Prefractionation techniques

Righetti PG, Castagna A, Herbert B, Reymond F, Rossier JS. Prefractionation techniques in proteome analysis. Proteomics 2003 3 1397-1407. 

To reduce the complexity of proteins expressed in the cell and overcome the differences in expression rates of proteins, prefractionation techniques have to be deployed before further protein characterization [116]. Two current approaches include electrophoretic and chromatographic methods. 

The prefractionation technique provides improvements in separation resolution, increased sensitivity, and enhanced ability of loading a much higher amount of sample in any narrow pH interval in gel electrophoresis. Once the complexity of proteins is reduced by prefractionation, individual fractions can be subjected to either 2D gel electrophoresis or multidimensional HPLC for further separation, followed by MS analysis. 

P. G. Righetti, A. Castagna, and B. Herbert, Prefractionation techniques in pro-teome analysis, A aZ. Chem. 73 (2001), 320A-326A. 

Righetti, P. G., Castagna, A, Antonioli, P., Sc Boschetti, E. (2005). Prefractionation techniques in proteome analysis The mining tools of the third millennium. Electrophoresis, 26, 297-319. 

Table 4.45 shows the main features of SEC. This technique has become an indispensable tool for polymer characterisation. SEC has some advantages over other LC methods, such as the predictability of the end of a chromatographic run and of the retention times in a calibrated chromatographic system. SEC is an attractive technique for prefractionation or sample clean-up prior to a more sensitive RPLC technique. This intermediate step is especially interesting for experimental purposes whenever polymer matrix interference cannot be separated from the peak of interest [647]. Disadvantages are that the whole separation must be eluted within the... 

The dynamic range of protein expression represents a main obstacle since abundant proteins are seldom of interest and others such as transcription factors are only present in a few copies. There is no detector that is able to visualize all proteins at the same time so that prefractionation and the investigation of subproteomes is required. In fact, pre-MS sample preparation techniques exploiting electrophoretic, chromatographic, or chemical properties of the analyte are often the bottleneck of proteomics. 

Different physical techniques can also be used for prefractionation of samples to be analyzed by 2-D gels. Corthals et al. have fractionated human serum by an electrokinetic technique [67], Fountoulakis et al. have used chromatofocus-ing as a prefractionation step in the analysis of low-abundance proteins in H. 

The MudPlT approach is not without problems. A fully automated procedure as described requires (almost) continuous MS data acquisition for more than 15 hours, resulting in several thousands of mass spectra that put high demands on computers and bioinformatics. The procedure is limited by the ability of the mass spectrometer to rapidly switch between MS and MS-MS analysis under DDA control. With current technology, certainly not all peptides present can be analysed with both modes (Ch. 17.7.2). The power of the approach is greatly enhanced by performing a protein prefractionation, either by RPLC [8, 45] or by means of a protein-specific enrichment technique such as AfC (Ch. 17.4.1). 

Another separation technique that has shown utility as a prefractionation method for dereplication is countercurrent chromatography (CCC) or centrifugal partition chromatography (CPC). This separation method is based on the... 

Fatty acids can be extracted either as FFAs or as BFAs. FFAs are obtained by the hydrolysis of BFAs. Liquid-liquid extraction has been successfully applied in the isolation of several classes of lipids, or of single lipids from complex mixtures. Popular extraction methods for lipids are the Folch extraction technique, or the Bligh and Dyer method. Solid-phase extraction (SPE), and solid-phase microextraction (SPME), are also available as simple and economical time- and solvent-efficient sample preparation methods. Prefractionation can be performed using SPE silica or aminopropyl-silica columns. 

Silver-ion TLC is often used with GC as part of a hyphenated technique for analysis of CLA isomers as FAME and, to a lesser extent, TAG. Precht and Molkentin utilized Ag-TLC to isolate trans 18 1 and CLA isomers (as FAME) before analysis by GC (100 m CP Sil 88 capillary column) and applied this technology to determine the frequency distributions of CLA in European bovine milk fats (24,25) and German human milk lipids (26). Prefractionation of the milk lipids by Ag-TLC was utilized to remove such FA as 9c-20 l and 21 1, FA that would otherwise coelute with the CLA isomers (as FAME) during analysis by GC. The procedure thus simplified the calculation of the percentage of CLA in these samples. Robert Ackman (27) apphed a similar technique to analyze the CLA isomers present in raw seafood. After the lipids were extracted and converted to FAME, Ag-TLC was utilized to remove 18 4n-3 and 18 4n-l, FAME isomers that co-eluted with 9c, 11/-18 2 and 10/,12c-18 2, respectively, on polyglycol-based capillary GC columns. 

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