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 &#62;98%</td>
			<td>Sigma</td>
			<td>A1542</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Bicarbonate &#62;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 &#181;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 &#181;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 &#181;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/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/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/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/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/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.

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

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 ...

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4.2K Views.  University of Birmingham. Here we present a protocol to characterize the complete biomolecular corona, proteins, and metabolites, acquired by nanomaterials from biofluids using a capillary electrophoresis – mass spectrometry approach.

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

Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1H/2H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. &#160;&#160;

Protein conformation and dynamics are key to understanding the relationship between protein structure and function. Hydrogen exchange coupled with high-resolution mass spectrometry is a versatile method to study the conformational dynamics of proteins as well as characterizing protein-ligand and protein-protein interactions, including contact interfaces and allosteric effects.

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique ...

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic acids containing three to six carbons), such as malate and citrate, are critical to the proper functioning of many biological systems, where they function in cellular respiration and can serve as indicators of cell health. In foods, organic acid content can have significant impact on taste, with increased SCCA levels resulting in a sour or &#34;acid&#34; taste. Because of this, methods for the rapid analysis of organic acid levels are of particular interest to the food and beverage industries. Unfortunately, however, most methods used for SCCA quantification are dependent on time-consuming protocols requiring the derivatization of samples with hazardous reagents, followed by costly chromatographic and/or mass spectrometric analyses. This method details an alternate method for the detection and quantification of organic acids from plant material and food samples using free zonal capillary electrophoresis (CZE), sometimes simply referred to as capillary electrophoresis (CE). CZE provides a cost-effective method for measuring SCCAs with a low limit of detection (0.005 mg/ml). This article details the extraction and quantification of SCCAs from plant samples. While the method provided focuses on measurement of SCCAs from coffee beans, the method provided can be applied to multiple plant-based food materials.

Using Capillary Electrophoresis to Quantify Organic Acids from Plant Tissue: A Test Case Examining Coffea arabica Seeds

This article presents a method for the detection and quantification of organic acids from plant material using free zonal capillary electrophoresis. An example of the potential application of this method, determining the effects of a secondary fermentation on organic acid levels in coffee seeds, is provided.

Carboxylic acids are organic acids containing one or more terminal carboxyl (COOH) functional groups. Short chain carboxylic acids (SCCAs; carboxylic ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog (Xenopus laevis) Embryos

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic development. To enable single-cell investigations directly in live embryos, new analytical approaches are needed, particularly those that are sensitive, selective, quantitative, robust, and scalable to different cell sizes. Here, we present a protocol that enables the in situ analysis of metabolism in single cells in freely developing embryos of the South African clawed frog (Xenopus laevis), a powerful model in cell and developmental biology. This approach uses a capillary microprobe to aspirate a defined portion from single identified cells in the embryo, leaving neighboring cells intact for subsequent analysis. The collected cell content is analyzed by a microscale capillary electrophoresis electrospray ionization (CE-ESI) interface coupled to a high-resolution tandem mass spectrometer. This approach is scalable to various cell sizes and compatible with the complex three-dimensional structure of the developing embryo. As an example, we demonstrate that microprobe single-cell CE-ESI-MS enables the elucidation of metabolic cell heterogeneity that unfolds as a progenitor cell gives rise to descendants during development of the embryo. Besides cell and developmental biology, the single-cell analysis protocols described here are amenable to other cell sizes, cell types, or animal models.

Microprobe Capillary Electrophoresis Mass Spectrometry for Single-cell Metabolomics in Live Frog Xenopus laevis Embryos

We describe steps that enable fast in situ sampling of a small portion of an individual cell with high precision and minimal invasion using capillary-based micro-sampling, to facilitate chemical characterization of a snapshot of metabolic activity in live embryos using a custom-built single cell capillary electrophoresis and mass spectrometry platform.

The quantification of small molecules in single cells raises new potentials for better understanding the basic processes that underlie embryonic ...

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

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

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

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

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...