Food Biochemistry and Food Processing (2 edition)

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22 Application of Proteomics to Fish Processing and Quality 411

this does not present a problem. However, when investigating,

for example, regulatory cascades, the proteins of interest are

likely to be present in very low abundance and may at times

be undetectable because of the dominance of high-abundance

ones. Simply increasing the amount of sample is usually not

an option, as it will give rise to overloading artifacts in the

gels (O’Farrell 1975). In transcriptomic studies, where a simi-

lar disparity can be seen in the abundance of RNA transcripts

present, this problem can be overcome by amplifying the low-

abundance transcripts using the polymerase chain-reaction, but

no such technique is available for proteins. The remaining op-

tion, then, is fractionation of the protein sample in order to weed

out the high-abundance proteins, allowing a larger sample of the

remaining proteins to be analyzed. A large number of fraction-

ation protocols, both specific and general, are available. Thus,

Østergaard and coworkers used acetone precipitation to reduce

the abundance of hordeins present in barley (Hordeum vulgare)

extracts (Østergaard et al. 2002) whereas Locke and cowork-

ers used preparative isoelectrofocussing to fractionate Chinese

snow pea (Pisum sativum macrocarpon) lysates into fractions

covering three pH regions (Locke et al. 2002). The fractionation

method of choice will depend on the specific requirements of

the study and on the tissue being studied. Discussion of some

fractionation methods can be found in Ahmed (2009), Bodzon-

Kulakowska et al. (2007), Butt et al. (2001), Canas et al. (2007),

Corthals et al. (1997), Dreger (2003), Fortis et al. (2008), Issaq

et al. (2002), Lee and Pi (2009), Lopez et al. (2000), Millea

and Krull (2003), Pieper et al. (2003), Righetti et al. (2005a, b)

Rothemund et al. (2003), von Horsten (2006).

Identification by Peptide Mass Fingerprinting

Identification of proteins on 2DE gels is most commonly

achieved via mass spectrometry (MS) of trypsin digests. Briefly,

the spot of interest is excised from the gel, digested with trypsin

(or another protease), and the resulting peptide mixture is ana-

lyzed by MS. The most popular MS method is matrix-assisted

laser desorption/ionization time-of-flight (MALDI-TOF) MS

(Courchesne and Patterson 1999), where peptides are suspended

in a matrix of small, organic, ultraviolet-absorbing molecules

(such as 2,5-dihydroxybenzoic acid) followed by ionization by

a laser at the excitation wavelength of the matrix molecules and

acceleration of the ionized peptides in an electrostatic field into

a flight tube where the time of flight of each peptide is measured,

giving its expected mass.

The resulting spectrum of peptide masses (Fig. 22.5) is then

used for protein identification by searching against expected pep-

tide masses calculated from data in protein sequence databases,

such as SwissProt or the National Centre for Biotechnology In-

formation (NCBI) nonredundant protein sequences data base,

using the appropriate software. Several programs are available,

many with a web-based open-access interface. The ExPASy

Intensity

Mass (m/z)

Figure 22.5.A trypsin digest mass spectrometry fingerprint of a rainbow trout liver protein spot, identified as apolipoprotein A I-1 (S. Martin,

unpublished). The open arrows indicate mass peaks corresponding to trypsin self-digestion products and were, therefore, excluded from the

analysis. The solid arrows indicate the peaks that were found to correspond to expected apolipoprotein A I-1 peptides.