Separation complex protein mixture

Gel electrophoresis provides a simple method for separating complex protein mixtures. Because proteins are visualized using stains that may not be linearly incorporated in the gel, the intensity of the stained bands may be poorly correlated with the amount of protein. For this reason, gel electrophoresis is at best a semiquantitative technique capable of generating relative purity results. In CE, separations are commonly performed in free solution, i.e., in the absence of any support such as gel matrices. This allows the replacement of the capillary s content in between analyses and therefore the automation of the process. The use of UV-transparent fused-silica capillaries enables direct on-line optical detection of focused protein zones, eliminating the requirement for sample staining. The detection systems available to CE provide true quantitative capabilities. 

Two-dimensional discrete (as opposed to continuous) electrophoresis has become a premier analytical tool for separating complex protein mixtures. Since this technique employs IEF, further discussion will be postponed to Section 8.10. 

Proteins are involved in all biological processes and can therefore be considered the functionally most important biological molecules and are crucial for the description of biological systems. The systematic identification and characterization of proteins is called proteomics. A predominant technology platform in proteomics, two-dimensional gel electrophoresis, is used to separate complex protein mixtures allowing individual protein spots on the gel to be identified by computer-operated mass spectrometry. Mass spectrometric data are then processed through a series of computer algorithms such as Mass Lynx and ProteinLynx software to determine the sequence identity of the proteins. 

The early days of proteomics were driven by the need to develop novel analytical techniques for the rapid and sensitive identification of gel-separated proteins. At that time, two-dimensional (2D) gel electrophoresis was the predominant technique for separating complex protein mixtures. Furthermore, mass spectrometry was increasingly used for protein analysis. However, serious challenges arose from the use of these technologies. Mass spectrometers and HPLC were developed primarily to analyze small molecules however, they were not adapted to consider the full nature of proteomic samples, including sample volume, sample lost and sample contamination. 

A wide variety of separation mechanisms have been combined in 2D-HPLC to separate complex protein mixtures. Size exclusion [66], affinity [67], lEX [68-70], and chromatofocusing [71] have been used as a first-dimension separation in combination with RP as a second-dimension separation. lEX-RP, the most extensively studied combination, has been coupled to EIS time-of-flight MS for separation and identification of ribosomal proteins from yeast [72]. Approximately 70% of the potential ribosomal subunits isoforms could be identified with an average mass error of 50ppm. In the experimental setup, two parallel RP columns were alternately switched in series with a strong anion-exchange column to reduce the analysis time. 

Many techniques can be used to separate complex protein mixtures in what at least approaches a... 

Opiteck et al. and Opiteck and Jorgenson " described the 2-D HPLC system for separating complex protein mixtures that used two columns connected in series— the SEC column for the first dimension and the RP column for the second dimension. Peaks eluting from the SEC column were automatically introduced into the RP column to separate similarly sized proteins on the basis of their hydrophobicity. 

ProSwift WAX IS can be used to separate complex protein mixtures such as pancreatin, which contains a variety of proteases (protein digesting enzymes such as trypsin or pepsin), amylases (carbohydrate digesting enzymes), and lipases (fat digesting enzymes). Figure 3.287 shows the separation of pancreatin on a 50 mm x 1 mm i.d. ProSwift WAX-IS into an unbound and a bound fraction, the latter one containing several well-resolved peaks. 

This chapter has presented several comprehensive 2DLC approaches combining a first-dimension IEX separation and a second-dimension RP separation for the analysis of complex protein mixtures typical in proteomics studies. Online ESI-TOF/MS detection provided sensitive detection and valuable qualitative information (MW) for proteins eluting from the MDLC system. Coordinated fraction collection and subsequent MS analysis of peptides produced by proteolysis of the fractions provided in-depth information on protein identification and a mechanism... 

PMF is generally used to identify proteins that have been previously separated by 2-D GE so that additional information including the molecular weights and isoelectric points can be used to supplement PMF identification. PMF is not well suited for searching expressed sequence tag (EST) databases that contain incomplete gene coding information for particular ESTs and it is not adequate for the analysis of complex protein mixtures in solution. 

Displacement Separations of More Complex Protein Mixtures.315... 

Displacement Separations oe More Complex Protein Mixtures... 

Fig. 3A, B Strategies for the analysis of complex protein mixtures. A Proteins are separated by 2D PAGE and the relevant protein spots excised from the gel, digested, and analyzed by mass spectrometry. B Proteins are first digested into small peptides and then fractionated by two rounds of chromatography before each peptide is analyzed by mass spectrometry...

In immunoelectrophoresis two sequential procedures are applied to the analysis of complex protein mixtures (1) separation of the protein mixture by agarose gel electrophoresis, followed by (2) interaction with specific antibodies to examine the antigenic properties of the separated proteins. 

Transfer of proteins onto nitrocellulose or PVDF membranes is usually performed after separation of complex protein mixtures by polyacrylamide gel electrophoresis (PAGE). Proteins can be separated on the basis of their molecular weight or isoelectric point, under reducing or nonreducing conditions, and it is therefore for the investigator to establish the most appropriate separation conditions for particular samples. Full details of PAGE can be found in refs. 4 and 5, and the reader is encouraged to use these as sources of further details... 

Much of the pressure to develop automated sequential HPLC separations has come from the necessity to separate complex biological mixtures, especially protein mixtures. Traditionally, complex mixtures of proteins have been separated using two-dimensional gel electrophoresis (2D GEP). The first dimension gel separation is carried out with electrophoresis buffers, the gel plate is rotated 90° and the second SDS-PAGE separation is carried out under denaturing conditions, using sodium dilauryl sulfate. The separated spots are then visualized, scraped off the plate, and then extracted for further analysis. Protein analysis by MALDI time-of-flight mass spectrometry starts with this time- and labor-intensive 2D GEP separation mode. 

Protocol 7 for CZE was adapted from Gordon et al.77 These authors used this protocol to analyze a variety of proteins including a complex protein mixture. It may provide a good starting point for protein impurity analysis. If insufficient separation is achieved then the analyst is advised to change the pH by using one of the other buffers discussed above. Such an approach showed markedly different separations for a number of recombinant proteins and product-related impurities.39,78... 

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