Proteomics separation

Chen, J., Lee, C.S., Shen, Y., Smith, R.D., Baehrecke, E.H. (2002). Integration of capillary isoelectric focusing with capillary reversed-phase liquid chromatography for two-dimensional proteomics separation. Electrophoresis 23, 3143-3148. 

Buch, J.S., Li, Y., Rosenberger, F., DeVoe, D.L., Lee, C.S., Two-dimensional genomic and proteomic separations in a plastic microfluidic network. Micro Total Analysis Systems 2003, Proceedings 7th U.TAS Symposium, Squaw Valley, CA, Oct. 5-9, 2003, 477-480. 

Proteomics—separating and identifying protons 2-D PAGE to separate, MALDI-TOF mass spectrometry to identify, pp. 705, 707... 

Efficient analytical columns must have a homogeneous stationary phase of small particle size. In recent years, to reduce the volumes of solvents used in chromatography and to speed up analysis times, there has been a drive towards the use of narrower, shorter columns (Table 3.1). The use of such columns reduces the flow rate of the mobile phase and, hence, the overall amounts of solvent consumed. It also reduces the time spent in the column by the analytes. Another useful factor is that less sample is usually injected onto these columns, which can be very important where samples are precious, such as in the case of human blood samples and with proteomic separations. And if the particle size decreases with the length, there does not have to be an associated loss in efficiency and resolution. Connections between injector, column and detector should be of low volume and the inside diameters of components should... 

Besides sensitive methods for the analysis of proteins, bioinformatics is one of the key components of proteome research. This includes software to monitor and quantify the separation of complex samples, e.g., to analyze 2DE images. Web-based database search engines are available to compare experimentally measured peptide masses or sequence ions of protein digests with theoretical values of peptides derived from protein sequences. Websites for database searching with mass spectrometric data may be found at http //www.expasy.ch/tools, http //prospector.ucsf. edu/ and http //www.matrixscience.com. 

Concerning function integration, for example, micro-flow membrane reactors can exhibit similar process intensification, as shown already for their large-scale counterparts [75]. Separation columns for proteomics, immobilizing enzymes, utilize the large surface-to-volume ratios. Surface tension differences can guide and transport liquids selectively. 

In E. Coli bacterial lysates, the proteome (i.e., the full array of proteins produced) was analyzed by isoelectric focusing and mass spectrometry.97 A comparison of capillary electrophoretic separation and slab gel separation of a recombinant monoclonal antibody demonstrated that the precision, robustness, speed, and ease-of-use of CE were superior.98 Seventy-five proteins from the yeast ribosome were analyzed and identified by capillary electrophoresis coupled with MS/MS tandem mass spectrometry.99 Heavy-chain C-terminal variants of the anti-tumor necrosis factor antibody DE7 have been separated on capillary isoelectric focusing.100 Isoforms differing by about 0.1 pi units represented antibodies with 0,1 or 2 C-terminal lysines. 

The beauty of 2D gel electrophoresis as a separation technique is the orthogonality of the two separation dimensions separation by charge (isoelectric point) in the first dimension, and separation by size in the second dimension. Two-dimensional gel electropheresis is the core separation technique for proteomics, along with HPLC (for preparative isolation). 

The second step in 2D electrophoresis is to separate proteins based on molecular weight using SDS-PAGE. Individual proteins are then visualized by Coomassie or silver staining techniques or by autoradiography. Because 2D gel electrophoresis separate proteins based on independent physical characteristics, it is a powerful means to resolve complex mixtures proteins (Fig. 2.1). Modem large-gel formats are reproducible and are the most common method for protein separation in proteomic studies. 

Figure 2.2. Fractionation of protein extracts before 2D gel electrophoresis. Crude lysates can be fractionated by affinity purification or by a number of chromatographic techniques. In addition, organelles or other cellular structures can be purified and lysates from these organelles can be fractionated or separated directly on 2D gels. By repeating this procedure using a number of conditions it may be possible to visualize a large fraction of a cell s proteome.

Mass spectrometers measure the mass-to-charge ratio (m/z) of ions. They consist of an ionization source that converts molecules into gas-phase ions and a mass analyzer coupled to an ion detector to determine the m/z ratio of the ion (Yates III, 2000). A mass analyzer uses a physical property such as time-of-flight (TOF) to separate ions of a particular m/z value that then strike the detector (Fig. 2.3). The magnitude of the current that is produced at the detector as a function of time is used to determine the m/z value of the ion. While mass spectrometers have been used for many years for chemistry applications, it was the development of reproducible techniques to create ions of large molecules that made the method appropriate for proteomics. 

Gauss, C., Kalkum, M., Lowe, M., Lehrach, H., and Klose, J. (1999). Analysis of the mouse proteome. (I) Brain proteins Separation by two-dimensional electrophoresis and identification by mass spectrometry and genetic variation. Electrophoresis 20, 575-600. 

Hamler, R. L. Zhu, K. Buchanan, N. S. Kreunin, P. Kachman, M. T. Miller, F. R. Lubman, D. M. A two-dimensional liquid-phase separation method coupled with mass spectrometry for proteomic studies of breast cancer and biomarker identification. Proteomics 2004,4, 562-577. 

Electrospray (ESI) ionization mass spectrometry also plays in important role in bacterial characterization. Because it typically includes a chromatographic separation step, the approach is not considered as rapid as MALDI approaches, which do not incorporate a separation. However, compared to the times needed to grow bacteria in culture prior to analysis, the time frame is not lengthy, and the addition of chromatographic separation provides many opportunities to increase specificity. ESI/MS has been used to characterize cellular biomarkers for metabolic, genomic, and proteomics fingerprinting of bacteria, and these approaches are reported in two chapters. 

Perhaps the biggest increase in the application and development of the MDLC technique since Cortes s book is in life sciences, which accounts for approximately half of this book. One reason for this may be due to the high level of interest in studying the human proteome (proteomics). Proteomics is such a demanding application that the separating power needed to resolve even the normal proteins in the body is so demanding that maximum separation power is needed to provide this capability. Many aspects of separations in proteomics are discussed in Chapters 9-13, 15 and 16. Chapter 14 discusses enantiomeric compound separations by MDLC. 

Frahm, J.L., Howard, B.E., Heber, S., Muddiman, D.C. (2006). Accessible proteomics space and its implications for peak capacity for zero-, one-, and two-dimensional separations coupled with FT-ICR and TOF mass spectrometry. J. Mass Spectrom. 41, 281-288. 

D-polyacrylamide gel electrophoresis) maps of protein mixtures is discussed. 2D PAGE is considered the classical and principal tool for protein separation—prior to mass spectrometry—to achieve the main goal of proteomics, that is, a comprehensive identification and quantification of every protein present in a complex biological sample that would allow analysis of an entire intact proteome (Wilkins et al., 1997 Righetti et al., 2001 Hamdan and Righetti, 2005). 

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