Proteomics protein-complex identification

A relatively common question addressed by proteomics is the identification of proteins that interact with a target of interest to produce a protein complex. If an antibody to one member of the complex is available it is theoretically possible to isolate and identify all the associated proteins. Alternatively, an epitope tagged (for example, FLAG or HA) version of a protein can be expressed in a cell and the complex isolated by use of an antibody raised against the tag. 

A. C. Gavin, M. Bosche, R. Krause et al. Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature, 415 (2002), 141 Y. Ho, A. Gruhler, A. Heilbut et al. Systematic identification of protein complexes in Saccharomyces cerevisiae by mass spcclrometry. Nature, 415 (2002), 180. 

Qualitative and quantitative comparison of proteomes under different conditions can now help to further unravel complex biological processes. This is due to drastic improvements in massively parallel protein separation, identification and characterization as well as in bioinformatics. 

Another approach to overcoming the limitations inherent in the lEF dimension of 2-DE is to use alternative types of 2-D separations. 2-D blue native (BN) electrophoresis (Schagger and von Jagow, 1991) can be used to separate membrane and other functional protein complexes as intact, enzymatically active complexes in the first dimension. This is followed with a second-dimension separation by Tricine-SDS-PAGE to separate the complexes into their component subunits. This method, combined with protein identification by MALDI PMF, has been applied to several studies of the mitochondrial proteome (Brookes et al., 2002 Kraft et al., 2001). In a study of human heart mitochondria using BN/SDS-PAGE, the individual subunits of all five complexes of the oxidative phosphorylation system were represented and a novel variant of cytochrome c oxidase subunit Vic was reported (Devreese et al., 2002). 

Identification of the functions of proteins and other polymeric complexes or cell proteomes, in which the achievements of proteomics contributes greatly, is the subject of intensive research [1], Modem technology now allows us to investigate not only individual protein molecules in living cell, but also to understand their interaction with other macromolecules and reveal their previously unknown functions. Several facts are determined participation of polyfunctional macromolecular protein complexes in the biosynthesis of fatty acids, involvement of erythrocyte membrane proteins macromolecular complexes in exchange of COJO, biological effects of some growth factors (polyfunctional proteins), which sometimes is achieved by interactions of other protein complexes, etc. [2-4],... 

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). 

---