A Brief Review of Methodology for the Analysis of Biochemical Reactions and Cells

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hundreds of compounds in one experiment. When considering a whole cell, this task is very challenging because thousands of different species, the metabolome, coexist, with very different chemical and physical characteristics. Generally, targeted methods are designed to quantify specific species within a class of related intermediates, such as sugar phosphates, hexosamines, purines, amino acids, and lipids. Since targeted methods are tailored to the specific chemical or spectral properties of a given class, they lead to improved resolution and detection limits.

To date, most studies of cellular dynamics have focused on subsystems with a limited number of reactions or linked pathways. Analytes of interest typically belong to the same class and, thus, are accessible by a single analytical system. A prominent example are the intermediates of the central carbon metabolism, which comprises glycolysis, the pentose phosphate pathway, and the Krebs cycle. All metabolites in these pathways are carbohydrates that carry phosphate or carboxylic groups. Because of its pivotal role in catabolism and anabolism, in lower and higher organisms, central carbon metabolism is the best-studied biochemical system. Its analysis usually includes nucleotide phosphates, such as ATP and GTP, that reflect the energy state of a cell, and the redox cofactors NAD+ and NADP +. These cofactors participate in a large number of reactions and interconnect metabolic pathways, acting as global regulators.

The analytical system of choice is expected to provide simultaneous access to the metabolites of interest and globally relevant cofactors. In addition, it is desirable to have a platform that can be flexibly extended for purposes of untargeted metabolome profiling. From this perspective, two conceptually different systems hold prime attention: high-pressure liquid chromatography and capillary electrophoresis.

3.2.1 High-Pressure Liquid Chromatography (HPLC)

Chromatography separates species dispersed in a mobile phase (gaseous or liquid) as a result of interactions with a static phase. The interactions can be of various kinds, for example, ionic or hydrophobic. Effective separations are obtained as a result of differences in retention on the stationary phase. Sugar phosphates (in glycolysis and the pentose phosphate pathway), carboxylic acids (in the Krebs cycle), and nucleotides are all negatively charged. This feature favors their simultaneous analysis by high-performance anion-exchange chromatography (HPAEC) [1–3]. In less than 1 h, these compound classes can be separated in complex samples such as those isolated from tissues, plant leaves, or microorganisms. Notably, in anion-exchange chromatography, the majority of substances with identical molecular weight in central metabolism have been successfully resolved. A possible drawback are the strong acidic or alkaline mobile phases, which can cause degradation of the labile phosphodiester bond in sugar phosphates [3] but are required to ensure the presence of all analytes in their ionic form.

Even more difficult than the separation is the detection of central carbon metabolites. Direct UV detection is suitable only for nucleotides and some Krebs cycle intermediates [1,2]. In contrast, amperometric detectors are efficient only for species bearing multiple hydroxylic groups such as sugars [3,4]. Refractometric, light scattering, or fluorescence and UV detection are valuable alternatives, but suffer from scarce versatility. The most comprehensive results have been obtained by conductometric detection, with detection limits in the low picomole range for both sugar phosphates and carboxylic acids [2,3].

This method allows the quantification of inorganic anions as well, although particular care is necessary to limit strong background signals originating from salt contaminations.

A breakthrough was achieved with the advent of electrospray interfaces for the coupling of HPLC to mass spectrometers (MS). We discuss MS in detail in a following section. In conjunction with chromatography, MS provides subpicomole sensitivity and high-resolution m/z-based separation of chromatographically unresolved analytes [5,6]. Historically, MS interfaces were first developed for gas chromatography (GC). GC-MS has in fact become a very robust system for the simultaneous analysis of several hundreds of compounds [7,8], and has been extensively used for profiling studies in biological extracts [9,10]. GC-MS has three major disadvantages: It degrades thermolabile compounds; it often requires derivatization steps because about 80% of endogenous metabolites are not volatile; and it is prone to in-source fragmentation.

Since these restrictions do not apply to HPLC-MS systems, efforts have been undertaken to extend the range of metabolites amenable to HPLC in response to the demand for alternatives to GC-MS. The synergy between HPAEC and MS has been successfully applied to the quantification of glycolytic intermediates [6]. However, ion-exchange chromatography involves high salt concentrations which generally interfere with the MS detection. To complement GC-MS, additional chromatographic modes have been adopted. Reverse-phase (RP) HPLC is perhaps the most important, and is widely used in the analysis of nonpolar compounds. In RP-HPLC, separation relies on hydrophobic interactions with aliphatic chains on the stationary phase in a mixed organic–aqueous mobile phase. RP-HPLC, with or without an ion-pairing reagent, was successfully employed to quantify the sugar phosphates in pentose phosphate pathway intermediates [5], and to profile plant metabolites [11,12]. On the other hand, MS-based analysis of highly polar analytes, including central carbon intermediates, peptides, oligosaccharides, or nucleic acids, can be achieved by hydrophilic-interaction chromatography (HILIC) [13].

3.2.2 Capillary Electrophoresis (CE)

CE refers to a group of related techniques that employ high DC voltages to drive within minutes the separation of the analytes in narrow-bore capillaries filled with weakly conductive buffers. In the classical configuration, a fused silica capillary is spanned between two buffer reservoirs, in each of which an electrode is immersed to apply an electric potential. In the presence of an electric field across the length of the capillary (up to 500 V/cm for 5–200 µm i.d. capillaries), separation occurs by two overlapping phenomena: electrophoresis and electro-osmotic flow (EOF). In electrophoresis, ions migrate toward the electrode with opposite charge dependent on their mass, shape, and extent of solvation. In addition, all analytes, including neutral species, are affected by the EOF. The underlying mechanism is well understood: (1) The ionizable silanol groups (pI about 1.5) on the silica inner surface become negatively charged in contact with the background electrolyte (BGE) (at pH > pI). (2) Cations from the BGE are then attracted to the walls, forming a double electrical layer. When a potential is applied at the ends of the capillary, the movement of the cations in the direction of the anode generates a flow of buffer solution. The strength of the EOF is a function of factors such as the ionic buffer strength, the density of charges on the capillary wall, the pH, the applied electric field, and the dielectric constant and the viscosity of the running buffer. In fact, the EOF

14 DETERMINATION OF COMPLEX REACTION MECHANISMS

can be controlled for the specific requirements of the different CE modes by modifying these parameters or by chemical modification of the capillary inner surface. Although the effects of electrophoretic migration and EOF overlap, at neutral to alkaline pH the latter is overwhelming (up to 2–3 mm/s in silica capillaries) and ensures that cations, uncharged molecules, and anions migrate toward the anode at different velocities.

Detection typically occurs in-line, through an optical window placed in the proximity of the capillary outlet, either by spectral analysis, with average concentration sensitivities lower than in HPLC because of the shorter light path, or by laser-induced fluorescence [14], with detection limits reaching zeptomoles for appropriate fluorophores. Coupling and applications of CE-MS systems have been thoroughly reviewed [15]. For the analysis of complex biological mixtures, this combination is capable of detecting analytes such as proteins or metabolites in the femtoto attomole range.

CE offers important advantages compared to liquid chromatography [16]. In contrast to the parabolic flow profiles characteristic of hydrodynamic flow, such as in pumped HPLC, the EOF generated in electrophoresis is uniformly distributed across the tubing section and forms a plug-shaped flow profile. This drastically reduces band-broadening in CE, and results in very high resolution and, consequently, increased peak capacity. The major limitations are set by molecular diffusion and the heat generated by the resistance of the buffer to the current [15]. In addition, low sample volumes in the range of a few nanoliters are required. In the extreme case, CE was used to analyze the content of a single cell [17], corresponding to a volume of picoliters. Micromanipulation is typically used to introduce by suction a single cell in a 10–20 µm i.d. capillary, in which cell lysis is induced by the hypotonic BGE and electrophoresis occurs [18]. Detection in the attomole range is required to measure species represented in the cells at concentrations of at least 1 mM, such as proteins [19,20], amino acids [21], and glycolytic endproducts [22].

In the family of CE, capillary zone electrophoresis (CZE) is the simplest and the most frequently used form of CE. The system is characterized by a homogeneous buffer solution and a constant field strength. CZE is routinely employed in the analysis of clinically relevant metabolites and drugs, inorganic ions, metals, and proteins [16,23,24]. Notably, Soga and coworkers combined three CZE-MS methods to isolate 1,692 compounds in bacterial extracts, of which 352 were identified and quantified [25] (fig. 3.1). Another form of CE is capillary isoelectric focusing (CIEF), in which the inner capillary wall is usually coated with polyacrylamide or methylcellulose to suppress the EOF. A series of zwitterionic species (the ampholytes) are used to generate a pH gradient in the capillary. In the absence of EOF, analytes and ampholytes migrate as long as they are charged. Consequently, they will separate and distribute along the gradient based on their isoelectric point (pI), where the net charge becomes zero and migration stops. In CIEF, high-resolution separations of proteins with pI differences < 0.01 units can be achieved. This renders CIEF particularly useful for monitoring variants of human and recombinant proteins or their glycosylation patterns. Micellar electrokinetic capillary chromatography (MECC) represents a unique chromatographic technique. In the BGE, surfactants (anionic, cationic, nonionic, or zwitterionic) are added in excess of their critical micellar concentration to form charged micelles. Analytes (also neutral) are separated based on their partitioning, and in the presence of chiral additives such as cyclodextrins, the separation is amenable to enantiomeric recognition [26]. Finally, affinity CE is frequently used in the characterization of biomolecular interactions between proteins, DNA, and