Dr. Rawi Ramautar would like to acknowledge the financial support of the Veni grant scheme of the Netherlands Organization of Scientific Research (NWO Veni 722.013.008).

NOTE: The protocol described here for the use of sheathless CE-MS for metabolic profiling studies is for laboratory use only. The procedures outlined below are based on recently published work4,5. Further experimental details can be found in these papers. Prior to using this protocol, consult all relevant material safety data sheets (MSDS). Please use all appropriate laboratory safety procedures, including safety glasses, lab coat and gloves, when conducting the experiments outlined in this protocol.
1. Preparation of Reagents Solutions and Samples
<ol>
	<li>Preparation of the background electrolyte (BGE)
	<ol>
		<li>Prepare a new BGE solution (10% (v/v) acetic acid, pH 2.2) every day.</li>
		<li>Add 9.0 ml of water into a 10 ml glass vial and add 1.0 ml of acetic acid to the water in a fume hood. Mix the solution thoroughly using a vortex.</li>
	</ol>
	</li>
	<li>Preparation of metabolite standard mixture
	<ol>
		<li>Dissolve 50 &#181;l of a 50 &#181;M cation standard mixture containing 60 cationic metabolites into 50 &#181;l of water and mix the solution thoroughly. Store at -80 &#176;C when not in use.</li>
		<li>Dissolve 50 &#181;l of a 50 &#181;M anion standard mixture containing 30 anionic metabolites into 50 &#181;l of water and mix the solution thoroughly. Store this solution, in aliquots to prevent freeze/thaw cycles of the same standard mixture, at -80 &#176;C when not in use. 
		NOTE: The metabolite standard mixtures are stable for 3 months when stored properly at -80 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of extracts from the glioblastoma cell line
	<ol>
		<li>Wash the adherent human U-87 MG glioblastoma cells three times with 1 ml ice-cold 0.9% sodium chloride solution21.</li>
		<li>Add 2 ml ice-cold methanol/water solution (8/2, v/v) to the adherent cells and scrape using a rubber tipped cell scraper21.</li>
		<li>Collect the methanol/water solution in a tube and ultrasonicate for 2 min.</li>
		<li>Add chloroform to the methanol/water fraction (final ratio 8/8/2, v/v/v) and centrifuge the sample for 10 min at 16,100 x g and 4 &#176;C.</li>
		<li>Collect the methanol/water layer and evaporate this fraction using a vacuum concentrator. Reconstitute the dried material in 50 &#181;l water for analysis by sheathless CE-MS. When not in use store the sample at -80 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Setting up the Sheathless CE-MS System
<ol>
	<li>Installation of the bare fused-silica cartridge with the porous tip emitter
	<ol>
		<li>Place a new bare fused-silica cartridge with a porous tip emitter (30 &#181;m inner diameter x 90 cm total length) in the CE instrument.</li>
		<li>Apply a forward rinse at 50 psi for 15 min with the software controlling the CE instrument using 100% methanol and visually check whether liquid is flowing out from the capillary outlet during this rinsing step. Also perform a rinse in the opposite direction at 50 psi for 5 min using the BGE solution to visually examine whether liquid is flowing out from the conductive capillary.</li>
		<li>Repeat step 2.1.2 at a pressure of 100 psi in case no liquid drop formation has been observed at the capillary outlet. Install a new bare fused-silica cartridge if no liquid drop formation was observed during this step.</li>
		<li>Rinse the separation capillary with water at 50 psi for 10 min, followed by 0.1 M NaOH at 50 psi for 10 min, then by water at 50 psi for 10 min and finally with BGE at 50 psi for 10 min. 
		NOTE: Steps 2.1.1 until 2.1.4 are only required for the installation of a new capillary cartridge.</li>
	</ol>
	</li>
	<li>Coupling of the capillary porous tip emitter to ESI-MS 
	NOTE: Prior to coupling the CE capillary to MS, ensure that the MS instrument has been calibrated and connected to the CE system. Set the ESI voltage to 0. Fit the MS instrument with a nanospray source. Gas 1, gas 2 and interface heater temperature were not applied as ESI at very low flow rates occurs by just applying the ion spray voltage set at 1500 V. Set the curtain at 5 psi.
	<ol>
		<li>Remove the sprayer tip of the fused-silica cartridge from the water tube and install it in the nanospray source adapter for coupling to the MS instrument. Ensure that the height of the BGE vials in the CE instrument match the height of the sprayer tip.</li>
		<li>Check for flow of liquid through the conductive capillary by rinsing with BGE at 50 psi for 5 min. During this rinsing step a drop formation at the base of the ESI sprayer needle should be observed.</li>
		<li>Flush the separation capillary with BGE at 50 psi for 10 min in the forward direction. Drop formation should be observed at the tip of the porous tip emitter (sprayer tip) during this step.</li>
		<li>Position the porous tip emitter to the entrance of the MS inlet at a distance of circa 2 to 3 mm.</li>
		<li>Apply a voltage of 30 kV using a ramp time of 1 min and start acquiring MS data in the m/z range from 65 to 1,000 m/z for metabolic profiling studies using first an ESI voltage of 0. 
		NOTE: The mass spectrum should be void of signal as there should be no electrospray.</li>
		<li>Set the ESI voltage to 1,000 V while carry on measuring data. Increase the ESI voltage with increments of 200 V until a constant background signal is observed for at least 15 min.</li>
		<li>Optimize the porous tip emitter position with respect to the center of the MS inlet by moving it in the x, y, or z-direction in order to see which position provides the maximal and most stable MS signal (total ion electropherogram).</li>
		<li>After optimizing the position of the porous tip emitter and determining the optimal ESI voltage, set the ESI voltage to 0 V and decrease the CE voltage from 30 kV to 1 kV using a ramp time of 5 min.</li>
		<li>Create a MS method using the optimal ESI voltage and a CE method on the CE instrument for the analysis of metabolite standards and biological samples.</li>
	</ol>
	</li>
</ol>
3. Analysis of Metabolite Standards and Biological Samples
<ol>
	<li>Performance evaluation of the sheathless CE-MS system
	<ol>
		<li>Transfer 20 &#181;l of the anionic metabolite standard mixture into an empty 100 &#181;l microvial (PCR vial) which fits into a CE vial and put this vial in the inlet sample tray. 
		NOTE: The minimum volume required in the microvial for a reliable injection is 2 &#181;l.
		<ol>
			<li>Start the MS acquisition negative ion mode method created during step 2.2.9 and subsequently start the CE sequence using the software controlling the CE instrument.</li>
			<li>Rinse the separation capillary with BGE at 50 psi for 3 min followed by injection at 2.0 psi for 60 sec (20 nl corresponding to 3% of the capillary volume) and then by BGE injection at 1.0 psi for 10 sec. 
			NOTE: Subsequently, MS data acquisition is triggered.</li>
			<li>Apply a voltage of -30 kV with a ramp time of 1.0 min with a pressure of 0.5 psi for 30 min at the inlet. After a 30 min electrophoretic separation, stop MS data acquisition and decrease the CE voltage to -1 kV using a ramp time of 5 min (a gradual decrease of the CE voltage after the electrophoretic separation improves the durability of the porous tip capillary emitter).</li>
		</ol>
		</li>
		<li>Between sample injections, rinse the capillary with water, 0.1 M sodium hydroxide, water and BGE each at 30 psi for 3 min.</li>
		<li>Analyze the recorded data by determining the migration times and the signal intensity of the analyzed anionic metabolite mixture.</li>
		<li>Assess whether the anionic metabolite standards appear in the region between 10 and 28 min.</li>
		<li>Check whether three structurally related isomers, i.e., D-Glucose-1-phosphate, D-Glucose-6-phosphate and D-Fructose-6-phosphate are partially separated, i.e., the resolution between the first two peaks is circa 0.75 and of the last two peaks is circa 0.50 (see Figure 1). 
		NOTE: A resolution of 1.5 indicates a baseline separation of two adjacent peaks.</li>
		<li>Repeat steps 3.1.1 and 3.1.2 for the cationic metabolite mixture. Ensure that MS detection is now in positive ion mode and CE voltage is +30 kV.</li>
		<li>Analyze the recorded data by determining the migration times and the signal intensity of the analyzed cationic metabolite mixture.</li>
		<li>Assess whether the cationic metabolite standards appear in the region between 8 and 22 min. Check whether isoleucine and leucine are migrating between 15 and 15.5 min and determine if the resolution is circa 0.5.</li>
	</ol>
	</li>
	<li>Analysis of biological samples
	<ol>
		<li>Repeat steps 3.1.1 and 3.1.2 for anionic metabolic profiling of the extract of the glioblastoma cell line. 
		NOTE: The metabolic content obtained after sample pretreatment corresponds to a cell density of circa 20 cells/nl, therefore, a 20 nl injection is the equivalent of 400 cells per analysis. .</li>
		<li>Create an extracted ion electropherogram for the metabolite lactic acid (m/z 89.0243) using 5 mDa mass accuracy and check whether the signal intensity is above 100,000 counts.</li>
		<li>Repeat steps 3.1.1 and 3.1.2 for cationic metabolic profiling of the extract of the glioblastoma cell line. 
		NOTE: A highly information-rich total ion electropherogram should be observed for a 20 nl injection in this mode.</li>
		<li>After the analyses or when not in use, rinse the capillary with water at 50 psi for 15 min and store the inlet part of the capillary in a vial containing water and the porous section (outlet part) in a tube also containing water.</li>
	</ol>
	</li>
</ol>

<ol>
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We have nothing to disclose.

A sheathless CE-MS method employing a porous tip emitter has been presented for the analysis of highly polar and charged metabolites. A unique feature of this approach is that anionic or cationic metabolites can be profiled by only switching the MS detection and CE voltage polarity. A wide range of highly polar and charged metabolites in biological samples can be analyzed with a high separation efficiency, which is crucial for structurally similar metabolites, and with limits of detection in the (low) nanomolar range. The presented protocol focused on the use of sheathless CE-MS for metabolic profiling of cell extracts in order to exemplify the utility of the method for metabolic profiling of a biological sample. The approach described here can also be used for metabolic profiling of other types of biological samples, such as human urine5, given that a proper sample pretreatment procedure is used.
The sheathless CE-MS method is based on a porous tip emitter which allows the usage of the intrinsically low-flow property of CE. In this context, a stable ESI signal is a pre-requisite for reproducible metabolic profiling studies. Thus, it is important that the sprayer tip is properly positioned in front of the MS inlet. In this set-up, the ESI process is mainly dependent on the nature of the BGE and therefore, BGE optimization is critical. The sheathless configuration is less versatile in comparison to sheath-liquid CE-MS systems where all kinds of sheath-liquid compositions can be added to improve the ionization efficiency. The sheathless ESI sprayer needle needs to be completely filled with conductive liquid (i.e., BGE solution). An unstable ESI signal may result from a partial or fully plugged capillary. Rinsing at high pressures with BGE may solve this issue. Otherwise the separation capillary needs to be replaced. Prior to assessment of the analytical performance, a stable ESI background signal should be generated first which is consistent from one day to another.
The analytical performance of the sheathless CE-MS method for metabolic profiling studies needs to be checked daily using metabolite standard mixtures. Under the same experimental conditions, consistent migration times, i.e., variation below 3% for within-day (n=10) and between-day (n=5) using a 20 nl injection of a metabolite standard mixture (12.5 &#181;M), peak heights/areas (variation below 15%) and plate numbers (ranging between 60,000 and 400,000) should be obtained. Limits of detection should be in the nanomolar range for most metabolite standards. Only when these criteria are met is the method ready for metabolic profiling of biological samples. If not, the MS instrument needs to be tuned and re-calibrated or the porous tip capillary emitter needs to be changed.
An effective rinsing step between CE-MS analyses is of high importance, not only to prevent potential carryover but also to maintain the separation performance. Potential carryover may be caused by BGE vials contaminated with the sample and therefore solved by replacement with new BGE vials. When the sheathless CE-MS method is not in use, it is important to disconnect the separation capillary and to store the inlet side of the capillary in water and the outside submerged with the protective sleeve in a tube containing water to prolong capillary lifetime.
In summary, the proposed sheathless CE-MS method shows a strong potential for metabolic profiling of biological samples when used according to the procedures reported in this protocol. At this stage, inter-laboratory comparison data are definitely needed for sheathless CE-MS in order to assess the (long-term) reproducibility and robustness of this approach for metabolomics. This protocol may stimulate such a study. Various analytical challenges still need to be considered. For optimal performance, the CE current should be kept preferably below 5 &#181;A and at this stage the capillary porous tip emitters are only provided at a length of 91 cm which may hamper the development of high-throughput assays. Moreover, a low pH separation buffer was used for anionic metabolic profiling which may not be the most optimal for achieving a baseline separation of structurally related sugar phosphates. Also important is that only anionic metabolites can be analyzed which are (partially) negatively charged under the used separation conditions. The next step is to assess the utility of the sheathless CE-MS method for clinical metabolic profiling studies as currently, a single porous tip capillary emitter can only be used for the analysis of up to 100 biological samples.
Overall, further development in the sheathless CE-MS approach will open a new direction in the field of metabolomics, i.e., towards a deeper understanding of biological functions in sample-restricted cases.

In contemporary metabolomics, high end analytical separation techniques are used to analyze a wide range of metabolite classes in order to obtain a representative read-out of the physiological status of an organism1. The ultimate objective of a metabolomics study is to obtain an answer to a given biological/clinical question. At present, the Human Metabolome Database is comprised of more than 40,000 metabolite entries representing both endogenous and exogenous compounds (the latter originating from nutrients, microbiota, drugs and other sources)2. Given the huge diversity in physico-chemical properties and concentration range of these metabolites, multiple analytical techniques with different separation mechanisms should be used in conjunction in order to profile as many metabolites as possible in a given biological sample. For example, Psychogios et al. used a combination of five analytical separation techniques for metabolic profiling of human serum resulting in the detection of more than 4,000 chemically diverse metabolites3.
In this paper, attention will be paid to recently developed CE-MS strategies for metabolic profiling of biological samples4,5. In CE, more specifically capillary zone electrophoresis (CZE; normally referred as CE), compounds are separated on the basis of their charge-to-size ratio and, therefore, this analytical technique is highly suited for the analysis of polar and charged metabolites. The separation mechanism of CE is fundamentally different from chromatographic-based techniques, thereby providing a complementary view on the metabolic composition of biological samples6-8. Soga and co-workers were the first to show the utility of CE-MS for the global profiling of metabolites in biological samples9,10. Until now, the feasibility and usefulness of CE-MS for metabolomics has been widely demonstrated11-15. CE is generally coupled to MS via a sheath-liquid interfacing technique16,17; however, due to dilution of the capillary effluent by the sheath-liquid, the detection sensitivity is intrinsically compromised.
Recently, it was demonstrated that the use of a sheathless interface significantly improved the detection coverage of metabolites present in various biological samples as compared to CE-MS utilizing a classical sheath-liquid interface5,18,19. For example, circa 900 molecular features were detected in human urine by sheathless CE-MS whereas about 300 molecular features were observed with sheath-liquid CE-MS5. The sheathless interface used was based on a porous tip emitter, which was invented by Moini20, allowing the effective use of the intrinsically low-flow property of CE in combination with nano-ESI-MS.
In order to stimulate the use of sheathless CE-MS in the field of metabolomics, a protocol is presented describing how this approach can be used for the analysis of highly polar metabolites in biological samples, as exemplified for the analysis of extracts from the glioblastoma cell line. It is shown that the sheathless CE-MS method for the profiling of cationic metabolites can also be used for the profiling of anionic metabolites using exactly the same capillary and separation conditions, thereby reducing analysis time and providing one single analytical platform for the global profiling of charged metabolites. The protocol also describes a strategy for the effective alignment of the sheathless porous tip emitter with the MS instrument.

<table border="0" cellpadding="0" cellspacing="0" width="958">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CESI 8000 instrument</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">A98089</td>
			<td style="width:395px;">OptiMS adapter required to couple CESI to MS</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">OptiMS Fused-Silica Cartridge, 30 &#956;m ID x 90 cm total length</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">B07367</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">OptiMS Adapter for Sciex Nanospray III source</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">B07363</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CESI vials</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">B11648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro vials</td>
			<td style="width:167px;">Sciex</td>
			<td style="width:181px;">144709</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glacial acetic acid</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:181px;">A6283</td>
			<td>Use in fume hood</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Cationic metabolite mixture</td>
			<td style="width:167px;">Human Metabolome Technologies</td>
			<td style="width:181px;">H3304-3034</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Anionic metabolite mixture</td>
			<td style="width:167px;">Human Metabolome Technologies</td>
			<td style="width:181px;">H3304-1031</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Methanol (LC-MS Ultra Chromasolv)</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:181px;">14262</td>
			<td>Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium hydroxide solution</td>
			<td style="width:167px;">Sigma</td>
			<td style="width:181px;">72079</td>
			<td>0.1 M</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">U-87 MG Glioblastoma cell line</td>
			<td style="width:167px;">Sigma</td>
			<td>89081402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:167px;">Sigma</td>
			<td align="right" style="width:181px;">650498</td>
			<td>Toxic; use in fume hood</td>
		</tr>
	</tbody>
</table>

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.

The proposed sheathless CE-MS method is capable of providing highly efficient, i.e., plate numbers ranging from 60,000 to 400,000, profiles for anionic and cationic metabolites at nanomolar detection limits using 10% acetic acid (pH 2.2) as BGE. The separation performance of the method for the analysis of highly polar anionic metabolites is demonstrated for three structurally related sugar phosphate isomers (Figure 1). Though a baseline separation was not obtained for these three analytes, a partial separation is sufficient to allow their selective detection by MS as these analytes have the same exact mass. The potential of the sheathless CE-MS method for metabolic profiling of limited number of cells, i.e., a 20 nl injection corresponds to 400 cells (cell density is circa 20 cells/nl), is demonstrated for the analysis of cationic metabolites in an extract of the glioblastoma cell line (Figure 2), in which more than 300 molecular features were detected above a S/N-ratio &#8805; 5.
<img alt="Figure 1" src="/files/ftp_upload/54535/54535fig1.jpg" /> 
Figure 1. Analysis of sugar phosphate isomers by sheathless CE-MS. Extracted ion electropherogram for three sugar phosphate isomers (25 &#181;M) obtained with sheathless CE-MS in negative ion mode. Experimental conditions: BGE, 10% acetic acid (pH 2.2); separation voltage, -30 kV (+0.5 psi applied at the inlet of the CE capillary); sample injection, 2.0 psi for 60 sec. Reproduced with permission4. <a href="https://www.jove.com/files/ftp_upload/54535/54535fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54535/54535fig2.jpg" /> 
Figure 2. Analysis of isoleucine and leucine by sheathless CE-MS. Extracted ion electropherogram of two amino acid isomers (25 &#181;M) obtained with sheathless CE-MS in positive ion mode. Experimental conditions: BGE, 10% acetic acid (pH 2.2); separation voltage, +30 kV; sample injection, 2.0 psi for 60 sec. <a href="https://www.jove.com/files/ftp_upload/54535/54535fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54535/54535fig3.jpg" /> 
Figure 3. Potential of sheathless CE-MS for profiling cationic metabolites in a cell line extract. Metabolic profile (total ion electropherogram) observed in an extract of a glioblastoma cell line with sheathless CE-MS in positive ion mode. Experimental conditions: BGE, 10% acetic acid (pH 2.2); separation voltage, +30 kV; sample injection, 2.0 psi for 60 sec. Reproduced with permission4. <a href="https://www.jove.com/files/ftp_upload/54535/54535fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Sheathless Capillary ElectrophoresisÃ¢â‚¬â€œMass Spectrometry for Metabolic Profiling of Biological Samples at JoVE.com

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

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 ...

sheathless-capillary-electrophoresismass-spectrometry-for-metabolic

Nanyang Technological University

Research

JoVE Journal

Biochemistry

11.6K Views.  Leiden University. A protocol for metabolic profiling of biological samples by capillary electrophoresis–mass spectrometry using a sheathless porous tip interface design is presented.

Video: Sheathless Capillary Electrophoresis–Mass Spectrometry for Metabolic Profiling of Biological Samples

Capillary Electrophoresis Separation of Monoclonal Antibody Isoforms Using a Neutral Capillary

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.

Here, we present a comprehensive capillary zone electrophoresis protocol for the assessment of intrinsic physicochemical heterogeneity of monoclonal antibodies as a quality attribute.

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

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

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

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

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

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Use of Electron Paramagnetic Resonance in Biological Samples at Ambient Temperature and 77 K

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of redox-regulated signaling in biological settings. Electron paramagnetic resonance spectroscopy (EPR) is the only direct method to assess free radicals unambiguously. Its advantage is that it detects physiologic levels of specific species with a high specificity, but it does require specialized technology, careful sample preparation, and appropriate controls to ensure accurate interpretation of the data. Cyclic hydroxylamine spin probes react selectively with superoxide or other radicals to generate a nitroxide signal that can be quantified by EPR spectroscopy. Cell-permeable spin probes and spin probes designed to accumulate rapidly in the mitochondria allow for the determination of superoxide concentration in different cellular compartments.
In cultured cells, the use of cell permeable 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine (CMH) along with and without cell-impermeable superoxide dismutase (SOD) pretreatment, or use of cell-permeable PEG-SOD,&#160;allows for the differentiation of extracellular from cytosolic superoxide. The mitochondrial 1-hydroxy-4-[2-triphenylphosphonio)-acetamido]-2,2,6,6-tetramethyl-piperidine,1-hydroxy-2,2,6,6-tetramethyl-4-[2-(triphenylphosphonio)acetamido] piperidinium dichloride (mito-TEMPO-H) allows for measurement of mitochondrial ROS (predominantly superoxide).
Spin probes and EPR spectroscopy can also be applied to in vivo models. Superoxide can be detected in extracellular fluids such as blood and alveolar fluid, as well as tissues such as lung tissue. Several methods are presented to process and store tissue for EPR measurements and deliver intravenous 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine (CPH) spin probe in vivo. While measurements can be performed at room temperature, samples obtained from in vitro and in vivo models can also be stored at -80 &#176;C and analyzed by EPR at 77 K. The samples can be stored in specialized tubing stable at -80 &#176;C and run at 77 K to enable a practical, efficient, and reproducible method that facilitates storing and transferring samples.

Electron paramagnetic resonance (EPR) spectroscopy is an unambiguous method to measure free radicals. The use of selective spin probes allows for detection of free radicals in different cellular compartments. We present a practical, efficient method to collect biological samples that facilitate treating, storing, and transferring samples for EPR measurements.

The accurate and specific detection of reactive oxygen species (ROS) in different cellular and tissue compartments is essential to the study of ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...