Name: sheathless capillary electrophoresis–mass spectrometry for metabolic profiling of biological samples
Uploaded: 2016-10-01T15:00:00.000+00:00
Duration: 7 min 46 s
Description: Watch this Scientific Journal Video about Sheathless Capillary Electrophoresisâ€“Mass Spectrometry for Metabolic Profiling of Biological Samples at JoVE.com

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>

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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><tbody><tr><td>CESI 8000 instrument</td><td>Sciex</td><td>A98089</td><td>OptiMS adapter required to couple CESI to MS</td></tr><tr><td>OptiMS Fused-Silica Cartridge, 30&amp;nbsp;&amp;mu;m ID x 90&amp;nbsp;cm total length</td><td>Sciex</td><td>B07367</td><td></td></tr><tr><td>OptiMS Adapter for Sciex Nanospray III source</td><td>Sciex</td><td>B07363</td><td></td></tr><tr><td>CESI vials</td><td>Sciex</td><td>B11648</td><td></td></tr><tr><td>Micro vials</td><td>Sciex</td><td>144709</td><td></td></tr><tr><td>Glacial acetic acid</td><td>Sigma</td><td>A6283</td><td>Use in fume hood</td></tr><tr><td>Cationic metabolite mixture</td><td>Human Metabolome Technologies</td><td>H3304-3034</td><td></td></tr><tr><td>Anionic metabolite mixture</td><td>Human Metabolome Technologies</td><td>H3304-1031</td><td></td></tr><tr><td>Methanol (LC-MS Ultra Chromasolv)</td><td>Sigma</td><td>14262</td><td>Use in fume hood</td></tr><tr><td>Sodium hydroxide solution</td><td>Sigma</td><td>72079</td><td>0.1 M</td></tr><tr><td>U-87 MG Glioblastoma cell line</td><td>Sigma</td><td>89081402</td><td></td></tr><tr><td>Chloroform</td><td>Sigma</td><td>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>

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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

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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

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

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

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

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

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

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

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

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

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.

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 ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

Bioengineering

A Strategy for Sensitive, Large Scale Quantitative Metabolomics

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously.&#160; Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. Utilizing normal-phased liquid chromatography coupled to high-resolution mass spectrometry with polarity switching and a rapid duty cycle, we describe a protocol to analyze the polar metabolic composition of biological material with high sensitivity, accuracy, and resolution.

Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting ...

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

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