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/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.

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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 ...

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4.4K 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

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

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

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

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

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

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

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 ...

Capillary Electrophoresis-based Hydrogen/Deuterium Exchange for Conformational Characterization of Proteins with Top-down Mass Spectrometry

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of protein therapeutics and the determination of the conformational transition pathways critical for biological functions, ranging from molecular recognition to enzymatic catalysis. Hydrogen/deuterium exchange (HDX) reaction coupled with top-down mass spectrometric (MS) analysis provides a means to characterize protein higher-order structures and dynamics in a conformer-specific manner. The conformational resolving power of this technique is highly dependent on the efficiencies of separating protein states at the intact protein level and minimizing the residual non-deuterated protic content during the HDX reactions. 
Here we describe a capillary electrophoresis (CE)-based variant of the HDX MS approach that aims to improve the conformational resolution. In this approach, proteins undergo HDX reactions while migrating through a deuterated background electrolyte solution (BGE) during the capillary electrophoretic separation. Different protein states or proteoforms that coexist in solution can be efficiently separated based on their differing charge-to-size ratios. The difference in electrophoretic mobility between proteins and protic solvent molecules minimizes the residual non-deuterated solvent, resulting in a nearly complete deuterating environment during the HDX process. The flow-through microvial CE-MS interface allows efficient electrospray ionization of the eluted protein species following a rapid mixing with the quenching and denaturing modifier solution at the outlet of the sprayer. The online top-down MS analysis measures the global deuteration level of the eluted intact protein species, and subsequently, the deuteration of their gas-phase fragments. This paper demonstrates this approach in differential HDX for systems, including the natural protein variants coexisting in milk.

Presented here is a protocol for a capillary electrophoresis-based hydrogen/deuterium exchange (HDX) approach coupled with top-down mass spectrometry. This approach characterizes the difference in higher-order structures between different protein species, including proteins in different states and different proteoforms, by conducting concurrent differential HDX and electrophoretic separation.

Resolving conformational heterogeneity of multiple protein states that coexist in solution remains one of the main obstacles in the characterization of ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

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

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

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

Biology

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

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

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

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

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Discovering Protein Interactions and Characterizing Protein Function Using HaloTag Technology

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous proteomes, including those from viruses, bacteria, and yeast.&#160; In comparison, analysis of the human proteome lags behind, partially due to the sheer number of proteins which must be studied, but also the complexity of networks and interactions these present. To specifically address the challenges of understanding the human proteome, we have developed HaloTag technology for protein isolation, particularly strong for isolation of multiprotein complexes and allowing more efficient capture of weak or transient interactions and/or proteins in low abundance. &#160;HaloTag is a genetically encoded protein fusion tag, designed for covalent, specific, and rapid immobilization or labelling of proteins with various ligands. Leveraging these properties, numerous applications for mammalian cells were developed to characterize protein function and here we present methodologies including: protein pull-downs used for discovery of novel interactions or functional assays, and cellular localization. We find significant advantages in the speed, specificity, and covalent capture of fusion proteins to surfaces for proteomic analysis as compared to other traditional non-covalent approaches. We demonstrate these and the broad utility of the technology using two important epigenetic proteins as examples, the human bromodomain protein BRD4, and histone deacetylase HDAC1.&#160; These examples demonstrate the power of this technology in enabling &#160;the discovery of novel interactions and characterizing cellular localization in eukaryotes, which will together further understanding of human functional proteomics. &#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;

HaloTag technology is a multifunctional technology which has shown significant success in isolation of both small and large protein complexes from mammalian cells.&#160; Here we highlight the advantages of this technology compared to existing alternatives and demonstrate its utility to study numerous aspects of protein function inside eukaryotic cells.

Research in proteomics has exploded in recent years with advances in mass spectrometry capabilities that have led to the characterization of numerous ...

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

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