Protein high-performance instrumentation

Tandem mass spectrometry (MS/MS) is very useful for the amino acid sequencing of peptides, and has been used widely in both protein biochemistry and pro-teomics to identify proteins, to deduce the sequence of a peptide, and to detect and locate post-translational modifications. Until around a decade ago, the concept of amino acid sequencing by MS-technologjes was synonymous with ESI-MS/MS, but today MALDI-MS/MS techniques are implemented in high-performance instruments such that the quality of MALDI tandem mass spectra is comparable with that of ESI-MS/MS spectra. Currently, MALDI tandem mass spectrometers exist in a number of geometries, including TOF-TOF, Q-TOF, ion trap and orbitrap analyzers that each provide unique analytical features for the sequencing of peptides and proteins by MS/MS (details of the instrumentation for different types of MS/MS are provided in Chapter 2). 

High performance capillary electrophoresis was introduced originally as an analytical tool. Now that instruments are equipped with automated fraction collection, however, capillary electrophoresis can be used for micropreparative collection of individual peaks separated from a mixture. Using the fraction collection feature, nanomolar amounts of solute such as proteins, peptides, oligonucleotides can be collected in amounts sufficient for microsequencing. An intersample washing procedure and use of well-formed capillaries aid in the prevention of artifacts.44... 

Capillary electrophoresis employing chiral selectors has been shown to be a useful analytical method to separate enantiomers. Conventionally, instrumental chiral separations have been achieved by gas chromatography and by high performance liquid chromatography.127 In recent years, there has been considerable activity in the separation and characterization of racemic pharmaceuticals by high performance capillary electrophoresis, with particular interest paid to using this technique in modem pharmaceutical analytical laboratories.128 130 The most frequently used chiral selectors in CE are cyclodextrins, crown ethers, chiral surfactants, bile acids, and protein-filled... 

Bushey, M.M., Jorgenson, J.W. (1990). Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins. Anal. Chem. 62, 161-167. 

High performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are two instrumental separation techniques that are applicable to the separation of proteins and peptides. The advantage of HPLC and CE techniques is that they afford the analyst the freedom to resolve a complex mixture by different routes employing different... 

The m/z values of peptide ions are mathematically derived from the sine wave profile by the performance of a fast Fourier transform operation. Thus, the detection of ions by FTICR is distinct from results from other MS approaches because the peptide ions are detected by their oscillation near the detection plate rather than by collision with a detector. Consequently, masses are resolved only by cyclotron frequency and not in space (sector instruments) or time (TOF analyzers). The magnetic field strength measured in Tesla correlates with the performance properties of FTICR. The instruments are very powerful and provide exquisitely high mass accuracy, mass resolution, and sensitivity—desirable properties in the analysis of complex protein mixtures. FTICR instruments are especially compatible with ESI29 but may also be used with MALDI as an ionization source.30 FTICR requires sophisticated expertise. Nevertheless, this technique is increasingly employed successfully in proteomics studies. 

With the use of high performance materials and automated instruments, protein separation is becoming a more controllable process. However, some problems persist even with the use of sophisticated instruments. Many difficulties are still found in determining the optimal extraction and purification conditions, as well as in selecting suitable methods for detecting the protein and quantifying its biological activity. 

Fluorescence was used to study the dynamic character of proteins. I lowever, these studies were limited to proteins having high fluorescence quantum yield. Due to the lack of performed instrumentations and the assumption that intrinsic fluorescence from hemoproteins does not exist attempts to detect fluorescence from these hemoproleins were never realized before 1976. The low hemoproteins fluorescence was explained by tlic presence of a very strong energy transfer from tlie tryptophan to the heme. 

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