stain after 5 seconds. Allow grid to dry at room temperature.</li>
	<li>View stained sample grids using a transmission electron microscope.</li>
</ol>
3. Identification of Complete Pre-endosomal Anthrax Toxin Complex (LFN-Paprepore-CMG2) and Transitioned Complex (LFN-Papore without CMG2) Using Mass Spectrometry.
<ol>
	<li>Dilute released samples to 20 &#181;L and incubate for 1 hour.</li>
	<li>Add 2 &#181;L of 55 mM iodoacetamide, 25 mM ammonium bicarbonate, pH 8.0, and incubate for 1 hour at room temperature in dark (covered with aluminum foil).</li>
	<li>Dilute the sample with 100 &#181;L of 25 mM ammonium bicarbonate, pH 8.0, to reduce the guanidine hydrochloride concentration below 1 M.</li>
	<li>Add 5 &#181;L of sequencing grade modified trypsin at 20 ng/&#181;L and incubate at 37 &#176;C overnight.</li>
	<li>Add glacial acetic acid to a final concentration of 5% to reduce the pH to &#60;3, then reduce the volume to 10 &#181;L in vacuum concentrator.</li>
	<li>Transfer the peptide solution to the sample plate on the autosampler of the nLC 1200 uHPLC.</li>
	<li>Load 5 &#181;L the peptide solution onto a uHPLC reversed phase column mounted on the ionization stage of the MS.</li>
	<li>Wash column with 15 &#181;L of 0.1% formic acid at a maximum rate of 5 &#181;L/min and/or maximum pressure of 800 psi.</li>
	<li>Elute peptides from the reversed-phase C18 column at a flow rate of 350 nL/min over a 90 min period using a lineal gradient of 5% to 40% of solvent B in A+B (solvent A: 0.1% formic acid; solvent B: 95% acetonitrile with 0.1% formic acid).</li>
	<li>Analyze eluting peptides on line using tandem MS.
	<ol>
		<li>Set the ionization source to 2500 volts and ion transfer temperature to 250 &#176;C.</li>
		<li>Operate the mass spectrometer under automatic control to perform continuously one MS scan followed by as many tandem MSMS as possible in a 3 s period, using CID and a normalized collision energy of 35.</li>
	</ol>
	</li>
	<li>Identify peptide and protein components using standard methods. For this work, two sets of peptide and protein analysis were performed.</li>
</ol>

<ol>
	<li>Bellon, S., Buchmann, W., Gonnet, F., Jarroux, N., Anger-Leroy, M., Guillonneau, F., Daniel, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyphenation+of+surface+plasmon+resonance+imaging+to+matrix-assisted+laser+desorption+ionization+mass+spectrometry+by+on-chip+mass+spectrometry+and+tandem+mass+spectrometry+analysis.">Hyphenation of surface plasmon resonance imaging to matrix-assisted laser desorption ionization mass spectrometry by on-chip mass spectrometry and tandem mass spectrometry analysis.</a> Analytical Chemistry. 81, 7695-7702 (2009).</li><li>Kim, Y. E., Yi, S. Y., Lee, C. S., Jung, Y., Chung, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+patterned+biochips+for+on-chip+immuno-MALDI-TOF+MS:+SPR+imaging+coupled+multi-protein+MS+analysis.">Gold patterned biochips for on-chip immuno-MALDI-TOF MS: SPR imaging coupled multi-protein MS analysis.</a> The Analyst. 137, 386-392 (2012).</li><li>Abdiche, Y., Malashock, D., Pinkerton, A., Pons, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+kinetics+and+affinities+of+protein+interactions+using+a+parallel+real-time+label-free+biosensor,+the+Octet.">Determining kinetics and affinities of protein interactions using a parallel real-time label-free biosensor, the Octet.</a> Analytical Biochemistry. 377, 209-217 (2008).</li><li>Abdiche, Y. N., Malashock, D. S., Pinkerton, A., Pons, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+blocking+assays+using+Octet,+ProteOn,+and+Biacore+biosensors.">Exploring blocking assays using Octet, ProteOn, and Biacore biosensors.</a> Analytical Biochemistry. , 172-180 (2009).</li><li>Naik, S., Brock, S., Akkaladevi, N., Tally, J., McGinn-Straub, W., Zhang, N., Gao, P., Gogol, E. P., Pentelute, B. L., Collier, R. J., Fisher, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+kinetics+of+the+pH-driven+transition+of+the+anthrax+toxin+prepore+to+the+pore+by+biolayer+interferometry+and+surface+plasmon+resonance.">Monitoring the kinetics of the pH-driven transition of the anthrax toxin prepore to the pore by biolayer interferometry and surface plasmon resonance.</a> Biochemistry. , (2013).</li><li>Akkaladevi, N., Hinton-Chollet, L., Katayama, H., Mitchell, J., Szerszen, L., Mukherjee, S., Gogol, E., Pentelute, B., Collier, R., Fisher, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+anthrax+toxin+pore:+Lethal-factor+complexes+into+lipid+nanodiscs.">Assembly of anthrax toxin pore: Lethal-factor complexes into lipid nanodiscs.</a> Protein Science. 22, 492-501 (2013).</li><li>Jin, H., Cantin, G. T., Maki, S., Chew, L. C., Resnick, S. M., Ngai, J., Retallack, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+periplasmic+production+of+human+granulocyte+colony-stimulating+factor+(G-CSF)+in+Pseudomonas+fluorescens.">Soluble periplasmic production of human granulocyte colony-stimulating factor (G-CSF) in Pseudomonas fluorescens.</a> Protein Expression and Purification. 78, 69-77 (2011).</li><li>Yamniuk, A. P., Edavettal, S. C., Bergqvist, S., Yadav, S. P., Doyle, M. L., Calabrese, K., Parsons, J. F., Eisenstein, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABRF-MIRG+benchmark+study:+Molecular+interactions+in+a+three-component+system.">ABRF-MIRG benchmark study: Molecular interactions in a three-component system.</a> Journal of Biomolecular Techniques. , 101-114 (2012).</li><li>Moore, J. D., Perez-Pardo, M. A., Popplewell, J. F., Spencer, S. J., Ray, S., Swann, M. J., Shard, A. G., Jones, W., Hills, A., Bracewell, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+biological+characterisation+of+a+sensor+surface+for+bioprocess+monitoring.">Chemical and biological characterisation of a sensor surface for bioprocess monitoring.</a> Biosensors &amp; Bioelectronics. 26, 2940-2947 (2011).</li><li>Feld, G. K., Thoren, K. L., Kintzer, A. F., Sterling, H. J., Tang, I. I., Greenberg, S. G., Williams, E. R., Krantz, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+unfolding+of+anthrax+lethal+factor+by+protective+antigen+oligomers.">Structural basis for the unfolding of anthrax lethal factor by protective antigen oligomers.</a> Nature Structural &amp; Molecular Biology. 17, 1383-1390 (2010).</li><li>Fairhead, M., Howarth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+biotinylation+of+purified+proteins+using+BirA.">Site-specific biotinylation of purified proteins using BirA.</a> Site-Specific Protein Labeling: Methods and Protocols. , 171-184 (2015).</li><li>Naik, S., Kumru, O. S., Cullom, M., Telikepalli, S. N., Lindboe, E., Roop, T. L., Joshi, S. B., Amin, D., Gao, P., Middaugh, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+structurally+altered+and+aggregated+states+of+therapeutically+relevant+proteins+using+GroEL+coupled+to+bio-layer+interferometry.">Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry.</a> Protein Science. 23, 1461-1478 (2014).</li><li>Bandyopadhyay, A., Gao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Iminoboronate-based+peptide+cyclization+that+responds+to+pH,+oxidation,+and+small+molecule+modulators.">Iminoboronate-based peptide cyclization that responds to pH, oxidation, and small molecule modulators.</a> Journal of the American Chemical Society. 138, 2098-2101 (2016).</li><li>Grantham, J., Llorca, O., Valpuesta, J. M., Willison, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partial+occlusion+of+both+cavities+of+the+eukaryotic+chaperonin+with+antibody+has+no+effect+upon+the+rates+of+beta-actin+or+alpha-tubulin+folding.">Partial occlusion of both cavities of the eukaryotic chaperonin with antibody has no effect upon the rates of beta-actin or alpha-tubulin folding.</a> The Journal of Biological Chemistry. 275, 4587-4591 (2000).</li><li>Ercius, P., Alaidi, O., Rames, M. J., Ren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+Tomography:+A+Three-Dimensional+Analytic+Tool+for+Hard+and+Soft+Materials+Research.">Electron Tomography: A Three-Dimensional Analytic Tool for Hard and Soft Materials Research.</a> Advanced Materials. 27, 5638-5663 (2015).</li></ol>

No disclosures at this time.

This demonstration illustrates how macromolecular complex formation can be easily monitored using biolayer interferometry, visualized using EM, and verified with MS, all with microvolumes in a short time span. Structural assembly and observed complexes follow the biologically relevant predictions, further validating this combined methodology. As mentioned in the results section, the key element for assembly success requires rationally engineered cysteine mutagenesis to ensure that the complex protein-protein interfaces are properly oriented away from the biosensor surface.
Previous systems have used BLI and MS techniques to evaluate protein binding of two and three component systems as well as integrity of receptor binding of expressed protein, but, in both instances, the methods were not developed to take advantage of the tandem EM/MS approach8,9. The only other interferometry system that combined MS analysis to help characterize interactions was a dual-polarization interferometry10. Unfortunately, this system is no longer available for general use. As mentioned in the introduction, there have been a number of studies completed where samples were formed on SPR-like biosensor surfaces and removed from MS analysis. None of those examples resulted in complexes visualized using EM.
The limitations of this sequential method can be numerous but are solvable. For the initial immobilization and therefore foundation step of the assembly, a lack of knowledge of the structural interaction surfaces would certainly impede progress associated with monitoring initial assembly phases. The lack of structure information can be addressed by designing an engineered foundation construct where attachment chemistries (e.g. sulfhydryl moiety for disulfide linkages/ His-tagged positioning) can be moved to various regions within the core assembly system. In the case of the anthrax toxin complex, it was fortunate that the structure of LFN bound to PAprepore is available11. Rational placement of the engineered cysteine was localized to regions away from the PAprepore binding face. With cysteine linkage chemistries, it is preferable that no other reactive cysteines are present on the protein surface.
There are various attachment chemistries which can be used to engineer specific attachment site on a protein&#39;s surfaces. One of the most popular specific attachments involves positioning a biotin moiety at a specifically defined location on the protein surface12. Unfortunately, biotin binding to a streptavidin or avidin coated surfaces is quite tight. Reversal of the binding interaction is not simple. The use of an engineered His-tag at the N- or C-terminus and the subsequent ease of attachment to Ni-NTA surfaces is a more universal application of affinity immobilization. Of course, one of the caveats for engineering assembly attachment sites with His-tagged systems is the requirement that the N- and C-termini of the core assembly protein remain exposed and separated so that the attachment is easy. As with all assembly processes, the interaction interface of the core assembly protein must remain available as the complex assembly progresses.
Perhaps the most common concern of using biosensor surface chemistries is non-specific binding. Streptavidin tips are often a source of significant non-specific binding effects. Disulfide linked biotin can be used to release very specific complexes leaving behind the reduced S-biotin linkage tightly bound to immobilized streptavidin biosensors13. There are other reversible chemistries becoming available such as iminoboronates and to a lesser extent ketoamide14. This field is currently underdeveloped, but there is high interest in further developing reversible covalent protocols to avoid off-target drug toxicity effects that commonly accompany the use of covalent targeted drug development.
A limitation of using EM to visualize complexes is interpretation, especially in instances where the structures of the assembled complexes are not initially known. The spatial location of components within an undefined assembled macromolecular complex can be identified using monoclonal antibodies (mAb) as specific kinetic and structural markers. For example, once a complex is formed, monoclonal antibodies can be added that bind to specific components. This method is frequently used in EM to identify specific components within large assemblies15. Another limitation is related to the size of the complex, although there have been instances where defined symmetric assemblies as small as 70 kDa (GroES heptamer) are easily resolved using negative stain EM. Assembled complexes that are analyzed by EM are typically in the size range of ~100 &#197; in diameter or above. Recently however, proteins as small as 20 kDa have been resolved and low resolution structures have been obtained when using superior staining methodologies16.
For MS analysis, the increased sensitivity of current MS instrumentation down to the femtomolar (attomoles) level can in some cases increase the sensitivity of BLI detection. It is highly conceivable that protein signals that show a minimal but repeatable rise in amplitudes will result in identification of the protein in question. In addition, probing protein-protein interactions with one of the partners attached on the biosensor and the other in a cellular milieu will in effect result in a purification and subsequently easier detection of the newly formed complex. One limitation that may be observed with the current highly sensitive MS systems is that the protein of interest may not be in the database, but this observation is rare (e.g., proteomes from rare species). If the sequences of the proteins of interest are known, this problem is easily solved by including the protein(s) amino acid sequence in a background protein database (as described in this work in the protocol section). Another potential limitation of the methodology results from the resistance of a protein to trypsinolysis. Trypsin digestion is typically the default method for bottom up protein identification. However, proteins may be resistant to trypsin if they lack Arg and Lys residues or access to these residues are restricted by the folded structure. These limitations are resolved, respectively, by using alternative or a combination of proteases or including an unfolding reagent (urea or guanidine HCl, as indicated in 1.1.14 and 1.2.6) before enzymatic digestion.
Possible expansions of this methodology include allowing the user to follow and identify cellular assembly complexes from crude cellular lysates. It turns out testing for assembly components in concentrated cellular extracts is easy to perform using the biolayer interferometry procedures. Unlike more commonly used microfluidic based methodologies which are prone to clogging and sensitive to aggregation, the BLI approach can be used to directly immerse biolayer sensor tips into crude extracts to potentially assemble specific complexes directly from these concentrated impure samples. Once assembled, it is entirely feasible to use specific antibody probes as a follow up to the BLI system to further identify and even quantitate suspected components in cellular extracts that were identified using the microvolume MS method. Again, the key here is to use defined, properly oriented core proteins as specific affinity probes.
The ability to view the prepore to pore transition process kinetically with BLI will be highly useful in identifying potential &#34;anti-toxin&#34; small molecule inhibitors of the protein transitions that specifically function under late endosomal, low pH (5.0) conditions. This pH induced prepore to pore transition is inhibited in the presence of folding stabilizer (osmolytes) such as glycerol or sucrose, and thus lends strong support for developing specific targeted folding stabilizers that prevent PApore formation. This specific approach avoids and replaces crude aggregation-based assays where pH drops lead to protein precipitation. This latter method, although good for primary prescreening methods, often leads to false positive results where specific compounds inhibit the aggregation rather than the actual molecular transitions.
The downstream observations of the structure and identification of the individual assembled components within small microvolume samples can also be useful in validating potential lead compounds. This can be applied in instances where either specific assembly stabilization or destabilization is the target outcome. This kinetic/structure/identification parallel approach is useful for directly confirming the validity of suspected lead compound effectors of assembly and serves as a reasonable secondary screening step or medium throughput approach.
Cryo-EM is a useful technique to study the atomic details of macromolecule complexes in various states of assembly. Prior to preparing cryo-EM samples, it is important to first verify a preparation contains reasonably pure homogenous complexes with negative stain EM. The work presented herein demonstrates assembly of protein complexes on BLI biosensor surfaces, release of these complexes for EM visualization, and identification of these components using MS. This particular methodology of controlled assembly and release can be useful in generating very specific protocols that enhance homogenous sequential sample preparation for negative stain EM, a necessary step that must be demonstrated before advancing to cryo-EM. To obtain low resolution 3D structure, only 30-50 particles of complex would be needed to perform a conical tilt series (70 different 2D image views per particle) provided there is orientation diversity (multiple different views).
With respect to enhancing MS methods, advances in sensitivity and reduction in sample volume continue to improve. Nano flows and ultra-high pressure liquid chromatography together with the development of mass spectrometers with a fast duty cycle, increased sensitivity and resolving power. Recent introduction of the orbitrap mass spectrometer, in particular the latest version (orbitrap Fusion Lumos, and its expected successor the Orbitrap Fusion Lumos 1M) as well as search algorithms greatly facilitate this process.
The current methodology monitors the kinetic assembly and disassembly of anthrax toxin components using label-free BLI methodologies and evaluates the structure and identity of these components using EM and MS, respectively. The use of a simple single channel BLI system coupled with routine negative staining EM analysis and elementary MS techniques are more than adequate to characterize an assembly process.

Identifying and understanding the specificity governing protein complex assembly in vivo is of extreme interest for biochemical researchers. Large heterogeneous protein assemblies are the norm rather than the exception. This notion is supported by spectroscopic monitoring of in vivo assembly, isolating complexes using gentler cell disruption techniques, evaluating products from affinity-based purification methods, and visualizing them using high resolution cryogenic electron microscopy (cryo-EM). To understand the control of assembly specificity within the cell, researchers must routinely isolate, identify, and ultimately characterize these dynamic assembling/disassembling structures. The most heavily used molecular tool to identify the components of these assemblies frequently requires antibody-based immunoprecipitation, which relies on maintaining complex stability during cell disruption. Various coupled analytical techniques were recently developed to capture complexes from cell samples using microfluidic based approaches, such as surface plasmon resonance (SPR). Following removal from the SPR surfaces, these samples were analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)1,2. Advancing this methodology using easier protocols will allow researchers to visualize and validate predicted complexes that occur in the cellular milieu. Since SPR is a microfluidics-based system, problems often arise from aggregate formation. Circumventing this problem requires sample dilution, which in turn, can decrease the integrity of concentration liable biological complexes.
A relatively recent advancement in label-free technologies is the development of the biolayer interferometry (BLI) system3. These particular light-based reflectance systems replicate, or best emulate, SPR binding and kinetic results at a fraction of the cost3,4 particularly if single channel units are used. BLI measures changes in reflected light interference patterns between a reference layer (control) and the biolayer surface (experimental). The resulting change in phase is measured in real time as a kinetic and quantitative readout5. The biosensor surface, containing specific immobilization chemistries, is physically transferred between solutions, as opposed to buffer changes by microfluidic approaches in SPR, for measurement via wavelength phase deflections. Mass transfer effects are prevented by agitating the solution. Unlike SPR, these systems are quite useful in evaluating complexes from crude biological samples. The physical parameter measured during BLI experiments primarily depends on a change in mass or thickness at the biosensor surface which results from protein complex assembly or disassembly.
These fiber optic biosensors are easy to use and relatively inexpensive. One of the emerging useful aspects of BLI is the facile removal of newly assembled protein complexes from the surface. A recent application of this method allowed our laboratory to observe the real time kinetics of large scale pH induced protein structure rearrangement of the anthrax toxin protective antigen (PA) component as the prepore (PAprepore) transitioned to its pore (PApore) form. This transition on the biosensor tip was verified using electron microscopy (EM)6. Removal of complexes from biosensor surfaces avoids larger volume dilution effects frequently encountered when releasing complexes from chip surfaces when using microfluidics systems.
In the current work, anthrax toxin complexes are assembled and disassembled on the biosensor surface and then released into microliter volumes. The resulting complex components are validated orthogonally using EM and mass spectrometry (MS).

<table><tbody><tr><td>(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)) (EDC)</td><td>Thermo Scientific</td><td>22980</td><td></td></tr><tr><td>(2-(2-pyridinyldithio)ethanamine) (PDEA)</td><td>GE Lifesciences</td><td>BR100058</td><td></td></tr><tr><td>0.5 mL black microtubes</td><td>Sigma-Aldrich</td><td>Z688304-500EA</td><td></td></tr><tr><td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)</td><td>Avanti</td><td>850457C-25mg</td><td></td></tr><tr><td>Acclaim PepMap 50 &amp;mu;m x 150mm, C18, 2 &amp;mu;m, 100 &amp;Aring;</td><td>ThermoFisher Scientific</td><td>164561</td><td></td></tr><tr><td>Acetic acid glacial</td><td>Fisher</td><td>A38SL-212</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>A955-4</td><td></td></tr><tr><td>Amine reactive 2nd generation (AR2G) tips</td><td>Forte Bio</td><td>18-5092</td><td></td></tr><tr><td>Ammonium bicarbonate</td><td>Fisher</td><td>A643-500</td><td></td></tr><tr><td>Anotop 10 syringe filter, 0.02um</td><td>GE Lifesciences</td><td>6809-1002</td><td></td></tr><tr><td>BLItz System</td><td>Pall Forte Bio</td><td>45-5000</td><td></td></tr><tr><td>Boric acid</td><td>Fisher Scientific</td><td>10043-35-3</td><td></td></tr><tr><td>Capillary Morphogenic Gene-2</td><td></td><td></td><td>Protein produced and purified in-house</td></tr><tr><td>Carbon coated Cu 300 grid</td><td>Electron Microscopy Sciences</td><td>CF300-CU-50</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>GoldBio</td><td>DTT25</td><td></td></tr><tr><td>Formic acids</td><td>JT Baker</td><td>0129-01</td><td></td></tr><tr><td>Guanidine hydrochloride (GuHCl)</td><td>Sigma-Aldrich</td><td>G4505-100G</td><td></td></tr><tr><td>Iodoacetamide</td><td>MP-BioMedicals,LLC</td><td>100-351</td><td></td></tr><tr><td>JEM-1400 Transmission Electron Microscope</td><td>JEOL</td><td></td><td></td></tr><tr><td>L-cystiene hydrochloride hydrate</td><td>Sigma-Aldrich</td><td>C121800-5G</td><td></td></tr><tr><td>Lethal Factor N-terminal domain</td><td></td><td></td><td>Protein produced and purified in-house</td></tr><tr><td>MS software version 2.2</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>N-hydroxysuccinimide (NHS)</td><td>Sigma-Aldrich</td><td>090M14531V</td><td></td></tr><tr><td>Orbitrap Fusion Lumos</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>PCR tubes</td><td>ThermoFisher Scientific</td><td>AB-0620</td><td></td></tr><tr><td>Pelco easiGlow glow discharge cleaning system</td><td>Ted Pella, Inc</td><td>91000</td><td></td></tr><tr><td>Protective Antigen prepore</td><td></td><td></td><td>Protein produced and purified in-house</td></tr><tr><td>Qualitative filter paper circles</td><td>Fisher Scientific</td><td>09-795C 5</td><td></td></tr><tr><td>Sequencing grade modified trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>Sequest HT search engine</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>Fisher Scientific</td><td>BP334-500</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>S271-10</td><td></td></tr><tr><td>Sodium cholate</td><td>Sigma-Aldrich</td><td>C1254-100G</td><td></td></tr><tr><td>Tris base</td><td>Fisher Scientific</td><td>BP152-10</td><td></td></tr><tr><td>Uranyl formate (UF)</td><td>Electron Microscopy Sciences</td><td>22450</td><td></td></tr><tr><td>Xcalibur</td><td>ThermoFisher Scientific</td><td>OPTON-30487</td><td></td></tr></tbody></table>

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.

The ability to monitor and validate the assembly of large macromolecular complexes is a crucial step towards understanding the specificity and functionality of large biomolecular assemblies. The results of the methods presented herein demonstrate the ease with which large protein complexes (&#62;150 kDa mass) can be assembled using biolayer interferometry, all while monitoring the kinetics and amplitude of assembly. The unique compact nature of the biosensor surface enables assembly analysis to be extended by releasing assembled complexes into small microvolumes. These microvolumes can be used to visualize the initial physical structure of the complexes using EM and to verify the identity of the complex components using MS. A schematic overview of this entire process is shown in Figure 1.
A key element for the successful assembly and verification of macromolecular complex on the biosensor surface involves the proper orientation of the initial seed protein. This ensures that the protein-protein interaction sites are accessible, not sterically blocked, and optimally positioned away from the biosensor surface. As shown in Figure 2, the proper orientation of the anthrax toxin complex is achieved by using a specifically engineered N-terminal fragment of lethal factor (LFN) so the LFN-PAprepore binding site is always positioned opposite of the biosensor covalent attachment site6,7. The subsequent buildup of the complex with binding of PA to the LFN BLI biosensor followed by soluble CMG2 binding to PA ultimately creates a translocation competent anthrax toxin complex.
The BLI sensogram trace is a real-time read out of the amplitude changes due to specific addition of the anthrax toxin components as they are added onto the biosensor. Figure 3 shows a representative trace paired with a model of the complex predicted to form at that step in the process. The first rise is LFN loading onto the tip. After quenching and baseline, PAprepore then binds to LFN followed by the addition of soluble CMG2 receptor resulting in the assembled pre-endosomal complex of LFN-PAprepore-CMG2. To progress toward the late endosome environment, the entire complex is subjected to a low pH pulse (pH 5.0) that weakens the receptor binding, allowing the prepore to transition to its extended membrane inserted pore conformation.6 Sensogram traces of the acidification step are shown in Figure 4. The initial increase or &#39;spike&#39; in amplitude is likely the pore extension6 that must occur prior to the decrease in the CMG2 receptor binding. The larger amplitude decline is most likely substantial or complete receptor dissociation due to diminished binding affinity. Previous work in this laboratory indicated CMG2 binding to the fully extended PApore is negligible compared to the CMG2-PAprepore interaction6. In addition, the sensogram kinetic trace, observed in all sensograms of LFN-PAprepore-CMG2 transitions to LFN-PApore with a pH drop, is reproducible over multiple runs.
Prior to and after acidification, the biosensor attached complexes are easily released for visualization by negative stain EM and identification by MS (Figure 5). Representative complexes from the EM results are shown in Figure 6. Pre-endosomal sample grids, show densities consistent with intact ternary complexes consisting of LFN-PAprepore-CMG2. Post-acidification complex grids, show PA transitioned to pore and solubilized by micelle inclusion with no obvious CMG2 density.
The identities of the pre- and post-acidification complexes were verified by MS. A database where the sequences of PA, P13423; LFN, P15917; and CMG2, P58335 were included in a background of a mouse proteins database derived from the NCBInr repository. Only the proteins of interest were obtained from this first database search, with the following amino acid coverage for the ternary and binary complexes: 54% and 22% for PA, 36% and 6% for LF and 43% for CMG2, respectively (CMG2 was not detected on the binary complex). In order to maximize the protein amino acid coverage, a second peptide/protein identification was performed using a protein database containing only the three proteins of interest. Pre-endosomal MS samples contained peptides from all three toxin components with 60.46%, 67.97%, and 54.15% coverage for LFN, PA, and CMG2, respectively (Figure 7). Post-endosomal results, shown in Figure 8, only contained peptides from LFN and PA (57.41% and 67.79% coverage, respectively). The lack of CMG2 in the post-endosomal samples are consistent with the observed BLI nm decrease during pore formation.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57902/57902fig1.jpg" /> 
Figure 1. Schematic overview for analysis of protein complexes assembled on and isolated from BLI biosensor using EM and MS. <a href="https://www.jove.com/files/ftp_upload/57902/57902fig1large.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/57902/57902fig2.jpg" /> 
Figure 2. Biosensor activation: First step in orientation specific assembly on BLI biosensor. The E126C Lethal Factor N-terminal domain, (LFN) is linked through a thio-linkage creating the properly orientated PAprepore binding interface. <a href="https://www.jove.com/files/ftp_upload/57902/57902fig2large.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/57902/57902fig3.jpg" /> 
Figure 3. Monitoring anthrax toxin assembly and disassembly with BLI: The sensogram shows amplitude changes as a function of time due to specific addition of the anthrax toxin components as they are added onto the biosensor starting with the LFN loading (step 4 in Table 1). PAprepore is then loaded onto the surface followed by the addition of soluble CMG2 receptor. The assembled pre-endosomal complex consists of an LF-PAprepore- CMG2. To progress toward the late endosome environment, the entire assemble functional anthrax toxin is subjected to a low pH pulse (pH 5.0) that weakens the receptor binding, allowing the prepore to transition to its extended membrane inserted pore conformation. Exposure of the membrane spanning pore is confirmed and solubilized by addition of micelles. <a href="https://www.jove.com/files/ftp_upload/57902/57902fig3large.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/57902/57902fig4.jpg" /> 
Figure 4. BLI sensogram of CMG2 release: Traces of the acidification step show an initial increase or &#39;spike&#39; in amplitude followed by a larger amplitude decline that are likely pore formation and receptor dissociation, respectively. Vertical dotted red lines denote start of a new step. Sensograms are aligned at the bolded dotted red line. <a href="https://www.jove.com/files/ftp_upload/57902/57902fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57902/57902fig5.jpg" /> 
Figure 5. Analyzing protein complexes assembled on and isolated from BLI biosensor using EM and MS: Biosensor attached complexes are easily released into 5 &#181;L of buffer containing DTT for visualization by negative stain EM and identification by MS. <a href="https://www.jove.com/files/ftp_upload/57902/57902fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57902/57902fig6.jpg" /> 
Figure 6. Visualization of protein complexes with EM: Pre-endosomal sample grids, show densities consistent with intact ternary complexes consisting of LFN-PAprepore-CMG2. Post-endosomal complex grids, show PA transitioned to pore and solubilized by micelle with no obvious CMG2 density. Models of predicted complexes (left hand side) are at the same scale as individual particles shown. Particles colorized with predicted protein (based on size of EM density) are shown below each particle. <a href="https://www.jove.com/files/ftp_upload/57902/57902fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57902/57902fig7.jpg" /> 
Figure 7. Verification of protein complexes with MS: Pre-endosomal MS samples contained peptides from all three toxin components with 60.46%, 67.97%, and 54.15% coverage for LFN, PA, and CMG2, respectively. Peptides detected at a false discovery rate (FDR) equal or higher than 5% shown in yellow, FDR equal or higher than 1% shown in green. <a href="https://www.jove.com/files/ftp_upload/57902/57902fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57902/57902fig8.jpg" /> 
Figure 8. Verification of protein complexes with MS: Post-endosomal samples contained peptides from LFN and PA (57.41% and 67.79% coverage, respectively), but not CMG2. FDR equal or higher than 5% shown in yellow, FDR equal or higher than 1% shown in green. <a href="https://www.jove.com/files/ftp_upload/57902/57902fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Step</td>
			<td>Time (s)</td>
			<td>Description&#160;</td>
		</tr>
		<tr>
			<td>1</td>
			<td>30</td>
			<td>Initial baseline</td>
		</tr>
		<tr>
			<td>2</td>
			<td>420</td>
			<td>Activation</td>
		</tr>
		<tr>
			<td>3</td>
			<td>300</td>
			<td>Activation</td>
		</tr>
		<tr>
			<td>4</td>
			<td>300</td>
			<td>LFN loading</td>
		</tr>
		<tr>
			<td>5</td>
			<td>240</td>
			<td>Cysteine quench</td>
		</tr>
		<tr>
			<td>6</td>
			<td>120</td>
			<td>Baseline</td>
		</tr>
		<tr>
			<td>7</td>
			<td>300</td>
			<td>PAprepore association&#160;</td>
		</tr>
		<tr>
			<td>8</td>
			<td>30</td>
			<td>Baseline</td>
		</tr>
		<tr>
			<td>9</td>
			<td>300</td>
			<td>CMG2 association</td>
		</tr>
		<tr>
			<td>10</td>
			<td>30</td>
			<td>Baseline</td>
		</tr>
		<tr>
			<td>11</td>
			<td>300</td>
			<td>Acid transition</td>
		</tr>
		<tr>
			<td>12</td>
			<td>30</td>
			<td>Baseline</td>
		</tr>
		<tr>
			<td>13</td>
			<td>300</td>
			<td>Micelle association</td>
		</tr>
	</tbody>
</table>
Table 1. Step times for single BLI run to assembly anthrax toxin complex. Note that baselines are used to wash away unbound protein from previous step and/or establish a new buffer baseline.

Watch this Scientific Journal Video about Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy at JoVE.c

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

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 ...

analyzing-dynamic-protein-complexes-assembled-on-released-from

Research

JoVE Journal

Biochemistry

9.3K Views.  University of Kansas Medical Center. Hier presenteren we een protocol voor het controleren van de montage en demontage van de toxine van de bloedzweer met behulp van biolayer interferometrie (BLI). Na montage/demontage op de biosensor oppervlak, de grote eiwitcomplexen worden vrijgegeven uit het oppervlak voor visualisatie en identificatie van onderdelen van de complexen met behulp van elektronenmicroscopie en massaspectrometrie, respectievelijk.

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

Bio-layer Interferometry for Measuring Kinetics of Protein-protein Interactions and Allosteric Ligand Effects

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia coli&#160;ATP synthase. Bacterial F-type ATP synthase is the target of a new, FDA-approved antibiotic to combat drug-resistant tuberculosis. Understanding bacteria-specific auto-inhibition of ATP synthase by the C-terminal domain of subunit &#949;&#160;could provide a new means to target the enzyme for discovery of antibacterial drugs. The C-terminal domain of &#949;&#160;undergoes a dramatic conformational change when the enzyme transitions between the active and inactive states, and catalytic-site ligands can influence which of &#949;&#39;s conformations is predominant. The assay measures kinetics of &#949;&#39;s binding/dissociation with the catalytic complex, and indirectly measures the shift of enzyme-bound &#949;&#160;to and from the apparently nondissociable inhibitory conformation. The Bio-layer Interferometry signal is not overly sensitive to solution composition, so it can also be used to monitor allosteric effects of catalytic-site ligands on &#949;&#39;s conformational changes.

The protocols here describe kinetic assays of protein-protein interactions with Bio-layer Interferometry. F-type ATP synthase, which is involved in cellular energy metabolism, can be inhibited by its &#949; subunit in bacteria. We have adapted Bio-layer Interferometry to study interactions of the catalytic complex with &#949;&#8217;s inhibitory C-terminal domain.

We describe the use of Bio-layer Interferometry to study inhibitory interactions of subunit &#949;&#160;with the catalytic complex of Escherichia ...

Chemistry

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

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 ...

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 ...

Biology

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. ...

Expanding the Mass Photometry Analysis Concentration Range with Microfluidics

Analysis of Protein Complex Formation at Micromolar Concentrations by Coupling Microfluidics with Mass Photometry

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without labels. Mass photometry is generally suited to analyzing samples in the 100 pM-100 nM concentration range. However, in many biological systems, it is necessary to measure more concentrated samples to study low-affinity or transient interactions. Here, we demonstrate a method that effectively expands the range of sample concentrations that can be analyzed by mass photometry from nanomolar to tens of micromolar. 
In this protocol, mass photometry is combined with a novel microfluidics system to investigate the formation of protein complexes in solution in the micromolar concentration range. With the microfluidics system, users can maintain a sample at a desired higher concentration followed by dilution to the nanomolar range - several milliseconds prior to the mass photometry measurement. Due to the speed of the dilution, data is obtained before the equilibrium of the sample has shifted (i.e., dissociation of the complex). 
The technique is applied to measure interactions between an immunoglobulin G (IgG) antibody and the neonatal Fc receptor, showing the formation of high-order complexes that were not quantifiable with static mass photometry measurements. 
In conclusion, the combination of mass photometry and microfluidics makes it possible to characterize samples in the micromolar concentration range and is proficient in measuring biomolecular interactions with weaker affinities. These capabilities can be applied in a range of contexts - including the development and design of biotherapeutics - enabling thorough characterization of diverse protein-protein interactions.

Author Spotlight: Characterization of Low-Affinity Protein Interactions in Solution Using MassFluidix Technology

This protocol combines mass photometry with a novel microfluidics system to investigate low-affinity protein-protein interactions. This approach is based on the rapid dilution of highly concentrated complexes in solution, which enables low-affinity measurements and broadens the applicability of mass photometry.

Mass photometry is a versatile mass measurement technology that enables the study of biomolecular interactions and complex formation in solution without ...

Quantitative Analysis of RPA-ssDNA Binding Kinetics Using Label-Free Methods

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of ...

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 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 ...

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

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