Food Biochemistry and Food Processing (2 edition)

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22 Application of Proteomics to Fish Processing and Quality 407

Figure 22.1.An overview over the “classic approach” in proteomics. First, a protein extract (crude or fractionated) from the tissue of choice is

subjected to two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Once a protein of interest has been identified, it is excised from

the gel, subjected to degradation by trypsin (or other suitable protease) and the resulting peptides analyzed by mass spectrometry (MS),

yielding a peptide mass fingerprint. In many cases, this is sufficient for identification purposes, but if needed, peptides can be dissociated into

smaller fragments and small partial sequences obtained by tandem mass spectrometry (MS/MS). See text for further details.

two-dimensional electrophoresis (2DE) followed by protein

identification via peptide mass fingerprinting of trypsin digests

(Fig. 22.1) remains the workhorse of most proteomics work,

largely because of its high resolution, simplicity, and mass accu-

racy. This “classic approach” will, therefore, be the main focus of

this chapter. A number of reviews on the advances and prospects

of proteomics within various fields of study are available. Some

recent ones include: Andersen and Mann (2006), Balestrieri

et al. (2008), Beretta (2009), Bogyo and Cravatt (2007), Drabik

et al. (2007), Ikonomou et al. (2009), Issaq and Veenstra (2008),

Jorrin-Novo et al. (2009), Latterich et al. (2008), L ́opez (2007),

Mamone et al. (2009), Malmstrom et al. (2007), Premsler et al.

(2009), Smith et al. (2009), Wang et al. (2006), Wilm (2009),

Yates et al. (2009).

Two-Dimensional Electrophoresis

2DE, the cornerstone of most proteomics research, is the si-

multaneous separation of hundreds, or even thousands, of pro-

teins on a two-dimensional polyacrylamide slab gel. The po-

tential of a two-dimensional protein separation technique was

realized early on, with considerable development efforts taking

place in the 1960s (Margolis and Kenrick 1969, Kaltschmidt

and Wittmann 1970). The method most commonly used today

was developed by Patrick O’Farrell. It is described in his seminal

and thorough 1975 paper (O’Farrell 1975) and is outlined briefly

later. It is worth emphasizing that great care must be taken that

the proteome under investigation is reproducibly represented on

the 2DE gels, and that individual variation in specific protein