We acknowledge financial support for the project from the French grant REPLIPOX ANR-13-BSV8-0014 and by research grants from the Service de Sant&#233; des Arm&#233;es and the D&#233;l&#233;gation G&#233;n&#233;rale pour l'Armement. We are thankful to the ESRF for the SAXS beam time. We thank Andrew McCarthy, Gordon Leonard, Wim Burmeister, and Guy Schoehn for financial and scientific support.

<ol>
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The authors have nothing to disclose.

For many macromolecules, a final purification step using chromatography is required prior to SAXS data collection to obtain a good quality data set. However, not all samples remain stable; they may be prone to aggregation or re-equilibration to a mixture of oligomerization states. Therefore, a final online purification step on the beamline is required to minimize the time between purification and data collection in order to obtain the best-quality SAXS data. Depending on the biophysical properties of the protein of interest, SEC-SAXS or IEC-SAXS might be chosen to obtain optimal sample quality. Here, on a protein construct derived from the helicase/primase D5, both techniques are explained and discussed.
Acquisition of SEC-SAXS data is becoming more and more standardized and is available on many BioSAXS beamlines. Data analysis, especially background subtraction, is relatively straightforward and easy. However, a stable buffer signal and the sufficient separation of the macromolecular species remains essential. Therefore, it is critical to reserve enough time to equilibrate the column thoroughly. Failure of this method can be due to persistent contaminants of similar size to the protein of interest, low concentrations, and radiation-sensitive buffers.
In practice, initially, SEC-SAXS is likely to be used as the method of choice for most macromolecular samples. Still, many purification protocols require a prior IEC step due to the presence of contaminants or aggregation. Given that each concentration and chromatography step is associated with losses of sample (estimated at 30-50%) and time, direct IEC-SAXS is advantageous. For samples that cannot be purified by SEC, be it due to the presence of similarly sized &#34;contaminants&#34; or because they severely aggregate at the necessary concentrations, IEC-SAXS would always be the better-suited approach. Also, the higher flow rates supported by many IEC columns can help to reduce the transit time between purification and measurement. In the example presented here, IEC was used with a step elution, which allows for the separation of the close peaks of D5323-785 from contaminants by carefully choosing the salt concentration steps. In principle, the number of steps is unlimited, but practically, at least 1 step per peak is required, and not too many should be chosen. For the background subtraction method described above, it is crucial to measure a relatively high number of different buffer compositions in order to find the matching one.
A shared downside of both techniques is the lack of precise protein concentration information. Due to this, precise mass determination based on forward scattering is not possible. For globular proteins such as D5323-785, the Porod volume provides an alternative, albeit less precise, mass estimate, but for highly flexible or disordered proteins, this approach would not be valid.
A variation of the step-wise gradient IEC-SAXS method presented here is the use of a linear gradient instead. While it is possible to work with as many steps as desired to isolate sub-peaks using a step-wise elution, in the linear gradient approach, it is required to optimize the gradient conditions carefully in order to separate the peaks entirely before starting the SAXS experiment. Background subtraction in this approach could be done frame-wise and could be verified by the comparison of the individual frames, but it requires more advanced data handling, and a dedicated software does not exist yet.
The choice of a suitable column is critical for both techniques, as it determines the separation of the macromolecular species. Size-exclusion columns differ in loading capacity, the size range of separable macromolecules, and resolution, while ion-exchange columns vary in the kind and the density of their immobilized charges.
While the protocol presented here is specific to the ESRF beamline BM29, adaptation to any other SAXS beamline is, in principle, straightforward. The main requirements are a sufficiently high X-ray flux and a suitable detector (ideally single-photon-counting), to acquire reasonable signal-to-noise data in the range of seconds or less, and an online liquid chromatography system capable of creating gradients. The exact implementation would, of course, depend on the local beamline environment.
The results obtained on D5323-785 using the two methods differ slightly. The radius of gyration is slightly smaller for the IEC data than for the SEC data, and the local minima of the scattering curve are shifted to slightly larger scattering vectors. This means that the D5323-785 measured with IEC-SAXS is slightly more compact than the D5323-785 measured with SEC-SAXS. This might be due to differences in the sample preparation, in the time between purification and measurement (IEC is faster), or, less likely, to a contaminant in the SEC-purified sample. The bead models obtained completely independently with both methods are comparable (Figure 1H). D5323-785 shows the expected hollow, hexameric structure18.
In conclusion, online ion-exchange and online size-exclusion chromatography are important biochemical purification methods that can be coupled directly to SAXS6,7,9-12,25,26. The background subtraction of IEC-SAXS data is slightly more difficult and ambiguous than for SEC-SAXS, but it is nevertheless possible. Depending on the biophysical properties of the protein of interest, both SEC and IEC-SAXS allow for the optimization of species separation with inherent advantages. Providing that the validation steps (as described) are correctly observed, the resulting data can be analyzed with confidence, and models can be determined using the standard tools available within the community. Together, both techniques allow online separation for a broad range of biological macromolecules, yielding data not accessible via standard static measurements.

BioSAXS is a powerful tool to determine the shape of nano-sized objects1-4. The scattering of X-rays by a solution containing macromolecules, sized in the nm range, is recorded at very low angles. This angular range contains information about global parameters: the radius of gyration; the largest intraparticle distance; the particle shape; and the degree of folding, denaturation, or disorder. The technique does not require crystals, and the macromolecule stays in solution and thus can be kept in conditions mimicking certain important parameters of the cell, such as ionic strength, pH, etc. The knowledge of these factors might help to determine, for example, the physiologically relevant oligomeric state of a protein of interest or to validate a proposed model of a complex. The characterization of protein-protein interactions in different buffer conditions, the creation of models of missing domains, the refinement of homology models, and the determination of discrete folded and unfolded states can be performed quickly and easily5.
As with any technique, BioSAXS has intrinsic weaknesses: aggregated or denatured samples, mixtures of particles, heterogeneous samples, radiation damage, and buffer mismatches may result in un-interpretable data. For many analysis methods, it is implicitly assumed that the sample is monodisperse, a requirement that is often difficult to obtain in practice. In many cases, the degradation of the sample is subtle and cannot be detected in the data on its own, and any attempt to interpret the data gives inaccurate or even misleading results. To overcome these obstacles, the combination of size-exclusion chromatography (SEC) and SAXS was implemented on many beamlines to ensure data quality and to make this technique more accessible for increasingly difficult samples6-11. Recently, we added a new method to the repertoire by developing online ion-exchange chromatography (IEC)-coupled SAXS12. Both techniques are opening SAXS to a wide range of biological particles formerly impossible to analyze. The choice of which method to use depends on the biophysical properties of the particles of interest.
SEC separates macromolecules by their size, whereby at least a 10% difference in apparent molecular mass is needed for the separation. Physical limitations of the column and physiological properties of the samples, like hydrophobic surfaces, flexibility, and lack of stability, also complicate data collection, analysis, and interpretation.
Ion-exchange chromatography, which separates molecules based on their charge and, hence, their binding affinity to the IEC column, can be used instead of or in addition to SEC. The total charge can be readily manipulated by changing the pH, or varying the salt concentration of the buffer, providing a relatively simple method for the controlled elution of the molecules from the IEC column. By using the charge, the separation of similar types and sizes of molecules, which would otherwise be difficult to separate, can be performed routinely with IEC. Additionally, IEC has the advantage of being able to deal with diluted samples, allowing one to avoid the concentration steps, which carry the potential risk of denaturing the protein. Unfortunately, as the charge distribution is highly sample-dependent, IEC requires optimization regarding the pH and the salt concentrations13,14.
For many proteins that are difficult to express, purify, or both, only low quantities of sample are available to study. It is important to be efficient and to minimize the number of purification steps and, therefore, the losses. For this reason, the last purification step is online directly prior to SAXS data acquisition, in order to increase the likelihood of collecting a good data set.
Here, we present and compare online SEC-SAXS and IEC-SAXS. Both techniques were implemented on the BioSAXS beamline BM29 at the European Synchrotron Radiation Facility (ESRF) in Grenoble, France15. As a test case, we used the D5N and helicase domain of the vaccinia virus protein D5, which was rather difficult to analyze structurally using other methods. The vaccinia virus is a member of the Poxviridae family and is 98% identical to the variola virus, the cause of small pox. Using the vaccinia system, we study the replication machinery, focusing here on the essential helicase-primase D5.
D5 is a 95-kDa protein with an N-terminal archeo-eukaryotic primase (AEP) domain16 followed by a cysteine cluster region (res. 240-345)16. Further towards the C-terminus comes a D5N domain (res. 340-460), which is always associated with D5-type helicases, and finally a superfamily 3 (SF3) helicase domain (res. 460-785) 17 (Figure 1A). The helicase domain of D5 builds a hexameric ring structure that is needed for tight binding to DNA. Thanks to recent SAXS and EM studies, the low-resolution structures of the primase and the helicase domains are now known18.
Here, we show how to use the implemented online chromatography techniques on the BioSAXS beamline BM29 at ESRF to gain insights into the structure of the C-terminal fragment (residue 323-785) of D5.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1,4 dithiothreitol [DTT]</td>
			<td>Euromedex</td>
			<td>EU0006-B</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Mini-PROTEAN TGX Precast Protein Gel</td>
			<td>Biorad</td>
			<td>4561033</td>
			<td></td>
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			<td>2x Phusion Flash PCR Master Mix</td>
			<td>ThermoFisher scientific</td>
			<td>F 548</td>
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		<tr>
			<td>acetic acid glacial</td>
			<td>VWR Chemicals (BDH Prolabo)</td>
			<td>20104.298</td>
			<td></td>
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		<tr>
			<td>Agarose D-5</td>
			<td>Euromedex</td>
			<td>LF45130653</td>
			<td></td>
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		<tr>
			<td>Amicon Ultra -15 Centrifugal filters Ultracel -30K</td>
			<td>Millipore Ltd</td>
			<td>UFC903024</td>
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			<td>Ampicillin sodium salt</td>
			<td>Euromedex</td>
			<td>EU0400-D</td>
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			<td>Benzonase</td>
			<td>Novagene</td>
			<td>70750-3</td>
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			<td>bromophenol blue&#160;</td>
			<td>MERCK</td>
			<td>8122</td>
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			<td>Complete protease inhibitor cocktail&#160;</td>
			<td>Roche</td>
			<td>11231400</td>
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			<td>Econo-Pac 10 DG Desalting column</td>
			<td>Bio-rad</td>
			<td>732-2010</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>E-5134</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>VWR Chemicals (BDH Prolabo)</td>
			<td>24388.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Euromedex</td>
			<td>26-128-6405-C</td>
			<td></td>
		</tr>
		<tr>
			<td>HindIII</td>
			<td>Roche</td>
			<td>656313</td>
			<td></td>
		</tr>
		<tr>
			<td>HisSelect HF Nickel Affinity Gel</td>
			<td>Sigma</td>
			<td>H0537</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid (HCl)</td>
			<td>VWR Chemicals (BDH Prolabo)</td>
			<td>20252.295</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>AppliChem Panreac</td>
			<td>A1073,0500</td>
			<td></td>
		</tr>
		<tr>
			<td>InstantBlue</td>
			<td>Expedeon</td>
			<td>ISB1L</td>
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			<td>LB broth Miller</td>
			<td>Fluka Analytical</td>
			<td>L3152</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>ICN Biomedicals Inc.</td>
			<td>191421</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>VWR Chemicals (BDH Prolabo)</td>
			<td>27808.297</td>
			<td></td>
		</tr>
		<tr>
			<td>NcoI</td>
			<td>Fermentas</td>
			<td>ER0571</td>
			<td></td>
		</tr>
		<tr>
			<td>One Shot BL21 Star (DE3)&#160;</td>
			<td>ThermoFisher scientific</td>
			<td>C6010-03</td>
			<td></td>
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			<td>pProEx HTb vector&#160;</td>
			<td>Addgene</td>
			<td>10711018</td>
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			<td>Primer</td>
			<td>&#160;Eurofins mwg operon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep kit</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick Gel extraction kit</td>
			<td>QIAGEN</td>
			<td>28706</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAquick PCR purification kid</td>
			<td>QIAGEN</td>
			<td>28106</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS&#160;</td>
			<td>Sigma</td>
			<td>75746</td>
			<td></td>
		</tr>
		<tr>
			<td>Superose 6 10/300 GL</td>
			<td>GE Healthcare</td>
			<td>17-5172-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR Safe DNA gel stain</td>
			<td>Invitrogen life technology</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>ThermoFisher scientific</td>
			<td>EL0011</td>
			<td></td>
		</tr>
		<tr>
			<td>TEV</td>
			<td></td>
			<td></td>
			<td>home made</td>
		</tr>
		<tr>
			<td>TOP 10</td>
			<td>Invitrogen life technology</td>
			<td>C404003&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Euromedex</td>
			<td>26-128-3094-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Uno Q 1R column&#160;</td>
			<td>Bio-rad</td>
			<td>720 0011</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>MPBiomedicals, LLC</td>
			<td>194834</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Beamline control software BsXCuBE</td>
			<td>ESRF</td>
			<td>Pernot et al. (2013), J. Synchrotron Rad. 20, 660-664</td>
			<td>local development</td>
		</tr>
		<tr>
			<td>Camserver software</td>
			<td>Dectris</td>
			<td>n.a.</td>
			<td>detector control software</td>
		</tr>
		<tr>
			<td>DAMAVER</td>
			<td>ATSAS 2.6.0</td>
			<td>http://www.embl-hamburg.de/biosaxs/download.html</td>
			<td>program to align ab initio models&#160;</td>
		</tr>
		<tr>
			<td>DAMMIF</td>
			<td>ATSAS 2.6.0</td>
			<td>http://www.embl-hamburg.de/biosaxs/download.html</td>
			<td>ab initio&#160; model reconstruction programm</td>
		</tr>
		<tr>
			<td>HPLC program Biologic Duo Flow</td>
			<td>Bio-rad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC program LabSolutions</td>
			<td>Shimadzu</td>
			<td>n.a.</td>
			<td></td>
		</tr>
		<tr>
			<td>ISPyB</td>
			<td>ESRF</td>
			<td>De Maria Antolinos et al. (2015). Acta Cryst. D71, 76-85.</td>
			<td>local development</td>
		</tr>
		<tr>
			<td>PRIMUS</td>
			<td>ATSAS 2.6.0</td>
			<td>http://www.embl-hamburg.de/biosaxs/download.html</td>
			<td>program, which performs the manipulation with experimental SAXS</td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>DeLano Scientific LLC</td>
			<td>https://www.pymol.org/</td>
			<td>software for visualization.</td>
		</tr>
		<tr>
			<td>SUPCOMB</td>
			<td>ATSAS 2.6.0</td>
			<td>http://www.embl-hamburg.de/biosaxs/download.html</td>
			<td>program to superimpose 3D structures</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioLogic Duo Flow</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioLogic Biofrac Fraction collector</td>
			<td>Biorad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HPLC system</td>
			<td>Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Labsonic P</td>
			<td>Sartorius Stedim biotech</td>
			<td>BBI8535108</td>
			<td></td>
		</tr>
	</tbody>
</table>

online size-exclusion and ion-exchange chromatography on a saxs beamline

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, particle size and shape, and surface-to-volume ratio of macromolecules. The technique is applicable to a very wide variety of solution conditions spanning a broad range of concentrations, pH values, ionic strengths, temperatures, additives, etc., but the sample is required to be monodisperse. This caveat led to the implementation of liquid chromatography systems on SAXS beamlines. Here, we describe the upstream integration of size-exclusion (SEC) and ion-exchange chromatography (IEC) on a beamline, different methods for optimal background subtraction, and data reduction. As an example, we describe how we use SEC- and IEC-SAXS on a fragment of the essential vaccinia virus protein D5, consisting of a D5N helicase domain. We determine its overall shape and molecular weight, showing the hexameric structure of the protein.

The results of the model-free invariant analysis are listed in Table 1. The analysis of the D5323-785 SEC-SAXS data showed a molecular mass estimate (from a Porod analysis) of 345 kDa versus 338 kDa, observed using IEC-SAXS. Both are in agreement with the expected mass of 6 times 53.5 kDa (321 kDa) for a hexamer. The ab initio modeling of both data sets was undertaken with no imposed symmetry (SEC-SAXS: &#967;2 = 0.88, IEC-SAXS: &#967;2 =3.1) and using C6 symmetry (SEC-SAXS: &#967;2 = 1.0, IEC-SAXS: &#967;2 = 4). As the overall fits to the scattering data for both reconstructions are comparable in both cases (Figure 1D and E), C6 symmetry can be assumed. Thus, the model corresponds to a hexagonal cone-like structure with a central channel, which appears partially obstructed (SEC: Figure 1F; IEC: Figure 1G, overlay Figure 1H). An examination of individual models before averaging shows that this obstruction is likely to be an artifact of the averaging process.
<img alt="Figure 1" src="/files/ftp_upload/54861/54861fig1x.jpg" /> 
Figure 1: SAXS data analysis and comparison of D5323-785 (figure adapted from References 12 and 18). A) Schematic domain structure of D5 protein and D5323-785. B) Offline ion-exchange chromatogram with the indication of the three peaks. Orange curve: Absorbance at 280 nm (au), blue curve: percentage of buffer B. Percentages of buffer B at the peaks are indicated. C) Online ion-exchange chromatogram. Color key as in B. Additionally, the measured intensity is indicated in green. The experimental scattering curve and calculated curve of the model obtained by SEC-SAXS D) and IEC E) data analysis of D5323-785. F) Bead model of D5323-785 based on SEC data and G) IEC data. H) Overlay of the SEC and IEC models. The panels in F) to H) were created using a molecular visualization software24. <a href="http://cloudflare.jove.com/files/ftp_upload/54861/54861fig1large.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>Data-collection parameters</td>
			<td></td>
		</tr>
		<tr>
			<td>Instrument:</td>
			<td>ESRF BM29</td>
		</tr>
		<tr>
			<td>Wavelength (&#197;)</td>
			<td>0.99</td>
		</tr>
		<tr>
			<td>q-range (&#197;-1)</td>
			<td>0.0032 &#8211; 0.49</td>
		</tr>
		<tr>
			<td>Sample-to-detector distance</td>
			<td>2.864 m</td>
		</tr>
		<tr>
			<td>Exposure time (sec)</td>
			<td>1 per frame</td>
		</tr>
		<tr>
			<td>Concentration range</td>
			<td>n.a.</td>
		</tr>
		<tr>
			<td>Temperature (K)</td>
			<td>293</td>
		</tr>
		<tr>
			<td>Detector</td>
			<td>Pilatus 1M (Dectris)</td>
		</tr>
		<tr>
			<td>Flux (photons/s)</td>
			<td>1 &#215; 1012</td>
		</tr>
		<tr>
			<td>Beam size (&#181;m2)</td>
			<td>700 &#215; 700</td>
		</tr>
		<tr>
			<td>Structural parameters for D5323-785, SEC</td>
			<td></td>
		</tr>
		<tr>
			<td>I0 (cm-1) [from Guinier]</td>
			<td>0.0237</td>
		</tr>
		<tr>
			<td>Rg (&#197;) [from Guinier]</td>
			<td>48</td>
		</tr>
		<tr>
			<td>qminRg &#8211; qmaxRg used for Guinier</td>
			<td>0.77 - 1.24</td>
		</tr>
		<tr>
			<td>Dmax (&#197;) [from p(r) ]</td>
			<td>145</td>
		</tr>
		<tr>
			<td>q-range used for p(r) (&#197;-1)</td>
			<td>0.02 - 0.17</td>
		</tr>
		<tr>
			<td>Porod volume Vp (&#197;3) [from Scatter]</td>
			<td>(570 &#177; 5) &#215; 103</td>
		</tr>
		<tr>
			<td>Molecular mass Mr (kDa) [from Vp]</td>
			<td>345</td>
		</tr>
		<tr>
			<td>Calculated monomeric Mr from sequence (kDa)</td>
			<td>321</td>
		</tr>
		<tr>
			<td>Structural parameters for D5323-785, IEC</td>
			<td></td>
		</tr>
		<tr>
			<td>I0 (cm-1) [from Guinier]</td>
			<td>0.386</td>
		</tr>
		<tr>
			<td>Rg (&#197;) [from Guinier]</td>
			<td>46.5 &#177; 0.1</td>
		</tr>
		<tr>
			<td>qminRg &#8211; qmaxRg used for Guinier</td>
			<td>0.44 - 1.29</td>
		</tr>
		<tr>
			<td>Dmax (&#197;) [from p(r) ]</td>
			<td>120</td>
		</tr>
		<tr>
			<td>q-range used for p(r) (&#197;-1)</td>
			<td>0.03 - 0.18</td>
		</tr>
		<tr>
			<td>Porod volume Vp (&#197;3) [from Scatter]</td>
			<td>(577 &#177; 5) &#215; 103</td>
		</tr>
		<tr>
			<td>Molecular mass Mr (kDa) [from Vp]</td>
			<td>339</td>
		</tr>
		<tr>
			<td>Calculated hexameric Mr from sequence (kDa)</td>
			<td>321</td>
		</tr>
	</tbody>
</table>
Table 1: SAXS data parameters. The table summarizes the parameters of SAXS data acquisition and analysis.

Watch this Scientific Journal Video about Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline at JoVE.com

Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

Biological small angle X-ray scattering (BioSAXS) is a powerful technique in molecular and structural biology used to determine solution structure, ...

online-size-exclusion-ion-exchange-chromatography-on-saxs

Research

JoVE Journal

Biochemistry

17.2K Views.  European Synchrotron Radiation Facility. The determination of the solution structure of a protein by small angle X-ray scattering (SAXS) requires monodisperse samples. Here, we present two possibilities to ensure minimal delays between sample preparation and data acquisition: online size-exclusion chromatography (SEC) and online ion-exchange chromatography (IEC).

Video: Online Size-exclusion and Ion-exchange Chromatography on a SAXS Beamline

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in solution, is a relative technique that relies on the elution volume of the analyte to estimate molecular weight. When the protein is not globular or undergoes non-ideal column interactions, the calibration curve based on protein standards is invalid, and the molecular weight determined from elution volume is incorrect. Multi-angle light scattering (MALS) is an absolute technique that determines the molecular weight of an analyte in solution from basic physical equations. The combination of SEC for separation with MALS for analysis constitutes a versatile, reliable means for characterizing solutions of one or more protein species including monomers, native oligomers or aggregates, and heterocomplexes. Since the measurement is performed at each elution volume, SEC-MALS can determine if an eluting peak is homogeneous or heterogeneous and distinguish between a fixed molecular weight distribution versus dynamic equilibrium. Analysis of modified proteins such as glycoproteins or lipoproteins, or conjugates such as detergent-solubilized membrane proteins, is also possible. Hence, SEC-MALS is a critical tool for the protein chemist who must confirm the biophysical properties and solution behavior of molecules produced for biological or biotechnological research. This protocol for SEC-MALS analyzes the molecular weight and size of pure protein monomers and aggregates. The data acquired serve as a foundation for further SEC-MALS analyses including those of complexes, glycoproteins and surfactant-bound membrane proteins.

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering SEC-MALS

This protocol describes the combination of size exclusion chromatography with multi-angle light scattering (SEC-MALS) for absolute characterization of proteins and complexes in solution. SEC-MALS determines the molecular weight and size of pure proteins, native oligomers, heterocomplexes and modified proteins such as glycoproteins.

Analytical size-exclusion chromatography (SEC), commonly used for the determination of the molecular weight of proteins and protein-protein complexes in ...

Cell Culture on Silicon Nitride Membranes and Cryopreparation for Synchrotron X-ray Fluorescence Nano-analysis

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions and their disturbed homeostasis is involved in various diseases. State-of-the-art synchrotron X-ray fluorescence nanoprobes provide the required sensitivity and spatial resolution to elucidate the two-dimensional (2D) and three-dimensional (3D) distribution and concentration of metals inside entire cells at the organelle level. This opens new exciting scientific fields of investigation on the role of metals in the physiopathology of the cell. The cellular preparation is a key and often complex procedure, particularly for basic analysis. Although X-ray fluorescence techniques are now widespread and various preparation methods have been used, very few studies have investigated the preservation of the elemental content of cells at best, and no stepwise detailed protocol for the cryopreparation of adherent cells for X-ray fluorescence nanoprobes has been released so far. This is a description of a protocol that provides the stepwise cellular preparation for fast cryofixation to enable synchrotron X-ray fluorescence nano-analysis of cells in a frozen hydrated state when a cryogenic environment and transfer is available. In case nano-analysis has to be performed at room temperature, an additional procedure for freeze-drying the cryofixed adherent cellular preparation is provided. The proposed protocols have been successfully used in previous works, most recently in studying the 2D and 3D intracellular distribution of an organometallic compound in breast cancer cells.

Presented here is a protocol for cell culture on silicon nitride membranes and plunge-freezing prior to X-ray fluorescence imaging with a synchrotron cryogenic X-ray nanoprobe. When only room temperature nano-analysis is provided, the frozen samples can be further freeze-dried. These are critical steps to obtain information on the intracellular elemental composition.

Very little is known about the distribution of metal ions at the subcellular level. However, those chemical elements have essential regulatory functions ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.