Published: October 1st, 2016
A protocol for metabolic profiling of biological samples by capillary electrophoresis–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 protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis–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.
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.
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
2. Setting up the Sheathless CE-MS System
3. Analysis of Metabolite Standards and Biological Samples
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 ≥ 5.
Figure 1. Analysis of sugar phosphate isomers by sheathless CE-MS. Extracted ion electropherogram for three sugar phosphate isomers (25 µM) 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. Please click here to view a larger version of this figure.
Figure 2. Analysis of isoleucine and leucine by sheathless CE-MS. Extracted ion electropherogram of two amino acid isomers (25 µM) 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µM), 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 µA 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.
We have nothing to disclose.
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).
|CESI 8000 instrument
|OptiMS adapter required to couple CESI to MS
|OptiMS Fused-Silica Cartridge, 30 μm ID x 90 cm total length
|OptiMS Adapter for Sciex Nanospray III source
|Glacial acetic acid
|Use in fume hood
|Cationic metabolite mixture
|Human Metabolome Technologies
|Anionic metabolite mixture
|Human Metabolome Technologies
|Methanol (LC-MS Ultra Chromasolv)
|Use in fume hood
|Sodium hydroxide solution
|U-87 MG Glioblastoma cell line
|Toxic; use in fume hood
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