Name: capillary electrophoresis mass spectrometry approaches for characterization of the protein and metabolite corona acquired by nanomaterials
Uploaded: 2020-10-27T14:00:00.000+00:00
Duration: 7 min 54 s
Description: Watch this Scientific Journal Video about Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials at JoVE.com

A.J.C, J.A.T and I.L acknowledge funding from the European Commission via Horizon 2020 project ACEnano (Grant no. H2020-NMBP-2016-720952). W.Z acknowledges PhD funding from the China Scholarship Council (CSC, No. 201507060011). R.R acknowledges the financial support of the Vidi grant scheme of the Netherlands Organization of Scientific Research (NWO Vidi 723.0160330).

Use of human biofluid was approved as per the IRB protocol guidelines from University of Leiden and Innsbruck Medical University. When human or animal biofluids are being investigated, ethical approval from the research institution is required, and has been obtained in the case of the results shown in this protocol. Furthermore adequate reporting should also be performed to ensure transparency and reusability of data in future work9,29,30.
1. Preparation of background electrolytes (BGE)
NOTE: All solvents should be prepared in a suitable fume hood and adequate personal protective equipment must be used for all steps (labcoat, gloves and goggles). In each step, low binding plastic vials are required to minimize analyte loss and contamination of sample with salts leached from glassware.
<ol>
	<li>Prepare 10% acetic acid BGE (v/v), pH 2.2 on the daily basis for metabolomics.
	<ol>
		<li>Into a 10 mL volumetric flask add 1 mL of acetic acid, followed by the addition of deionized (DI) water to the marked line and thorough mixing.</li>
	</ol>
	</li>
	<li>Prepare 100 mM acetic acid, pH 2.9 on the daily basis for proteomics.
	<ol>
		<li>Into a 10 mL volumetric flask add 57 &#181;L of acetic acid, followed by the addition of DI water to the marked line and thorough mixing.</li>
	</ol>
	</li>
</ol>
2. NM preparation
<ol>
	<li>Vigorously disperse NMs in water using published guidelines31,32,33. 
	NOTE: In the development of this protocol, 7 NMs were used as follows: 100 and 1,000 nm carboxylated polystyrene were used for protein and metabolite corona, respectively. 100 nm unmodified polystyrene NMs, 13 nm anatase TiO2 NMs which were unmodified or coated with either polyacrylate or PVP polymers and 22 nm unmodified SiO2 NMs were used for both protein and metabolite coronas. While the method was developed and demonstrated using these NMs, it should be noted that this approach would be applicable for a wide array of NM compositions, sizes, shapes, and morphologies.</li>
	<li>Characterize particle size using either dynamic light scattering34,35, single particle inductively coupled plasma mass spectrometry36, nanoparticle tracking analysis37, or transmission electron microscopy and surface charge as zeta potential using a zeta sizer34.</li>
</ol>
3. Biomolecular corona formation
<ol>
	<li>Split 2 mL of human plasma (or biofluid of choice) into 1 mL aliquots, one for the metabolite and protein corona and the second for plasma metabolome characterization.</li>
	<li>Incubate 1 mg/mL of NMs in the human plasma for 1 h at 37 &#730;C while gently mixing at 500 rpm in a thermomixer. Following incubation centrifuge the sample at 4,000 x g for 15 min at 4 &#730;C to pellet the NMs.</li>
	<li>Collect the plasma supernatant for metabolite corona analysis. Retain the NM-protein corona complex for protein corona characterization.</li>
</ol>
4. Protein corona isolation
<ol>
	<li>Resuspend the NM-protein complex (the pellet) in 1 mL of 10x PBS Buffer and vortex vigorously for 2 min to remove unbound proteins. Centrifuge the PBS solution at 4,000 x g for 15 min at 4 &#730;C to pellet the NMs, and carefully remove the supernatant without disturbing the pellet.</li>
	<li>Resuspend the pellet in 1 mL of ammonium bicarbonate buffer (ABC) (100 mM, pH 8.0) and vortex vigorously for 2 min to remove unbound proteins and undesired salts. Centrifuge at 4,000 x g for 15 min at 4 &#730;C to pellet the NMs; remove and discard the supernatant. Repeat this step.</li>
	<li>Reduce protein disulfide bonds by dissolving the NM-protein pellet in 20 &#181;L of ABC buffer (100 mM pH 8) containing 10 mM dithiotheritol. Incubate the reduction solution for 30 min at 56 &#730;C.</li>
	<li>To digest proteins, add 2 &#181;g of sequencing grade Trypsin to 20 &#181;L of ABC containing 0.1% surfactant. Incubate the digest solution for 16 h at 37 &#730;C.</li>
	<li>Alkylate the sample by the addition of 20 &#181;L of iodoacetamide at 55 mM in 100 mM ABC and incubate at room temperature for 20 min.</li>
	<li>Add 20 &#181;L of 0.1 M HCl to cleave the surfactant and leave for 10 min at room temperature.</li>
	<li>Enrich and desalt peptides using a C18 packed desalting pipette tip.
	<ol>
		<li>Wet the tip with 80 &#181;L 0.1% formic acid 50% acetonitrile 5 times, clean with 80 &#181;L 0.1% formic acid 5 times, draw up and elute sample 10 times, desalt sample by flushing 80 &#181;L 0.1 % formic acid 5 times and elute the sample 2 times with 80 &#181;L 0.1% formic acid 70% acetonitrile into a clean low binding PCR tube.</li>
	</ol>
	</li>
	<li>Lyophilize the samples and store at -20 &#730;C until analysis.</li>
</ol>
5. Metabolite corona isolation
<ol>
	<li>Take the control plasma and the supernatant from the NM plasma incubation from Step 3.3 and keep on ice during sample preparation.</li>
	<li>Take 50 &#181;L from each into separate vials and dilute 1 in 10 with DI water. Take 50 &#181;L of each diluted sample and move to new vials for step 5.3.</li>
	<li>Add 200 &#181;L chloroform, 250 &#181;L methanol and 350 &#181;L DI water and vigorously vortex mixture for 2 min. Centrifuge for 10 min at 20,800 x g at 4 &#730;C.</li>
	<li>Take 500 &#181;L of supernatant and filter through a 3 kDa centrifugal filter for 2 h at 10,000 x g at 4 &#730;C. Take 430 &#181;L of ultrafiltrate and dry in a speed vac. Samples can now be frozen prior to analysis.</li>
</ol>
6. Preparation of CESI-MS system38
NOTE: Prior to the installation of the capillaries, check that the inlet and outlet end of the capillaries are intact and that there are no visible breakages in the capillary.
<ol>
	<li>Installation of a neutral coated capillary for protein corona analysis39.
	<ol>
		<li>Place a new neutral coated capillary (90 cm length with 30 &#181;m internal diameter) into the CESI instrument following the manufacturer guidelines.</li>
	</ol>
	</li>
	<li>Preparation of CESI capillaries for proteomic analysis.
	<ol>
		<li>Flush the separation capillary in the forward direction using 100 mM of HCl for 5 min at 100 psi and check for droplet formation at the outlet of the capillary. Flush separation capillary with BGE at 100 psi for 10 min and then DI water at 100 psi for 30 min (this is only required for new capillaries).</li>
		<li>Flush both the separation capillary and the conductive capillary with BGE at 90 psi for 5 min and ensure droplets form at the outlet end of both capillaries.</li>
		<li>Flush the separation capillary with DI water at 90 psi for 10 min, then 50 psi for 10 min with 0.1 M HCl, followed again by 90 psi for 10 min with DI water and finally BGE at 90 psi for 10 min. Couple CESI to the MS using a nanospray source and adapter as previously described38.</li>
	</ol>
	</li>
	<li>Installation of bare fused silica capillary for metabolite corona characterization38.
	<ol>
		<li>Place a new bare fused silica capillary (90 cm length with a 30 &#181;m internal diameter) into the CESI instrument following manufacturer guidelines.</li>
	</ol>
	</li>
	<li>Preparation of CESI capillaries for metabolomics analysis.
	<ol>
		<li>Flush the separation capillary in the forward direction using 100% MeOH at 50 psi for 15 min and check for droplet formation at the outlet of the capillary. Flush both the separation capillary and the conductive capillary with BGE at 90 psi for 5 min and ensure droplets form at the outlet of both capillaries.</li>
		<li>Flush the separation capillary with DI water at 90 psi for 10 min, then 50 psi for 10 min with 0.1 M NaOH, followed again by 90 psi for 10 min with DI water and finally BGE at 90 psi for 10 min. Couple CESI to the ESI-MS using a nanospray source and adapter as previously described.38</li>
	</ol>
	</li>
</ol>
7. CE-MS for protein corona analysis
<ol>
	<li>Resuspend samples from step 4.8 in 20 &#181;L of 50 mM ammonium acetate pH 4.0.</li>
	<li>Perform 5 psi 10 s hydrodynamic injection of sample. Separate using a 30 kV separation voltage with the following pressure gradient: 0-10 min at 1 psi, 10-35 min at 1.5 psi and 35-45 min at 5 psi using 100 mM, pH 2.9 acetic acid as BGE.</li>
	<li>Collect mass spectra between 250-2000 m/z with top-10 data dependent fragmentation acquisition.</li>
	<li>Between samples, rinse capillary with 0.1 M HCl for 3 min at 100 psi and 10 min 100 psi of BGE for separation capillary and for 3 min at 100 psi for the conductive liquid capillary.</li>
</ol>
8. CESI-MS for metabolite corona analysis
NOTE: All samples must be analyzed twice, once in positive mode for the detection of cations and once in negative mode for the detection of anions. The control plasma samples need to be analyzed at least 5 times.
<ol>
	<li>Resuspend NM exposed and unexposed plasma samples from step 5.4 in 430 &#181;L of DI water and vigorously vortex for 2 min. Filter through a 0.1 &#181;m membrane filter.</li>
	<li>Take 95 &#181;L of filtrate and add 5 &#181;L of internal standards at 200 &#181;M for cations (L-methionine sulfone) and 400 &#181;M for anions (2,2,4,4-D4-citric acid) and vortex vigorously. Centrifuge at 4,000 x g at 4 &#730;C for 10 min prior to CESI-MS analysis.</li>
	<li>Inject sample hydrodynamically for 30 s (cations) and 40 s (anions) at 2 psi.</li>
	<li>Separate metabolites in 10% acetic acid with a separation voltage of 30 kV in forward (cations) and reverse (anions) mode. Reverse separation mode is assisted with 0.5 psi forward pressure on the separation capillary.</li>
	<li>Set mass spectrometer to collect data in positive (cations) or negative (anions) mode over the mass range 65-1000 m/z. Between samples, rinse capillary with 0.1 M HCl, 0.1 M NaOH, DI water and BGE at 50 psi for 2 min.</li>
</ol>

J.A.T is an employee of AB Sciex UK Ltd whom are participating as an industry partner on the ACEnano project, J.A.T and all other authors have no conflict of interest in this work.

This is the first method proposed to characterize the complete biomolecular corona incorporating proteins and metabolites, from the same sample, using CESI-MS. The ability to characterize both the proteins and metabolites from the same sample will significantly increase the amount of data collected from a single experimental exposure enabling new insights into the process of corona formation and its impacts for NMs toxicity and efficacy as nanomedicines. Furthermore, CESI-MS is an ideal platform to characterize both classes of biomolecules with just a change of capillary required. Moving forward, new methods developed for non-aqueous CE will enable lipidomics to be performed using CE-MS40 thus it is now possible to analyze proteins, metabolites and lipids using a single platform. The current conventional approaches would require two dedicated LC-MS platforms, one for the protein corona and one for the metabolite corona. Thus, this approach can collect twice as much data using a single analytical platform.
There are some caveats to this approach particularly with the metabolite corona as this protocol proposes an indirect measurement of the corona. As such, problem may arise when the metabolite levels are present at high concentrations relative to the NMs and the subsequent drop in metabolite concentration in the matrix is small. However, unlike with the protein corona, it is unlikely that a &#8220;one size fits all&#8221; approach to isolating the metabolite corona will be developed due to each NM having unique surface chemistries. Going forward it is more likely that NM specific approaches to isolate the metabolite corona will be developed and validated however; this will be applicable to only that specific NM. Despite this, the CESI-MS will still be a highly suitable platform for the characterization of the polar charged metabolites regardless of how they are isolated. Also noteworthy is that if a pellet does not form during the centrifugation steps designed to pellet the NMs, a higher centrifugal force may be required; this is most likely to occur with less dense NMs. It is also critical that all filtering steps are adhered to within the protocol due to the narrow bore of the capillaries these can easily become blocked if any particulate matter has not been adequately eliminated from the sample.
While the protein corona has been an intense area of research for a number of years the ability to combine that with the metabolite corona is a new field of interest for scientists investigating the bio-nano interface6,14. To date, the majority of these studies have focused upon the non-polar, lipid fraction of the metabolite corona9,28. This current method enables the characterization of the highly polar and charged metabolites in the corona which includes important metabolites involved in energy metabolism, glycolysis, and DNA synthesis. As a result, when combined with the proteomics workflow, a more holistic characterization of the biomolecular corona is possible. This enables a more in-depth analysis of bio-nano interactions moving forward. This method enables enhanced mechanistic insights into receptor-mediated endocytosis via protein and metabolite interactions with membrane receptors. Furthermore, inter and intra corona interactions between proteins and small molecules may be explored; for example, it has been recently shown that the proteins in the corona play a key role in the recruitment of metabolites15. It is worth noting that the representative results in Figure 3 and Figure 4 are absolute quantification of metabolites, however, an untargeted approach using multivariate data analysis to compare all CE-MS features in controls compared to exposed plasma would also elucidate metabolite binding. Thus, there is a significant scope to investigate how, and which, metabolites and proteins influence their respective recruitment into NM coronas. These additional studies may help design coronas to optimize delivery of nanomedicines or uncover further hurdles to their development such as the suppression of pharmaceutical action because of the biomolecular corona blocking or masking targeting functionalities.
CESI-MS with its nano scale flow rates of around 20 nL/min and minimal use of organic solvents offers an improvement in green and economic credentials over standard LC-MS and nanoLC-MS in terms of solvent use, the main challenges for metabolomics and proteomics studies, respectively. CESI-MS offers highly reproducible results for both migration time and peak area which compare favorably with LC-MS based methods11,15. Furthermore, compared to nanoLC-MS, carry-over is not an issue in CESI-MS. Therefore, extensive system clean-up between sample analyses is not required, which greatly improves sample throughput11.
In summary, the proposed workflow is the first to detail an approach to characterize both the protein and metabolite components of the complete biomolecular corona of NMs. This unlocks a new aspect of bio-nano interactions, the metabolite corona, thus enabling the collection of twice as much data from the same sample, enabling a more complete understanding of interactions at the bio-nano interface.

Bio-nano interactions, at the interface between nanomaterial (NM) surfaces and biological matrices from which biomolecules adsorb to the NM, is an intense area of research underpinning nanosafety and nanomedicine1. This layer of adsorbed biomolecules confers a biological identity to NMs, thus cells identify them as &#8220;self&#8221;, subsequently influencing cellular uptake, distribution and biological endpoints2,3,4. The vast majority of bio-nano studies have focused upon the proteins within the corona, and have demonstrated that proteins vary quantitatively and qualitatively between NMs of different compositions5. This variation is dependent upon both the properties of the NM such as size, functionalization, and material in addition to the properties of the biological matrix including composition and salt content5. A rising area of interest in the bio-nano interface are the metabolites that adsorb to NMs6. Several studies have demonstrated that, as with the proteins, NM properties are a key factor in determining the composition of the metabolites in the corona and that they can influence biological outcomes of NM exposure6,7,8.
In studies of the biomolecular corona there are four distinct phases to its characterization, corona formation, the isolation of the biomolecules within the corona, their detection via qualitative or quantitative mass spectrometry and identification9. To date, a range of techniques have been adopted to isolate the protein corona including boiling in SDS-PAGE running buffer, solvent and salt extractions or direct digestion of proteins in situ on the surface of NMs. In terms of the metabolites in the corona, the isolation is more complex with various methods using solvents or even the dissolution of the NMs proposed as solutions8,10,11. However, unlike the protein corona a &#8220;one size fits all&#8221; approach is unlikely to apply to the isolation of metabolites from NMs, due to the wide chemical space occupied by the metabolome and the diversity of possible NMs. An alternative approach is to characterize the biological matrix before and after NM exposure with the difference in metabolite concentrations attributed to the adsorption of metabolites to NMs.12
This alternative approach would be applicable to all biofluids and NMs and although it does require twice as much analysis, it consequently offers a much more precise measure of the metabolite corona and has no risk of low metabolite recoveries from the NM surface being mistaken for low binding of the specific metabolite.
The second step to the biomolecular corona analysis is the detection of the biomolecules. Traditionally for the proteins in the corona, this has been the remit of nano-LC-MS, the workhorse of proteomics; other approaches such as NMR13, 1D and 2D SDS-PAGE have also been applied. In terms of the metabolite corona LC-MS7, GC-MS8 and direct infusion mass spectrometry have been utilized14. However, a new approach has recently started to gain traction, namely capillary electrophoresis-mass spectrometry (CE-MS)11,15 and has been present in NM labs as a standalone technique to characterize various physical and chemical properties of NMs16. CE-MS is an orthogonal separation technique to nanoLC-MS and GC-MS and is capable of enabling the detection of highly polar and charged metabolites17,18. Furthermore, CE-MS is well suited for the analysis of proteins and their posttranslational modifications such as phosphorylation and glycosylation19,20,21,22. The final step, identification and data analysis can be performed in several ways depending upon if a quantitative / qualitative or targeted / untargeted approach is being used, this, however, falls outside the remit of this protocol.
CESI-MS, a combination of CE and a nanoESI interface, is a recent advance in CE technology utilizing a sheathless interface. This enables direct connection of ultra-low flow CE (&#60;20 nL/min) to a high resolution mass spectrometer with no dilution, resulting in significantly improved detection sensitivity23,24,25. CESI-MS enables volume limited samples (&#60; 10 &#181;L) to be analyzed while maintaining a highly sensitive analysis26. CESI-MS also compares favorably to traditional low flow rate nanoLC-MS approaches used in proteomics and metabolomics in terms of reproducibility27,28, throughput, and carry over making it an exciting prospect for the complete biomolecular corona characterization.
To highlight the potential of CESI-MS for the analyses of bio-nano interactions this work describes the sample preparation required to isolate the NM biomolecular corona from a single human plasma sample in such a way as to maximize data from both the protein and metabolite aspects of the complete biomolecular NM corona. While we report on the use of human plasma, this protocol would be equally appropriate for other biofluids such as blood serum, complete cell culture medium, cell lysate, cerebrospinal fluid or urine. The analyses of these two components (proteins and metabolites) will then be described using neutral coated CESI capillaries for the protein corona and bare fused silica capillaries for both cationic and anionic metabolites.

<table><tbody><tr><td>1,4-Dithiothreitol</td><td>Sigma</td><td>10197777001</td><td></td></tr><tr><td>2,2,4,4-D4-citric</td><td>Simga</td><td>485438-1G</td><td>Internal standard anion</td></tr><tr><td>Ammonium Acetate &gt;98%</td><td>Sigma</td><td>A1542</td><td></td></tr><tr><td>Ammonium Bicarbonate &gt;99.5%</td><td>Sigma</td><td>09830</td><td></td></tr><tr><td>Capillary cartridge coolant</td><td>Sciex</td><td>359976</td><td></td></tr><tr><td>CESI 8000 instrument</td><td>Sciex</td><td>A98089</td><td></td></tr><tr><td>CESI Cap</td><td>Sciex</td><td>B24699</td><td></td></tr><tr><td>CESI Vials</td><td>Sciex</td><td>B11648</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>650498</td><td>Toxic, use in a fume hood</td></tr><tr><td>Glacial Acetic acid</td><td>Sigma</td><td>A6283</td><td></td></tr><tr><td>Hydrochloric acid 1mol/L</td><td>Sigma</td><td>43617</td><td></td></tr><tr><td>Iosoacetamide</td><td>Sigma</td><td>I1149</td><td></td></tr><tr><td>L-methionine sulfone</td><td>Sigma</td><td>M0876-1G</td><td>Internal standard cation</td></tr><tr><td>Methonal (LC-MS Ultra Chromasolve)</td><td>Sigma</td><td>14262</td><td>Use in a fume hood</td></tr><tr><td>Microvials</td><td>Sciex</td><td>144709</td><td></td></tr><tr><td>nanoVials</td><td>Sciex</td><td>5043467</td><td></td></tr><tr><td>Neutral Surface Cartridge 30 &amp;micro;M ID x 90 cm total length</td><td>Sciex</td><td>B07368</td><td></td></tr><tr><td>OptiMS Adapter for Sciex Nanospray III source</td><td>Sciex</td><td>B07363</td><td>B07366 and B83386 for Thermo mass spectrometers, B85397 for Waters mass spectrometers, B86099 for Bruker mass spectrometers</td></tr><tr><td>OptiMS Fused-Silica Cartridge 30 &amp;micro;m ID x 90 cm total length</td><td>Sciex</td><td>B07367</td><td></td></tr><tr><td>Phospahte buffered saline 10x</td><td>Sigma</td><td>P7059</td><td></td></tr><tr><td>Pierce C18 Tips, 100 &amp;micro;L bed</td><td>Thermo Fisher Scientific</td><td>87784</td><td></td></tr><tr><td>Polystyrene 100 nm</td><td>Polysciences Inc</td><td>876</td><td></td></tr><tr><td>Polystyrene carboxylated 100 nm</td><td>Polysciences Inc</td><td>16688</td><td></td></tr><tr><td>Polystyrene carboxylated 1000 nm</td><td>Polysciences Inc</td><td>08226</td><td></td></tr><tr><td>Rapigest SF (surfactant)</td><td>Waters</td><td>186001860</td><td></td></tr><tr><td>Sequencing Grade modified Trypsin</td><td>Promega</td><td>V5111</td><td></td></tr><tr><td>SiO2 Ludox TM-40</td><td>Sigma</td><td>420786</td><td></td></tr><tr><td>Sodium Hydroxide solution</td><td>Simga</td><td>72079</td><td></td></tr><tr><td>TiO2 Dispex coated</td><td>Promethion particles</td><td>Bespoke particles</td><td></td></tr><tr><td>TiO2 PVP coated</td><td>Promethion particles</td><td>Bespoke particles</td><td></td></tr><tr><td>TiO2 uncoated</td><td>Promethion particles</td><td>Bespoke particles</td><td></td></tr></tbody></table>

capillary electrophoresis mass spectrometry approaches for characterization of the protein and metabolite corona acquired by nanomaterials

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the past decade. Interest in the bio-nano interface arises from the fact that the biomolecular corona confers a biological identity to NMs and thus causes the body to identify them as &#8220;self&#8221;. For example, previous studies have demonstrated that the proteins in the corona are capable of interacting with membrane receptors to influence cellular uptake and established that the corona is responsible for cellular trafficking of NMs and their eventual toxicity. To date, most research has focused upon the protein corona and overlooked the possible impacts of the metabolites included in the corona or synergistic effects between components in the complete biomolecular corona. As such, this work demonstrates methodologies to characterize both the protein and metabolite components of the biomolecular corona using bottom-up proteomics and metabolomics approaches in parallel. This includes an on-particle digest of the protein corona with a surfactant used to increase protein recovery, and a passive characterization of the metabolite corona by analyzing metabolite matrices before and after NM exposures. This work introduces capillary electrophoresis &#8211; mass spectrometry (CESI-MS) as a new technique for NM corona characterization. The protocols outlined here demonstrate how CESI-MS can be used for the reliable characterization of both the protein and metabolite corona acquired by NMs. The move to CESI-MS greatly decreases the volume of sample required (compared to traditional liquid chromatography &#8211; mass spectrometry (LC-MS) approaches) with multiple injections possible from as little as 5 &#181;L of sample, making it ideal for volume limited samples. Furthermore, the environmental consequences of analysis are reduced with respect to LC-MS due to the low flow rates (&#60;20 nL/min) in CESI-MS, and the use of aqueous electrolytes which eliminates the need for organic solvents.

The described method is the first to characterize both the proteins and metabolites in the NM biomolecular corona using the same human plasma sample. This method is capable of detecting &#62;200 proteins and &#62;150 metabolites, thus enabling the most comprehensive overview of the complete biomolecular corona to be determined, enabling enhanced understanding of NMs cellular attachment, uptake and impacts.
The use of CESI-MS for both proteomics (Figure 1) and metabolomics (Figure 2) separation and detection shows good separation windows on both capillaries for each approach.
This method is capable of distinguishing between the proteins in the corona across a wide range of NMs compositions, thus demonstrating the methods&#8217; applicability to NMs across a broad range of physical and chemical characteristics (Table 1).
The unique fingerprints of the metabolite corona can also be distinguished using the metabolomic branch of this methodology (Figure 3). Although this approach utilizes a passive approach to characterize the NM metabolite corona it is still capable of uncovering interesting insights into the role of metabolites in the biomolecular corona such as the differential adsorption of isomers (Figure 4).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61760/61760fig01.jpg" /> 
Figure 1: Exemplar electropherogram of a SiO2 protein corona separation using CESI-MS following an on-particle digest. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61760/61760fig01large.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/61760/61760fig02.jpg" /> 
Figure 2: Example of extracted ion electropherograms obtained from endogenous compounds in plasma by CE-MS. The numbered compounds are as follows: 1. Ornithine; 2. Lysine; 3. Arginine; 4. Histidine; 5. Creatine; 6. Glycine; 7. Alanine; 8. Valine; 9. Isoleucine; 10. Leucine; 11. Serine; 12. Threonine; 13. Asparagine; 14. Methionine; 15. Glutamine; 16. Glutamic acid; 17. Phenyl-D5-alanine; 18. Phenylalanine; 19. Tyrosine; 20. Proline; 21. Methionine sulfone (internal standard). Published under an open access Creative Commons CC BY license and reproduced from Zhang et al17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61760/61760fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Analysis of protein corona across an array of 6 NMs. Heat map of protein classes identified on silica (SiO2), titania (TiO2), PVP capped titania (TiO2-PVP), dispex capped titania (TiO2-Dispex), polystyrene and carboxylated polystyrene NMs. Percentages refer to the proteins&#8217; abundance relative to total protein content based upon top 3 peptides abundance for each protein. Given the differences observed in the relative abundances of proteins in the different NM coronas, it is clear that this proposed protocol is sufficiently sensitive to differentiate between the protein corona of different NMs. Figure reproduced from a CESI-MS analysis of the protein corona, published under an open access Creative Commons CC BY license11. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61760/Table_1.xlsx" target="_blank">Please click here to download this table.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61760/61760fig03.jpg" /> 
Figure 3: Targeted analysis of the cationic metabolites in the NM corona. Adsorption of cations to SiO2 and TiO2 NMs. Red refers to the initial concentration of metabolites whereas blue refers to the concentration of metabolites following a 1 h incubation with the NMs at 37 &#730;C. The reduction in metabolite concentration in the solution is a result of metabolite adsorption to the NM surface. No significant adsorption of metabolites was observed for the polystyrene NMs. Box plots represent the minimum and maximum values, median and interquartile ranges. Figure reproduced from a CESI-MS targeted analysis of the metabolite corona published under an open access Creative Commons CC BY license15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61760/61760fig03large.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/61760/61760fig04.jpg" /> 
Figure 4: Targeted analysis of the anionic metabolites in the NM corona with a focus on isomer adsorption. Adsorption of anions to a range of titania NMs shows clear differences between various isomers such as the sugar phosphates. Red refers to the initial concentration of metabolites whereas blue refers to the concentration of metabolites following a 1 h incubation with the NM at 37 &#730;C. The reduction in metabolite concentration in solution is a result of metabolite adsorption to the NM surface. No significant adsorption of metabolites was observed for the SiO2 and the polystyrene NMs. Box plots represent the minimum and maximum values, median and interquartile ranges. Figure reproduced from a CESI-MS targeted analysis of the metabolite corona published under an open access Creative Common CC BY license15. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61760/61760fig04large.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>Analyte</td>
			<td>Mean RSD peak area</td>
			<td>Mean RSD migration time</td>
		</tr>
		<tr>
			<td>1928 peptides intraday (n=3)</td>
			<td>15%</td>
			<td>0.30%</td>
		</tr>
		<tr>
			<td>27 cations intra-day (n=16)</td>
			<td>5.80%</td>
			<td>2.20%</td>
		</tr>
		<tr>
			<td>27 cation inter-day (n=36)</td>
			<td>8.60%</td>
			<td>1.80%</td>
		</tr>
	</tbody>
</table>
Table 2: Reproducibility of CESI-MS technical replicates for peptides and cationic metabolites. RSD: relative standard deviation, as a measure of reproducibility. The reproducibility of CESI-MS in-terms of peak areas and migration times is very good, with all analytes having a mean RSD &#60;15% and migration times &#60;2.2%. These results demonstrate the high degree of confidence that can be attributed to the data collected using this technique and confirm that variations detected are due to genuine differences in sample composition (enrichment on NMs) rather than analytical variation. Table 2 is generated from results presented previously11,15.

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Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis &#8211; mass spectrometry approach.

The adsorption of biomolecules from surrounding biological matrices to the surface of nanomaterials (NMs) to form the corona has been of interest for the ...

capillary-electrophoresis-mass-spectrometry-approaches-for

Nanyang Technological University

Research

JoVE Journal

Chemistry

4.3K Views.  University of Birmingham. Hier presenteren we een protocol om de volledige biomoleculaire corona, eiwitten en metabolieten te karakteriseren, verkregen door nanomaterialen uit biofluïden met behulp van een capillaire elektroforese - massaspectrometriebenadering.

Video: Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Biochemistry

Extraction of Aqueous Metabolites from Cultured Adherent Cells for Metabolomic Analysis by Capillary Electrophoresis-Mass Spectrometry

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but also to identify biomarkers for early-stage cancer detection and prediction of chemotherapy response in cancer patients. Preparation of uniform samples for metabolomic analysis is a critical issue that remains to be addressed. Here, we present an easy and reliable protocol for extracting aqueous metabolites from cultured adherent cells for metabolomic analysis using capillary electrophoresis-mass spectrometry (CE-MS). Aqueous metabolites from cultured cells are analyzed by culturing and washing cells, treating cells with methanol, extracting metabolites, and removing proteins and macromolecules with spin columns for CE-MS analysis. Representative results using lung cancer cell lines treated with diamide, an oxidative reagent, illustrate the clearly observable metabolic shift of cells under oxidative stress. This article would be especially valuable to students and investigators involved in metabolomics research, who are new to harvesting metabolites from cell lines for analysis by CE-MS.

The purpose of this article is to describe a protocol for extraction of aqueous metabolites from cultured adherent cells for metabolomic analysis, particularly, capillary electrophoresis-mass spectrometry.

Metabolomic analysis is a promising omics approach to not only understand the specific metabolic regulation in cancer cells compared to normal cells but ...

Cancer Research

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 ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Bioengineering

Digital Microfluidics for Automated Proteomic Processing

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics.
Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work.
Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Digital Microfluidics is a technique characterized by the manipulation of discrete droplets (~nL - mL) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. Here, we report a platform capable of automating several proteomic processing steps.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and ...

Biology

UV-Vis Spectroscopic Characterization of Nanomaterials in Aqueous Media

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the nanoscale, i.e. 1&#8211;100 nm), dynamic nature, and diverse properties. At the same time, reliable and repeatable characterization is paramount to ensure safety and quality in the manufacturing of NM-bearing products. There are several methods available to monitor and achieve reliable measurement of nanoscale-related properties, one example of which is Ultraviolet-Visible Spectroscopy (UV-Vis). This is a well-established, simple, and inexpensive technique that provides non-invasive and fast real-time screening evaluation of NM size, concentration, and aggregation state. Such features make UV-Vis an ideal methodology to assess the proficiency testing schemes (PTS) of a validated standard operating procedure (SOP) intended to evaluate the performance and reproducibility of a characterization method. In this paper, the PTS of six partner laboratories from the H2020 project ACEnano were assessed through an interlaboratory comparison (ILC). Standard gold (Au) colloid suspensions of different sizes (ranging 5&#8211;100 nm) were characterized by UV-Vis at the different institutions to develop an implementable and robust protocol for NM size characterization.

This study presents the benchmarking results for an interlaboratory comparison (ILC) designed to test the standard operating procedure (SOP) developed for gold (Au) colloid dispersions characterized by ultraviolet-visible Spectroscopy (UV-Vis), amongst six partners from the H2020 ACEnano project for sample preparation, measurement, and analysis of the results.

The physicochemical characterization of nanomaterials (NMs) is often an analytical challenge, due to their small size (at least one dimension in the ...

Engineering

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 ...

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 ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

Multimodal Analytical Platform on a Multiplexed Surface Plasmon Resonance Imaging Chip for the Analysis of Extracellular Vesicle Subsets

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter and are used as a means of intercellular communication. They are emerging as promising diagnostic and therapeutic tools for a variety of diseases. There are two main biogenesis processes used by cells to produce EVs with differences in size, composition, and content. Due to their high complexity in size, composition, and cell origin, their characterization requires a combination of analytical techniques. This project involves the development of a new generation of multiparametric analytical platforms with increased throughput for the characterization of subpopulations of EVs. To achieve this goal, the work starts from the nanobioanalytical platform (NBA) established by the group, which allows an original investigation of EVs based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy (AFM) of vesicular targets trapped on a microarray biochip. The objective was to complete this EV investigation with a phenotypic and molecular analysis by Raman spectroscopy. These developments enable the proposal of a multimodal and easy-to-use analytical solution for the discrimination of EV subsets in biological fluids with clinical potential

This paper proposes a new generation of multiparametric analytical platforms with increased throughput for the characterization of extracellular vesicle subsets. The method is based on a combination of multiplexed biosensing methods with metrological and morphomechanical analyses by atomic force microscopy, coupled with Raman spectroscopy, to qualify vesicular targets trapped on a microarray biochip.

Extracellular vesicles (EVs) are membrane-derived, tiny vesicles produced by all cells that range from 50 to several hundreds of nanometers in diameter ...

Capillary Electrophoresis Mass Spectrometry Approaches for Characterization of the Protein and Metabolite Corona Acquired by Nanomaterials

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