Fast performance protein liquid chromatography

High-performance liquid chromatography (HPLC) and fast protein liquid chromatography (FPLC) rely on the same separation principles as the traditional chromatography columns, but tend to be much faster because of high flow rates that are possible due to the uniform bead size and the mechanical strength of the beads. See also Chapter 4, section 1.2.2. 

There are several problems requiring careful attention. Lysozyme has a tendency to form complexes with many substances [e.g., alkyl sulfates, fatty acids, aliphatic alcohols (Smith and Stocker, 1949), cephalins (Brusca and Patrono, 1960), and other proteins]. Of particular importance is its tendency to form complexes with transferrins [e.g., ovotrans-ferrin (Ehrenpreis and Warner, 1956)]. These interactions lead to difficulties in the isolation of lysozyme. Some recent workers have used fast protein liquid chromatography (FPLC) and high-performance liquid chromatography (HPLC) (e.g., Ekstrand and Bjorck, 1986). The resolution in these procedures may not always be satisfactory, and in HPLC pressure and solvent effects must be monitored carefully if the product is to be suitable for conformation and activity studies. 

The following sections will describe some of the various methods of liquid chromatography suitable for separation and analysis of biological (macro)molecules. Such systems often use high pressures and rapid flow rates, and are sometimes loosely described as HPLC (high performance liquid chromatography) or FPLC (fast protein liquid chromatography). 

The by far largest application area of high-performance ion-exchange chromatography is the separation of biopolymers proteins and nucleic acids. Although a special term, fast protein liquid chromatography (FPLQ, is occa-... 

The initial PIA purification method was developed by Mack et al. (3). These authors used a different, two-step chromatography protocol involving size-exclusion and ion exchange chromatography on Sephadex G-200, Q-Sepharose, and S-Sepharose. A similar purification method has been described recently to isolate a PIA-related polysaccharide polymer in E. coli (7). Briefly, E. coli cells were incubated in 50 raM Tris-HCL buffer (pH 8.0), 100 mg lysozyme, and 0.1 M EDTA at room temperature for 2 h. Phenol/chloroform extraction steps were performed to separate protein and debris contamination from the polysaccharide. Samples were concentrated by ultrafiltration devices (10,000 MW cut off) and fractionated on a fast protein liquid chromatography (FPLC) system with a Sephacryl S-2000 column (equilibration and elution buffer 0.1 MPBS, pH 7.4). 

Fast protein liquid chromatography (FPLC) is a term applied to several chromatography techniques which are used to purify proteins. Many of these techniques are identical to those carried out under high performance liquid chromatography, however use of FPLC techniques are typically for preparing large scale batches of a purified product. 

Nimura, N., Itoh, H., Kinoshita, T., Nagae, N., Nomura, M. (1991). Fast protein separation by reversed-phase high-performance liquid-chromatography on octadecylsilyl-bonded non-porous silica-gel — effect of particle-size of column packing on column efficiency. J. Chromatogr. 585(2), 207-211. 

Reversed-phase HPLC is widely utilized to generate a peptide map from digested protein, and the MS online method provides rapid identification of the molecular mass of peptides. The HPLC-MS-FAB online system is a sensitive and precise method for low-MW peptides (<3000 Da) even picomol quantities can be detected. However, as the MW of the analytes increases, the ionization of peptides becomes more difficult and decreases the sensibility of the FAB-MS (112). Electrospray ionization (ESI-MS) was found to be an efficient method for the determination of molecular masses up to 200,000 Da of labile biomolecules, with a precision of better than 0.1%. Molecular weights of peptide standards and an extensive hydrolysate of whey protein were determined by the HPLC-MS-FAB online system and supported by MALDI-TOF (112). Furthermore, HPLC-MS-FAB results were compared with those of Fast Performance Liquid Chro-motography (FPLC) analysis. Mass spectrometry coupled with multidimensional automated chromatography for peptide mapping has also been developed (9f,l 12a). 

Fast-protein or high-performance liquid chromatography systems using columns such as Mono Q, Poros HQ or TSK DEAE 5PW are suitable for the purification of immunoglobulins following this protocol. 

Adam et ai, 1995 Childs and Mak, 1993 Katoch and Moreland, 1995). In chicken gizzard and rat aorta, immunoprecipitated MAPK exhibits phosphotransferase activity toward myelin basic protein and purified caldesmon. When crudely extracted from porcine carotid arteries, MAPK exhibits phosphotransferase activity that has been quantitated using a peptide substrate (APRTPGGRR). To determine if the peptide kinase activity detected in crude tissue extracts is specific for MAPK (the peptide may detect other protein kinases), proteins in the extracts have been separated on a Mono-Q fast-performance liquid chromatography column as shown in Fig. 2, and further characterized. Only two peaks of phosphotransferase activity... 

FIGU R E 2 Line graph (A) and immunoblots (B,C) showing separation of MAPK isoforms by Mono-Q fast-performance liquid chromatography (Adam et al., 1995). Extracts of porcine carotid arteries were separated on a 1-ml Mono-Q column. Aliquots from each fraction were assayed for MAPK activity (A). Only fractions containing activity above background are presented for clarity. Proteins from specified fractions were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and assayed by immu-noblot for the presence of either MAPK (B) or phosphotyrosine (C). Reprinted with permission. "Circulation Research." Copyright 1995 American Heart Association. 

The spectroscopic and photochemical properties of the synthetic carotenoid, locked-15,15 -cA-spheroidene, were studied by absorption, fluorescence, CD, fast transient absorption and EPR spectroscopies in solution and after incorporation into the RC of Rb. sphaeroides R-26.1. High performance liquid chromatography (HPLC) purification of the synthetic molecule reveal the presence of several Ai-cis geometric isomers in addition to the mono-c/x isomer of locked-15,15 -c/x-spheroidene. In solution, the absorption spectrum of the purified mono-cA sample was red-shifted and showed a large c/x-peak at 351 nm compared to unlocked all-spheroidene. Spectroscopic studies of the purified locked-15,15 -mono-c/x molecule in solution revealed a more stable manifold of excited states compared to the unlocked spheroidene. Molecular modeling and semi-empirical calculations revealed that geometric isomerization and structural factors affect the room temperature spectra. RCs of Rb. sphaeroides R-26.1 in which the locked-15,15 -c/x-spheroidene was incorporated showed no difference in either the spectroscopic properties or photochemistry compared to RCs in which unlocked spheroidene was incorporated or to Rb. sphaeroides wild type strain 2.4.1 RCs which naturally contain spheroidene. The data indicate that the natural selection of a c/x-isomer of spheroidene for incorporation into native RCs of Rb. sphaeroides wild type strain 2.4.1 was probably more determined by the structure or assembly of the RC protein than by any special quality of the c/x-isomer of the carotenoid that would affect its ability to accept triplet energy from the primary donor or to carry out photoprotection. 

Henzel W J, Bourell J H, Stults, J T (1990). Analysis of protein digests by capillary high-performance liquid chromatography and on-line fast atom bombardment mass spectrometry. Anal. Biochem. 187(2) 228-233. 

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