INTRODUCTION
There are experimental methods for studying proteins (e.g., for detecting proteins, for isolating and purifying proteins, and for characterizing the structure and function of proteins, often requiring that the protein first be purified). Isolation, analysis, and characterization of proteins utilizes a wide variety of chromatographic techniques such as size exclusion, hydrophobic interaction, ion exchange, reversed phase, and others. These techniques are applied for both the analytical and industrial-scale uses, and the detection systems are critical for the proper determination of analytes. Most proteins are colorless and this limits the use of detection systems based on visible light, although it should be noted that detection in the visible light range may be very useful for some protein specific modifications such as the Maillard reaction (Grandhee, S. K.and Monnier,V. M. (1991) Mechanism of formation of the Maillard protein cross-link pentosidine. J. Biol. Chem. 266, 11,649–11,653). The predominant detection system for proteins is based on the fact that all proteins and peptides absorb UV light at the 215-nm wavelength. At this wavelength, proteins can be detected, without any chemical modification and with little interference from many of the solvents/buffers used for chromatographic separations. Proteins containing tryptophan, tyrosine, or phenylalanine residues show additional UV absorption at 280 nm, which permits specific identification of peptides containing these amino acid residues. Current analytical procedures for characterization and analysis of proteins require not only their detection but also substantial analytical work to detect post translational modifications, confirm some of the structural properties such as disulfide bonds, and, in the case of biopharmaceutical products, detect process residuals. The compounds that have to be detected/analyzed include non-UV absorbing carbohydrates, polyethylene glycols, and a number of non-protein process impurities, which frequently absorb very little or no UV light. In some cases, the non-UV absorbing compounds may also be chemically unreactive, which complicates the possible chemical tagging with the UV absorbing or fluorescent tag. In such cases, detection techniques are based on different principles that may be used for sensitive detection of proteins and related compounds. Examples of such techniques are western blotting, electrochemical detectors, conductometric detectors, specialized detectors. Explanation is given below.
WESTERN BLOT
“The western blot is a widely accepted analytical technique used to detect specific proteins”.
It uses gel electrophoresis to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are stained with antibodies specific to the target protein ( Towbin et al.(1979). “Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications are given here.( Proceedings of the National Academy of Sciences USA 76 (9): 4350–54). Gel electrophoresis step is included in Western blot analysis to resolve the issue of the cross-reactivity of antibodies. An improved immunoblot method, Zestern analysis, is able to address this issue without the electrophoresis step, thus significantly improving the efficiency of protein analysis.
In order to make the proteins accessible to antibody detection they are moved from within the gel onto a membrane made of nitrocellulose or polyvinylidene difluoride (PVDF).
Detection
During the detection process the membrane is “probed” for the protein of interest with a modified antibody which is linked to a reporter enzyme; when exposed to an appropriate substrate this enzyme drives a colourimetric reaction and produces a color.
Analysis
After the unbound probes are washed away, the western blot is ready for detection of the probes that are labeled and bound to the protein of interest. In practical terms, not all westerns blot results reveal protein only at one band in a membrane. Size approximations are taken by comparing the stained bands to that of the marker or ladder loaded during electrophoresis. The process is repeated for a structural protein, such as actin or tubulin that should not change between samples. The amount of target protein is normalized to the structural protein to control between groups. This practice ensures correction for the amount of total protein on the membrane in case of errors or incomplete transfers.
Colorimetric Detection
The colorimetric detection method depends on incubation of the western blot with a substrate that reacts with the reporter enzyme (such as peroxidase) that is bound to the secondary antibody. This converts the soluble dye into an insoluble form of a different color that precipitates next to the enzyme and thereby stains the membrane. Development of the blot is then stopped by washing away the soluble dye. Protein levels are evaluated through densitometry (how intense the stain is) or spectrophotometry.
Chemiluminescent Detection
Chemiluminescent detection methods depend on incubation of the western blot with a substrate that will luminesce when exposed to the reporter on the secondary antibody. The light is then detected by photographic film, and more recently by CCD cameras which capture a digital image of the western blot. The image is analysed by densitometry, which evaluates the relative amount of protein staining and quantifies the results in terms of optical density.
ELECTROCHEMICAL DETECTORS
Electrochemical detection may be used for the analysis of many different groups of chemical compounds such as carbohydrates, aliphatic alcohols, aminoalcohols, sulfurcontaining compounds, and others. In some cases, these compounds do not absorb UV light and may be difficult to “tag” with fluorescent and/or UV absorbing tags. Electrochemical detection may be used for analysis of many different compounds, but is rarely used if UV and/or fluorescence detection are possible. One notable exception is carbohydrates, which are frequently analyzed by electrochemical detection despite the fact that fluorescent tagging permits for very sensitive determination. Both UV-Vis and fluorescence detection are undeniably the most common modes of detection in protein chemistry. These modes of detection permit the analysis of most compounds related to protein characterization and the determination of most compounds that are potential process residuals in protein manufacturing. In some specific cases, the compounds of interest may have very low (or no) UV absorbance and may be very difficult to chemically “tag” with the UV absorbing or fluorescent tag. Examples of such compounds are oxidized dithiothreitol (DTT), isopropyl-b-galactosidase (IPTG), quaternary ammonium salts, urea, guanidine, and polysorbates (Tweens), all of which may be process-related impurities in protein manufacturing. In such cases, electrochemical detection may provide an easy and very sensitive means of detection. Moreover, in the case of electrochemical detection, most samples can be analyzed “as is” without initial sample preparation and/or modification simplifying analytical procedures. Carbohydrates are another example of compounds frequently detected electrochemically, although in this case, fluorescent tags and chemistry for tagging in most cases is readily available. Electrochemical detection utilizes reduction/oxidation of compounds enforced by an externally applied voltage. The detector measures the current (charge) changes that result from the electron flow resulting from the oxidation or reduction that occurs at the electrode . (Lisman, J. A., Underberg W. J. M., and Lingeman, H. (1990) Electrochemical derivatization, in Detection-Oriented Derivatization Techniques in Liquid Chromatography (Cazes, J. ed.), Marcel Dekker, New York, pp. 283–322. 23. LaCourse, W. (1997) Pulsed Electrochemical Detection in High Performance Liquid Chromatography, Wiley, New York.). In practice, the most popular modes of electrochemical detection are pulsed amperometric detection and integrated pulsed amperometric detection (IPAD). In these modes, voltage is delivered to the electrodes in pulses (cycles) and it changes according to the preprogrammed pattern (waveform). The changes in the current flow are integrated over the selected part of the waveform and sent to the recording device. Each pulse (cycle) may last from a fraction of a second to several seconds. Integrated pulsed amperometric detection sometimes utilizes complex waveforms that permit sensitive detection and “self-cleaning” of the electrode. Most electrochemical detectors are limited to three-step waveforms permitting for time-controlled three changes in voltage and selection of the integration part of the cycle. The voltage changes correspond to the detection, oxidation, and reduction potential selected for the analyte. Some electrochemical detectors such as ED50 manufactured by Dionex permit for programming of complex waveforms apparently increasing the sensitivity and reliability of detection. It should be noted that the development and optimization of a waveform time-consuming, requires specialized instrumentation, and frequently needs additional optimization by “trial and error.” Most modern electrochemical detectors offer very low-volume flow cells with exchangeable electrodes. Most common electrodes are gold, platinum, silver, and glassy carbon. Some manufacturers offer porous carbon electrodes designed for the detection of selected groups of compounds such as catecholamines. The linear dynamic range and the accuracy of electrochemical detectors are comparable to UV-Vis detectors and the detection limit is in the low-nanogram range. An additional advantage of electrochemical detectors is a possibility of selective detection of analytes that may be achieved by manipulating potential of the electrode(s).
General Considerations for Electrochemical Detection
High-performance liquid chromatography system used for the separation should provide solvent(s) flow free of metal ions. Some HPLC systems used with electrochemical detectors may require initial cleaning/passivation. The electrode should be chosen based on the published reports. For many applications related to protein analysis, a gold electrode is the most universal and versatile. Proper waveform should be selected based on existing information reported for the specific or structurally similar compounds. The electrode and flow cell should be clean. This is a very critical issue and both the electrode(s) and the cell should be frequently cleaned and polished (mechanically) using very fine sandpaper (400 or greater) and polishing pads. Reference pH electrode, if used , should be checked frequently for possible mechanical damage. The electrode should be partially filled with electrolyte. After each cleaning, the flow cell should be rinsed with alcohol and water to remove any solid particles, which could affect the cell’s performance.
Any spacers and gaskets used to assemble the cell should be inspected and have to be in excellent condition. A freshly cleaned cell may require several blank HPLC runs until it fully equilibrates. After completion of the analysis, the cell should be disconnected from power to avoid damage by extensive oxidation. Properly maintained electrochemical cell should provide very reproducible and reliable results even with HPLC runs lasting several days. In many cases, electrochemical detectors will be able to detect analytes at a very low concentration (parts per billion [ppb] level), which may be useful for analysis of process residuals.
CONDUCTOMETRIC DETECTION
Conductometric detection is usually a separate mode of detection available with most electrochemical detectors (Stickel, J. J. and Fotopoulos, A. (2001) Pressure–flow relationships for packed beds of compressible chromatography media at laboratory and production scale. Biotechnol. Prog. 17, 744–751). This mode of detection requires special instrumentation and supplies and is applicable to ionizable compounds. The most common application is detection of low levels of anions or cations. An example may be the detection of chloride or bromide ions. Conductometric detection has limited use in the analysis of protein and protein-related compounds. An example of conductometric detection in protein-related analyses might be the detection of guanidine. Guanidine is frequently used for protein isolation and its viral deactivation and can be detected at a low level as process impurity. Mass spectrometry (MS) as applied to protein analysis is a rapidly evolving technology. Initially, mass spectrometers were designed as very specialized (and expensive) analytical tools for structural analysis of proteins. Technological progress led to a large variety of mass spectrometers suitable for many tasks related to protein analysis. At the same time, the cost of relatively simple instruments kept going down and the physical dimensions of the mass spectrometer have decreased. This created the possibility of using mass spectrometers as universal detectors in chromatographic separations. Total ion current measured by mass spectrometers yields a profile similar to that obtained with UV or other detectors and molecular-mass information is invaluable for protein chemist. For this reason, mass spectrometers have a good chance of becoming universal detectors in protein analysis and chromatography in general. Among their several applications in protein analysis is the determination of molecular mass of intact proteins, determination of post translation modifications like glycosylation, truncation, deamidation, and so forth, and the determination of primary structure of recombinant proteins (Reinhold, V. N. (1998) Evaluation of glycosylation, in Development in Biological Standardization (Brown, F, Lubiniecki, A., and Murano, G., eds.), Karger, Basel, Vol 96, pp. 49–53. Harris, R. J., Molony, M. S., Kwong, M. J., and Ling, V. T. (1996), Identifying unexpected protein modifications, in Mass Spectrometry in the Biological Sciences (Burlingame, A. L. and Carr, S. A. eds.), Humana, Totowa, NJ., pp. 333–350.).
Multiple vendors (Perkin Elmer Sciex, Micromass, Thermo Finnigan, etc.) offer a large variety of detectors based on different principles of operation (triple–quadrupole, ion trap, time-of-flight, etc.). Therefore, it would be very difficult to provide any universal set of suggestions regarding implementation and optimization of mass-spectrometry-based detectors in chromatographic separations. Properly installed and optimized MS detectors provide chromatographers with a great deal of information, and within several years, they will probably become standard detectors for many HPLC applications. It should be noted, though, that most mass spectrometry detectors have very low tolerance to salts, detergents, and certain solvents and chemicals (e.g., organic amines or TFA), limiting the choice of chromatographic systems that may be used with such detectors.
SPECIALIZED DETECTORS
A separate group of detectors originated from gas chromatography and detect specific elements. Examples of such detectors are HPLC nitrogen and sulfur detectors offered by ANTEK (www.antekhou.com/product/chrom/hplc). These detectors may be very useful in specific cases where UV, fluorescence, and other detectors cannot be readily used, especially if the percent content of nitrogen (or sulfur) in the compound of interest is relatively high. However, these detectors require oxygen and inert gas (argon or helium) supplies and cannot be used with solvent systems containing nitrogen (or sulfur) and/or high salt concentration. In addition, these detectors cannot accept flow rates exceeding 0.3 mL/min, which further limits chromatographic options. It is highly recommended that the HPLC system used with nitrogen/sulfur detectors be dedicated for this specific purpose and not be used with any nitrogen- and/or sulfur-containing solvents (acetonitrile, DMSO, trimethylamine, etc). Despite all of the limitations, these detectors offer a selective and sensitive means of detection of specific compounds difficult to determine by any other technique.
Another example of specialized detectors is the Radiomatic series of detectors for measuring radioactivity in isotopically labeled proteins and peptides. These detectors are made by Perkin-Elmer and are available in three models (150TR, 610TR, and 625 TR). The detectors can work in stand-alone or in-line with HPLC system and are capable of monitoring single or dual-labeled radio isotopic samples. The detectors are equipped with preset energy windows for 3H, 14C, 35S, 32P, 125I, and several other radioactive isotopes. This class of detectors is especially useful for identifying and quantifying sites of radioactive isotope incorporation in a protein. For example, when a protein is labeled with the 125I isotope using IODO-GEN chemistry, the site most commonly labeled is tyrosine. The specific tyrosine residues that contain radioactive iodine and the extent of its incorporation could be determined using this detector. Another common application of this detector is in drug metabolism and pharmacokinetic analysis, where isotopically labeled metabolites are detected in physiological fluids.
REFERENCES
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- Dionex Corp. (1995) ED40 Electrochemical Detector Operator’s Manual Dionex Corp document# 034855, Sunnyvale, CA Rev. 03.
- Reinhold, V. N. (1998) Evaluation of glycosylation, in Development in Biological Standardization (Brown, F, Lubiniecki, A., and Murano, G., eds.), Karger, Basel, Vol 96, pp. 49–53.
- Harris, R. J., Molony, M. S., Kwong, M. J., and Ling, V. T. (1996), Identifying unexpected protein modifications, in Mass Spectrometry in the Biological Sciences (Burlingame, A. L. and Carr, S. A. eds.), Humana, Totowa, NJ., pp. 333–350.
Author:
Maheen Zaka
BS (Hon) Biotechnology
GCU Lahore, Pakistan
assignmentworld 