18 Proteomics: Methodology and Application in Fish...

18Proteomics: Methodology andApplication in Fish Processing

O. T. Vilhelmsson*, S. A. M. Martin, B. M. Poli, and D. F. Houlihan

401

IntroductionProteomics Methodology

Two-Dimensional ElectrophoresisSome Problems and Their Solutions

Identification by Peptide Mass FingerprintingSeafood Proteomics and Their Relevance to Processing and

QualityTracking Quality Changes Using ProteomicsAntemortem Effects on Quality and Processability

Antemortem Metabolism and Postmortem Qualityin Trout

Potential for Further Antemortem ProteinDegradation Studies

Can Antemortem Proteomics Shed Light on GapingTendency?

Species AuthenticationIdentification and Characterization of AllergensReferences

INTRODUCTION

Proteomics is most succinctly defined as “the studyof the entire proteome or a subset thereof,” the pro-teome being the expressed protein complement ofthe genome. Unlike the genome, the proteome variesamong tissues, as well as with time, in reflection ofthe organism’s environment and its adaptation there-to. Proteomics can therefore give a snapshot of theorganism’s state of being and, in principle at least,map the entirety of its adaptive potential and mecha-nisms. As with all living matter, foodstuffs are inlarge part made up of proteins. This is especially

true of fish and meat, where the bulk of the foodmatrix is constructed from proteins. Furthermore,the construction of the food matrix, both on the cel-lular and tissuewide levels, is regulated and broughtabout by proteins. It stands to reason, then, that pro-teomics is a tool that can be of great value to thefood scientist, giving valuable insight into the composition of the raw materials; quality involutionwithin the product before, during, and after process-ing or storage; and the interactions of the proteinswith one another, with other food components, orwith the human immune system after consumption.In this chapter, a brief overview of “classical” pro-teomics methodology is presented, and present andfuture applications in relation to fish and seafoodprocessing and quality are discussed.

PROTEOMICS METHODOLOGY

Unlike nucleic acids, proteins are an extremely var-iegated group of compounds in terms of their chem-ical and physical properties. It is not surprising,then, that a field that concerns itself with “the sys-tematic identification and characterization of pro-teins for their structure, function, activity and mo-lecular interactions” (Peng et al. 2003) shouldpossess a toolkit containing a wide spectrum ofmethods that continue to be developed at a briskpace. While high-throughput, gel-free methods, forexample, those based on liquid chromatography tan-dem mass spectrometry (LC-MS/MS) (Peng et al.2003), surface-enhanced laser desorption/ionization(Hogstrand et al. 2002), or protein arrays (Lee and*Corresponding author.

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402 Part III: Muscle Foods

Figure 18.1. 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. Once a protein of inter-est has been identified, it is excised from the gel and subjected to degradation by trypsin (or other suitable protease).The resulting peptides are analyzed by mass spectrometry, yielding a peptide mass fingerprint. In many cases this issufficient for identification purposes, but if needed, peptides can be dissociated into smaller fragments, and smallpartial sequences can be obtained by tandem mass spectrometry. See text for further details.

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18 Proteomics in Fish Processing 403

Nagamune 2004), hold great promise and are de-serving of discussion in their own right, the classicprocess of two-dimensional electrophoresis (2DE)followed by protein identification via peptide massfingerprinting of trypsin digests (Fig. 18.1) remainsthe workhorse of most proteomics work, largelybecause of its high resolution, simplicity, and massaccuracy. This “classic approach” will therefore bethe main focus of this chapter. A number of reviewson the advances and prospects of proteomics withinvarious fields of study are available. Some recentones include Aebersold and Mann (2003), Cash(2002), Cash and Kroll (2003), Graves and Haystead(2003), Huber et al. (2003), Kvasnicka (2003), Phi-zicky et al. (2003), Piñeiro et al. (2003), Pusch et al.(2003), Takahashi et al. (2003), and Tyers and Mann(2003).

TWO-DIMENSIONAL ELECTROPHORESIS

Two-dimensional electrophoresis (2DE), the corner-stone of most proteomics research, is the simultane-ous separation of hundreds, or even thousands, ofproteins on a 2D polyacrylamide slab gel. The po-tential of a 2D protein separation technique wasrealized early on, and considerable developmentefforts took place in the 1960s (Kaltschmidt andWittmann 1970, Margolis and Kenrick 1969). Themethod most commonly used today was developedby Patrick O’Farrell and is described in his seminaland thorough 1975 paper (O’Farrell 1975). O’Far-rell’s method first applies a process called isoelectricfocusing, where an electric field is applied to a tubegel on which the protein sample and carrier am-pholytes have been deposited. This separates theproteins according to their molecular charge. Thetube gel is then transferred onto a polyacrylamideslab gel, and the isoelectrically focused proteins arefurther separated according to their molecular massby conventional sodium dodecyl sulfate–polyacry-lamide gel electrophoresis (SDS-PAGE), yielding atwo-dimensional map (Fig. 18.2) rather than the fa-miliar banding pattern observed in one-dimensionalSDS-PAGE. The map can be visualized and individ-ual proteins quantified by radiolabeling or by usingany of a host of protein dyes and stains, such asCoomassie blue, silver stains, or fluorescent dyes.By comparing the abundance of individual proteinson a number of gels (Fig. 18.3), up- or downregula-tion of these proteins can be inferred. It is worth

emphasizing that great care must be taken that theproteome under investigation is reproducibly repre-sented on the 2DE gels, and that individual variationin specific protein abundance is taken into consider-ation by running gels from a sufficient number ofsamples and performing the appropriate statistics.Pooling samples may also be an option, dependingon the type of experiment. Although a number ofrefinements have been made to 2DE since O’Far-rell’s paper was published, most notably the intro-duction of immobilized pH gradients (IPGs) for iso-electrofocusing (Görg et al. 1988), the procedureremains essentially as outlined above. For more de-tailed, up-to-date descriptions of methods, the read-er is referred to any of a number of excellent booksand laboratory manuals, such as Berkelman andStenstedt (1998), Link (1999), Walker (2002), andWestermeier and Naven (2002).

Some Problems and Their Solutions

The high resolution and good sensitivity of 2DE are what make it the method of choice for most pro-teomics work, but the method nevertheless has

Figure 18.2. A 2DE protein map of rainbow trout(Oncorhynchus mykiss) liver proteins with pH between4 and 7 and molecular mass about 10–100 (S. Martin,unpublished). The proteins are separated according totheir pH in the horizontal dimension and according totheir mass in the vertical dimension. Isoelectrofocusingwas by pH 4–7 immobilized pH gradient (IPG) strip, andthe second dimension was in a 10–15% gradient poly-acrylamide slab gel.

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several drawbacks. The most significant of thesehave to do with the diversity of proteins and theirexpression levels. For example, hydrophobic pro-teins do not readily dissolve in the buffers used forisoelectrofocusing. This problem can be overcome,though, using nonionic or zwitterionic detergents,allowing for 2DE of membrane and membrane-associated proteins (Babu et al. 2004, Chevallet etal. 1998, Henningsen et al. 2002, Herbert 1999).Vilhelmsson and Miller (2002), for example, wereable to use “membrane protein proteomics” to de-monstrate the involvement of membrane-associatedmetabolic enzymes in the osmoadaptive response ofthe foodborne pathogen Staphylococcus aureus. A2DE gel image of S. aureus membrane-associatedgels is shown in Figure 18.4.

Similarly, resolving alkaline proteins, particularlythose with pH above 10, on 2D gels has been prob-lematic in the past. Although the development ofhighly alkaline, narrow-range IPGs (Bossi et al.1994) allowed reproducible two-dimensional reso-lution of alkaline proteins (Guorg et al. 1997), theirrepresentation on wide-range 2DE of complex mix-tures such as cell extracts remained poor. Improve-ments in resolution and representation of alkalineproteins on wide-range gels have been made (Guorg

Figure 18.3. A screenshot from the 2DE analysis program Phoretix 2-D (NonLinear Dynamics, Gateshead, Tyne andWear, United Kingdom) showing some steps in the analysis of a two-dimensional protein map. Variations in abun-dance of individual proteins, as compared with a reference gel, can be observed and quantified.

Figure 18.4. A 2DE membrane proteome map fromStaphylococcus aureus, showing proteins with pHbetween 3 and 10 and molecular mass about 15–100(O. Vilhelmsson and K. Miller, unpublished).Isoelectrofocusing was in the presence of a mixture ofpH 5–7 and pH 3–10 carrier ampholytes, and the sec-ond dimension was in a 10% polyacrylamide slab gelwith a 4% polyacrylamide stacker.

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et al. 1999); nevertheless an approach that involvesseveral gels, each of a different pH range, from thesame sample is advocated for representative inclu-sion of alkaline proteins when studying entire pro-teomes (Cordwell et al. 2000). Indeed, Cordwell etal. were able to significantly improve the representa-tion of alkaline proteins in their study on the rela-tively highly alkaline Helicobacter pylori proteomeby using both pH 6–11 and pH 9–12 IPGs (Bae et al.2003).

A second drawback of 2DE has to do with theextreme difference in expression levels of the cell’svarious proteins, which can be as much as 10,000-fold. This leads to swamping of low abundance pro-teins by high abundance ones on the two-dimensionalmap, rendering analysis of low abundance proteinsdifficult or impossible. For applications such as spe-cies identification or the study of major biochemicalpathways, where the proteins of interest are presentin relatively high abundance, this does not present aproblem. However, when investigating, for example,regulatory cascades, the proteins of interest are likelyto be present in very low abundance and may at timesbe undetectable because of the dominance of highabundance proteins. Simply increasing the amount ofsample is usually not an option, as it will give rise tooverloading artifacts in the gels (O’Farrell 1975). Intranscriptomic studies, where a similar disparity canbe seen in the abundance of RNA transcripts present,this problem can be overcome by amplifying the lowabundance transcripts using the polymerase chainreaction (PCR), but no such technique is available forproteins. The remaining option, then, is fractionationof the protein sample in order to weed out the highabundance proteins, allowing a larger sample of theremaining proteins to be analyzed. A large number offractionation protocols, both specific and general, areavailable. Thus, Østergaard et al. used acetone pre-cipitation to reduce the abundance of hordeins pres-ent in barley (Hordeum vulgare) extracts (Østergaardet al. 2002), whereas Locke et al. used preparativeisoelectrofocusing to fractionate Chinese snow pea(Pisum sativum macrocarpon) lysates into fractionscovering three pH regions (Locke et al. 2002). Thefractionation method of choice will depend on thespecific requirements of the study and on the tis-sue being studied. Discussion of some fractionationmethods can be found in Butt et al. (2001), Corthalset al. (1997), Dreger (2003), Issaq et al. (2002), Lo-pez et al. (2000), Millea and Krull (2003), Pieper etal. (2003), and Rothemund et al. (2003).

IDENTIFICATION BY PEPTIDE MASS

FINGERPRINTING

Identification of proteins on 2DE gels is most com-monly achieved via mass spectrometry of trypsindigests. Briefly, the spot of interest is excised fromthe gel and digested with trypsin (or another pro-tease), and the resulting peptide mixture is analyzedby mass spectrometry. The most popular mass spec-trometry method is matrix-assisted laser desorp-tion/ionization –time-of-flight (MALDI-TOF) massspectrometry (Courchesne and Patterson 1999),where peptides are suspended in a matrix of small,organic, UV-absorbing molecules (such as 2,5-dihydroxybenzoic acid), followed by ionization by alaser at the excitation wavelength of the matrix mol-ecules and acceleration of the ionized peptides in anelectrostatic field into a flight tube where the time offlight of each peptide is measured, giving its expect-ed mass.

The resulting spectrum of peptide masses (Fig.18.5) is then used for protein identification by search-ing against expected peptide masses calculated fromdata in protein sequence databases, such as SwissProt or the National Center for Biotechnology In-formation (NCBI) nonredundant protein sequencesdata base, using the appropriate software. Severalprograms are available, many with a web-basedopen-access interface. The ExPASy Tools website(http://www.expasy.org/tools) contains links to mostof the available software for protein identificationand several other tools. Attaining a high identifica-tion rate is problematic in fish and seafood pro-teomics due to the relative paucity of available pro-tein sequence data for these animals. As can be seenin Table 18.1, this problem is surprisingly acute forspecies of commercial importance. To circumventthis problem, it is possible to take advantage of theavailable nucleotide sequences, which in many cas-es are more extensive than the protein sequencesavailable, to obtain a tentative identity. How usefulthis method is will depend on the length and qualityof the available nucleotide sequences. It is importantto realize, however, that an identity obtained in thismanner is less reliable than that obtained throughprotein sequences and should be regarded only astentative in the absence of corroborating evidence(such as two-dimensional immunoblots, correlatedactivity measurements, or transcript abundance). Intheir work on the rainbow trout (Oncorhynchusmykiss) liver proteome, Martin et al. (2003b) and

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Vilhelmsson et al. (2004) were able to attain anidentification rate of about 80% using a combinationof search algorithms that included the open-accessMascot program (Perkins et al. 1999) and a licensedversion of Protein Prospector MS-Fit (Clauser et al.1999), searching against both protein databases anda database containing all salmonid nucleotide se-quences. In those cases where both the protein andnucleotide databases yielded results, a 100% agree-ment was observed between the two methods.

A more direct, if rather more time consuming,way of obtaining protein identities is by direct se-quence comparison. Until recently, this was accom-plished by N-terminal or internal (after proteolysis)sequencing by Edman degradation of eluted or elec-troblotted protein spots (Erdjument-Bromage et al.1999, Kamo and Tsugita 1999). Today, the methodof choice is tandem mass spectrometry (MS/MS). Inthe peptide mass fingerprinting discussed above,each peptide mass can potentially represent any of alarge number of possible amino acid sequence com-binations. The larger the mass (and longer the se-quence), the higher is the number of possible combi-

nations. In MS/MS one or several peptides are sepa-rated from the mixture and dissociated into frag-ments that then are subjected to a second round ofmass spectrometry, yielding a second layer of infor-mation. Correlating this spectrum with the candidatepeptides identified in the first round narrows downthe number of candidates. Furthermore, several shortstretches of amino acid sequence will be obtainedfor each peptide, which, when combined with thepeptide and fragment masses obtained, enhances thespecificity of the method even further (Chelius et al.2003, Wilm et al. 1996, Yu et al. 2003b). Mass spec-trometry methods in proteomics are reviewed inYates (1998).

SEAFOOD PROTEOMICS ANDTHEIR RELEVANCE TOPROCESSING AND QUALITY

Two-dimensional electrophoresis–based proteomicshave found a number of applications within food sci-ence. Among early examples are such applicationsas characterization of bovine caseins (Zeece et al.

Figure 18.5. A trypsin digest mass spectrometry fingerprint of a rainbow trout liver protein spot, identified asapolipoprotein 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 werefound to correspond to expected apolipoprotein A I-1 peptides.

18CH_Hui_277065 8/22/05 9:37 AM Page 406

407

Tab

le 1

8.1.

Som

e C

omm

erci

ally

or

Sci

entifi

cally

Impo

rtan

t Fis

h an

d S

eafo

od S

peci

es a

nd th

e A

vaila

bilit

y of

Pro

tein

and

Nuc

leot

ide

Seq

uenc

e D

ata

as o

f Jun

e 7,

200

4

Prot

ein

Nuc

leot

ide

Prot

ein

Nuc

leot

ide

Sequ

ence

sSe

quen

ces

Sequ

ence

sSe

quen

ces

Act

inop

tery

gii(

ray-

finne

d fis

hes)

77,3

961,

586,

862

Tetr

aodo

ntif

orm

es(p

uffe

rs a

nd

29,3

8730

5,44

9fil

efish

es)

Elo

pom

orph

a1,

215

1,47

3Pu

ffer

fish

(Tak

ifug

u ru

brip

es)

948

89,9

01A

ngui

llif

orm

es(e

els

and

mor

ays)

966

1,35

4G

reen

puf

ferfi

sh(T

etra

odon

28

,149

215,

158

nigr

ovir

idis

)E

urop

ean

eel (

Ang

uill

a an

guil

la)

114

199

Zei

form

es(d

orie

s)17

157

Clu

peom

orph

a18

033

7Jo

hn D

ory

(Zeu

s fa

ber)

3429

Clu

peif

orm

es(h

erri

ngs)

180

337

Scor

paen

ifor

mes

(sco

rpio

nfish

es/

634

1,38

8fla

thea

ds)

Atla

ntic

her

ring

(C

lupe

a ha

reng

us)

2935

Red

fish

(Seb

aste

s m

arin

es)

37

Eur

opea

n pi

lcha

rd (

Sard

ina

1744

Lum

psuc

ker

(Cyc

lopt

erus

lum

pus)

314

pilc

hard

us)

Ost

ario

phys

ii21

,562

771,

661

Cyp

rini

form

es(c

arps

)18

,890

722,

727

Cho

ndri

chth

yes

(car

tila

geno

us

2,38

92,

224

fishe

s)Z

ebra

fish

(Dan

io r

erio

)13

,659

704,

204

Car

char

hini

form

es(g

roun

d sh

arks

)48

039

9Si

luri

form

es(c

atfis

hes)

1,67

447

,635

Les

ser

spot

ted

dogfi

sh (

Scyl

iorh

inus

208

104

cani

cula

)C

hann

el c

atfis

h (I

ctal

urus

pun

ctat

us)

532

35,2

40B

lue

shar

k (P

rion

ace

glau

ca)

84

Pro

taca

ntho

pter

ygii

4,39

225

7,95

3L

amni

form

es(m

ackr

el s

hark

s)17

823

9Sa

lmon

ifor

mes

(sal

mon

s)4,

230

257,

923

Bas

king

sha

rk (

Cet

orhi

nus

max

imus

)16

16A

tlant

ic s

alm

on (

Salm

o sa

lar)

686

90,5

77R

ajif

orm

es(s

kate

s)27

530

4R

ainb

ow tr

out (

Onc

orhy

nchu

s m

ykis

s)1,

480

159,

907

Tho

rny

skat

e (R

aja

radi

ata)

396

Arc

tic c

harr

(Sa

lvel

inus

alp

inus

)90

251

Blu

e sk

ate

(Raj

a ba

tis)

10

Para

cant

hopt

eryg

ii1,

880

2,33

5L

ittle

ska

te (

Raj

a er

inac

ea)

162

152

Gad

ifor

mes

(cod

s)1,

445

1,52

8A

tlant

ic c

od (

Gad

us m

orhu

a)90

593

6M

ollu

sca

(mol

lusk

s)11

,229

35,1

87A

lask

a po

llock

(T

hera

gra

124

136

Biv

alvi

a3,

072

15,9

26ch

alco

gram

ma)

Saith

e (P

olla

chiu

s vi

rens

)16

26B

lue

mus

sel (

Myt

ilus

edu

lis)

535

591

(Con

tinue

s)

18CH_Hui_277065 8/22/05 9:37 AM Page 407

408

Tab

le 1

8.1.

(Con

tinue

d)

Prot

ein

Nuc

leot

ide

Prot

ein

Nuc

leot

ide

Sequ

ence

sSe

quen

ces

Sequ

ence

sSe

quen

ces

Had

dock

(M

elan

ogra

mm

us a

egle

finus

)56

61B

ay s

callo

p (A

rgop

ecte

n ir

radi

ans)

992,

106

Lop

hiif

orm

es(a

ngle

rfish

es)

197

82G

astr

opod

a7,

036

17,4

84M

onkfi

sh (

Lop

hius

pis

cato

rius

)6

9C

omm

on w

helk

(B

ucci

num

und

atum

)4

15A

cant

hopt

eryg

ii45

,732

550,

100

Aba

lone

(H

alio

tis

tube

rcul

ata)

1115

8Pe

rcif

orm

es(p

erch

-lik

es)

9,53

260

,715

Cep

halo

poda

931

1,49

0G

ilthe

ad s

ea b

ream

(Sp

arus

aur

ata)

139

325

Nor

ther

n E

urop

ean

squi

d (L

olig

o 30

39fo

rbes

i)E

urop

ean

sea

bass

(D

icen

trac

hus

150

264

Com

mon

cut

tlefis

h (S

epia

offi

cina

lis)

5244

labr

ax)

Atla

ntic

mac

krel

(Sc

ombe

r sc

ombr

us)

823

Com

mon

oct

opus

(O

ctop

us v

ulga

ris)

5879

Alb

acor

e (T

hunn

us a

lalu

nga)

4012

4B

luefi

n tu

na (

Thu

nnus

thyn

nus)

8517

8C

rust

acea

(cru

stac

eans

)6,

295

24,6

38Sp

otte

d w

olffi

sh (

Ana

rhic

has

min

or)

3016

Car

idea

689

916

Ber

ycif

orm

es (

saw

bell

ies)

345

181

Nor

ther

n sh

rim

p (P

anda

lus

bore

alis

)11

8O

rang

e ro

ughy

(H

oplo

stet

hus

012

Ast

acid

ea(l

obst

ers

and

cray

fishe

s)64

63,

507

atla

ntic

us)

Ple

uron

ecti

form

es(fl

atfis

hes)

957

7,39

2A

mer

ican

lobs

ter

(Hom

erus

16

02,

140

amer

ican

us)

Atla

ntic

hal

ibut

(H

ippo

glos

sus

3869

9E

urop

ean

cray

fish

(Ast

acus

ast

acus

)26

11hi

ppog

loss

us)

Witc

h (G

lypt

ocep

halu

s cy

nogl

ossu

s)5

22L

ango

ustin

e (N

ephr

ops

norv

egic

us)

1818

Plai

ce (

Ple

uron

ecte

s pl

ates

sa)

5021

6B

rach

yura

(sho

rt-t

aile

d cr

abs)

556

1,21

3W

inte

r flo

unde

r (P

seud

ople

uron

ecte

s 13

11,

347

Edi

ble

crab

(C

ance

r pa

guru

s)34

7am

eric

anus

)T

urbo

t (Sc

opht

halm

us m

axim

us)

4911

2B

lue

crab

(C

alli

nect

es s

apid

us)

4530

Sour

ce:

NC

BI

TaxB

row

ser

(http

://w

ww

.ncb

i.nlm

.nih

.gov

/Tax

onom

y/ta

xono

myh

ome.

htm

l/).

18CH_Hui_277065 8/22/05 9:37 AM Page 408


1989), wheat flour baking quality factors (Dough-erty et al. 1990), and soybean protein bodies (Leiand Reeck 1987). In recent years, proteomic investi-gations on fish and seafood products, as well as infish physiology, have gained considerable momen-tum, as can be seen in recent reviews (Parringtonand Coward 2002, Piñeiro et al. 2003). Herein, re-cent and future developments in fish and seafoodproteomics as relates to issues of concern in fishprocessing or other quality considerations are dis-cussed, paying particular attention to the as yet littleexploited potential for investigating the antemortemproteome for the benefit of postmortem quality invo-lution.

TRACKING QUALITY CHANGES USING

PROTEOMICS

A persistent problem in the seafood industry is post-mortem degradation of fish muscle during chilledstorage, which has deleterious effects on the fishflesh texture, yielding a tenderized muscle. Thisphenomenon is thought to be primarily due to autol-ysis of muscle proteins, but the details of this proteindegradation are still somewhat in the dark. However,degradation of myofibrillar proteins by calpains andcathepsins (Ladrat et al. 2000, Ogata et al. 1998) anddegradation of the extracellular matrix by the matrixmetalloproteases and matrix serine proteases, whichare capable of degrading collagens, proteoglycans,and other matrix components (Lødemel and Olsen2003, Woessner 1991), are thought to be among themain culprits. Whatever the mechanism, it is clearthat these quality changes are species dependent(Papa et al. 1996, Verrez-Bagnis et al. 1999) and,furthermore, appear to display seasonal variations(Ingólfsdóttir et al. 1998, Ladrat et al. 2000). Forexample, whereas desmin is degraded postmortemin sardine and turbot, no desmin degradation wasobserved in sea bass and brown trout (Verrez-Bagniset al. 1999). Of further concern is the fact that sever-al commercially important fish muscle processingtechniques, such as curing, fermentation, and pro-duction of surimi and conserves, occur under condi-tions conducive to endogenous proteolysis (Pérez-Borla et al. 2002). As with postmortem proteindegradation during storage, autolysis during pro-cessing seems to be somewhat specific. Indeed, themyosin heavy chain of the Atlantic cod was shownto be significantly degraded during processing of

“salt fish” (bachalhau), whereas actin was lessaffected (Thorarinsdottir et al. 2002). Problems ofthis kind, where differences are expected to occur inthe number, molecular mass, and pH of the proteinspresent in a tissue, are well suited to investigationusing 2DE-based proteomics. It is also worth notingthat protein isoforms other than proteolytic ones,whether they be encoded in structural genes orbrought about by posttranslational modification,usually have a different molecular weight or pH andcan, therefore, be distinguished on 2DE gels. Thus,specific isoforms of myofibrillar proteins, many ofwhich are correlated with specific textural propertiesin seafood products, can be observed using 2DE orother proteomic methods (Martinez et al. 1990,Piñeiro et al. 2003).

Several 2DE studies have been performed onpostmortem changes in seafood flesh (Kjærsgårdand Jessen 2003, Martinez and Jakobsen Friis 2004,Martinez et al. 2001a, Martinez et al. 1990, Mor-zel et al. 2000, Verrez-Bagnis et al. 1999) and havedemonstrated the importance and complexity of pro-teolysis in seafood during storage and processing.For example, Martinez et al. (1992) used a 2DEapproach to demonstrate different protein composi-tions of surimi made from prerigor versus postrigorcod, and they found that 2DE could distinguish be-tween the two. Kjærsgård and Jessen, who used2DE to study changes in the abundance of severalmuscle proteins during storage of the Atlantic cod(Gadus morhua), proposed a general model for post-mortem protein degradation in fish flesh in whichinitially calpains are activated due to the increase incalcium levels in the muscle tissue. Later, as pHdecreases and ATP is depleted, with the consequentonset of rigor mortis, cathepsins and the proteasomeare activated sequentially (Kjærsgård and Jessen2003).

ANTEMORTEM EFFECTS ON QUALITY AND

PROCESSABILITY

Malcolm Love started his 1980 review paper on bio-logical factors affecting fish processing (Love 1980)with a lament for the easy life of poultry processorswho, he said, had the good fortune to work on aproduct reared from hatching under strictly con-trolled environmental and dietary conditions “so thatplastic bundles of almost identical foodstuff for mancan be lined up on the shelf of a shop.” Since the

18CH_Hui_277065 8/22/05 9:37 AM Page 409


time of Love’s review, the advent of aquaculture hasmade attainable, in theory at least, just such a utopi-an vision. As every food processor knows, the quali-ty of the raw material is among the most crucialvariables that affect the quality of the final product.In fish processing, therefore, the animal’s own indi-vidual physiological status will to a large extent dic-tate where quality characteristics will fall within theconstraints set by the species’ physical and bio-chemical makeup. It is well known that an organ-ism’s phenotype, including quality characteristics, isdetermined by environmental as well as genetic fac-tors. Indeed, Huss noted in his review (Huss 1995)that product quality differences within the same fishspecies can depend on feeding and rearing condi-tions, differences wherein can affect postmortembiochemical processes in the product, which in turn,affect the involution of quality characteristics in thefish product. The practice of rearing fish in aquacul-ture, as opposed to catching wild fish, therefore raisesthe tantalizing prospect of managing the qualitycharacteristics of the fish flesh antemortem, whereindividual physiological characteristics, such as thosegoverning gaping tendency, flesh softening duringstorage, and so on, are optimized. To achieve thatgoal, the interplay between these physiological para-meters and environmental and dietary variablesneeds to be understood in detail. With the ever-increasing resolving power of molecular techniques,such as proteomics, this is fast becoming feasible.

Antemortem Metabolism and PostmortemQuality in Trout

In mammals, antemortem protease activities havebeen shown to affect meat quality and texture (Kris-tensen et al. 2002, Vaneenaeme et al. 1994). Forexample, an antemortem upregulation of calpainactivity in swine (Sus scrofa) will affect postmortemproteolysis and, hence, meat tenderization (Kris-tensen et al. 2002). In beef (Bos taurus), a correla-tion was found between ante- and postmortem activ-ities of some proteases, but not others (Vaneenaemeet al. 1994). As discussed in the above section, post-mortem proteolysis is a matter of considerable im-portance in the fish and seafood industry, and anyantemortem effects thereon are surely worth investi-gating.

In a recent study on the feasibility of substitutingfishmeal in rainbow trout (Oncorhynchus mykiss)

diets with protein from plant sources, 2DE-basedproteomics were among techniques used (Martin etal. 2003a,b; Vilhelmsson et al. 2004). Concomit-antly, various quality characteristics of fillet and bodywere also measured (De Francesco et al. 2004, Parisiet al. 2004). Among the findings was that, accordingto a triangular sensory test using a trained panel,cooked trout that had been fed the plant protein diethad higher hardness, lower juiciness, and lower odorintensity than those fed the fishmeal-containing diet,indicating an effect of antemortem metabolism onproduct texture. Furthermore, the amount and com-position of free amino acids in the fish flesh was sig-nificantly affected by the diet, as was the post-mortem development of the free amino acid pool.For example, while abundance of arginine was foundto decrease during storage of flesh from fishmeal-fed fish, it increased during storage of plant protein–fed fish (Table 18.2). The diets had been formulatedto have a nearly identical amino acid composition,and therefore these results may be taken to indicatealtered postmortem proteolytic activity in the plantprotein–fed fish as compared with the fishmeal-fedones.

In the proteomics part of the study, the liver pro-teome was chosen for investigation, since the liver isthe primary seat of many of the fish’s key metabolicpathways. This makes a direct comparison of theproteomic and quality characteristics results diffi-cult; nevertheless, some interesting observations canbe made. The study identified a number of metabol-ic pathways sensitive to plant protein substitution inrainbow trout feed, for example, pathways involvedin cellular protein degradation, fatty acid break-down, and NADPH metabolism (Table 18.3). In thecontext of this chapter, the effects on the proteasomeare particularly noteworthy. The proteasome is amultisubunit enzyme complex that catalyzes prote-olysis via the ATP-dependent ubiquitin-proteasomepathway, which in mammals, is thought to be re-sponsible for a large fraction of cellular proteolysis(Craiu et al. 1997, Rock et al. 1994). In rainbowtrout, the ubiquitin-proteasome pathway has beenshown to be downregulated in response to starvation(Martin et al. 2002) and to have a role in regulatingprotein deposition efficiency (Dobly et al. 2004).

Correlating the findings of these two parts of thestudy, it seems likely that the difference in textureand postmortem free amino acid pool developmentare affected by antemortem proteasome activity,

18CH_Hui_277065 8/22/05 9:37 AM Page 410


Table 18.2. Free Amino Acids and NH3 Levels in Muscle of Trout Fed a Fishmeal–ContainingDiet (FM) or a Plant Protein–Containing Diet (PP) at 0 and 9 Days after Death

0 d FM 9 d FM 0 d PP 9 d PPFAA � NH3 (n � 3) (n � 3) (n � 3) (n � 3)

(mg/100 g fresh muscle) (Mean � SD) (Mean � SD) (Mean � SD) (Mean � SD)

D,O-phosphoserine 0.52�0.19 0.47�0.22 0.49�0.05 1.10�0.25O-phosphoethanolamine NDa 0.22�0.44 ND NDTaurine 91.05�48.32 96.68�26.21 66.01�22.48 95.00�27.40Aspartic acid 0.52�0.30 0.32�0.18 0.21�0.22 0.74�0.30Threonine 6.36�1.24 6.81�1.37 6.94�2.65 11.94�1.91Serine 4.57�1.66 6.41�2.03 2.92�1.39 4.00�0.40Asparagine 7.33�2.43 ND 5.03�0.83 NDGlutamic acid 8.61�2.87 13.03�2.61 6.41�1.33 13.92�5.51Glutamine 22.74�2.51 3.24�3.78 14.79�3.27 NDProline 2.03�0.90 5.12�2.27 17.10�2.14 14.59�10.11Glycine 61.18�7.43 93.99�21.81 76.50�42.51 81.85�30.06Alanine 11.48�6.80 16.88�2.18 13.33�1.73 24.33�5.14Citrulline 0.22�0.30 0.22�0.26 1.76�0.94 0.19�0.242-amino-n-butyric acid 0.21�0.08 0.19�0.06 0.41�0.12 0.38�0.06Valine 3.55�0.85 4.35�1.02 3.84�0.62 5.64�1.08Cystine ND 0.03�0.06 ND NDMethionine 1.99�0.63 2.60�0.67 1.82�0.21 2.16�0.26Cystathionine ND 0.17�0.06 0.13�0.05 0.17�0.02Leucine 1.79�0.54 2.35�0.61 1.67�0.37 2.88�0.65Isoleucine 2.88�0.55 3.72�0.77 2.81�0.43 4.49�0.68Phenylalanine 1.06�0.45 1.89�0.72 1.26�0.15 2.02�0.28Tyrosine 1.65�0.74 2.11�060 1.46�0.75 2.03�0.23�-alanine 3.03�1.72 4.09�0.88 5.64�2.29 8.63�3.78D-2-amino-isobutyric acid 0.09�0.16 ND 0.06�0.11 0.12�0.21D-homocystine 0.02�0.03 0.03�0.06 0.15�0.15 0.09�0.08D-4-amino-butyric acid 1.65�0.12 1.36�0.38 4.10�3.92 5.22�1.30Tryptophane ND ND ND NDEthanolamine ND ND ND NDAllo-hyd ND ND ND NDD-hydroxylysine ND ND ND NDNH3 43.48�1.34 47.68�5.12 47.16�5.17 52.33�1.37Ornithine 2.74�1.29 1.55�0.66 1.85�0.53 5.08�5.00Lysine 31.54�24.53 18.17�6.74 15.43�5.37 22.46�3.09Histidine 32.03�15.42 39.29�3.91 147.49�15.71 127.40�21.453-methyhistidyne 0.06�0.02 0.06�0.05 0.14�0.25 0.08�0.141-methyhistidyne 0.57�0.35 3.88�1.25 0.39�0.20 5.58�2.34Anserine�L-carnosine 466.46�50.44 398.46�23.85 515.03�132.25 451.93�21.90Arginine 9.50�4.64 5.60�2.68 4.77�1.20 7.86�1.35� 873.62 834.55 1022.85 1013.70aND � identity not determined.

18CH_Hui_277065 8/22/05 9:37 AM Page 411

412

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18CH_Hui_277065 8/22/05 9:37 AM Page 412

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18CH_Hui_277065 8/22/05 9:37 AM Page 413


although further studies are needed to verify thatstatement.

Potential for Further Antemortem ProteinDegradation Studies

We are not aware of any proteomic studies, otherthan that discussed above, into the link between an-temortem protein metabolism and postmortem qual-ity in fish and seafood. However, given the substan-tial importance of protein degradation to the qualityand processability of fish and seafood, it may beworthwhile to consider the potential for applicationof proteomics within this field of study. In additionto having a hand in controlling autolysis determi-nants, protein turnover is a major regulatory engineof cellular structure, function, and biochemistry.Cellular protein turnover involves at least two majorsystems: the lysosomal system and the ubiquitin-proteasome system (Hershko and Ciechanover 1986,Mortimore et al. 1989). The 20S proteasome hasbeen found to have a role in regulating the efficiencywith which rainbow trout deposit protein (Dobly etal. 2004). It seems likely that the manner in whichprotein deposition is regulated, particularly in mus-cle tissue, has profound implications for the qualityand processability of the fish flesh.

Protein turnover systems, such as the ubiquitin-proteasome or the lysosome systems, are suitable forrigorous investigation using proteomic methods. Forexample, lysosomes can be isolated and the lyso-some subproteome queried to answer the question ofwhether and to what extent lysosome compositionvaries among fish expected to yield flesh of differentquality characteristics. Proteomic analysis on lyso-somes has been successfully performed in mam-malian (human) systems (Journet et al. 2000, Journetet al. 2002).

An exploitable property of proteasome-mediatedprotein degradation is the phenomenon of polyubiq-uitination, whereby proteins are targeted for destruc-tion by the proteasome by covalent binding to multi-ple copies of ubiquitin (Ciechanover 1994, Hershkoand Ciechanover 1986). By targeting these ubiquitin-labeledproteins, it ispossible toobserve theubiquitin-proteasome “degradome,” that is, to observe whichproteins are being degraded by the proteasome at agiven time or under given conditions. Gygi et al.have developed methods to study the ubiquitin-pro-teasome degradome in the yeast Saccharomyces

cerevisiae using multidimensional LC-MS/MS (Penget al. 2003).

Some proteolysis systems, such as that of thematrix metalloproteases, may be less directly amen-able to proteomic study. Activity of matrix metallo-proteases is regulated via a complex network of spe-cific proteases (Brown et al. 1993, Okumura et al.1997, Wang and Lakatta 2002). Monitoring of theexpression levels of these regulatory enzymes andhow they vary with environmental or dietary vari-ables may be more conveniently carried out usingtranscriptomic methods.

Can Antemortem Proteomics Shed Light onGaping Tendency?

A well-known quality issue when farmed fish arecompared with wild catch is that of gaping, a phe-nomenon caused by cleavage by matrix proteases ofmyocommatal collagen cross-links, which results inweakening and rupturing of connective tissue (Bør-resen 1992, Foegeding et al. 1996). Gaping can be aserious quality issue in the fish processing industryas, apart from the obvious visual defect, it causesdifficulties in mechanical skinning and slicing (Love1992) of the fish. Weakening of collagen, and hence,gaping, is facilitated by low pH. Well-fed fish, suchas those reared in aquaculture, tend to yield flesh ofcomparatively low pH, which thus tends to gape(Einen et al. 1999, Foegeding et al. 1996). Gaping istherefore a cause for concern with aquaculture-reared fish, particularly of species with high naturalgaping tendency, such as the Atlantic cod. Gapingtendency varies considerably among wild fishcaught in different areas (Love et al. 1974), and thus,it is conceivable that gaping tendency can be con-trolled with dietary or other environmental manip-ulations. Proteomics and transcriptomics, with theircapacity to monitor multiple biochemical processessimultaneously, are methodologies eminently suit-able to finding biochemical or metabolic markersthat can be used for predicting features such as gap-ing tendendency of different stocks reared under dif-ferent dietary or environmental conditions.

SPECIES AUTHENTICATION

Food authentication is an area of increasing impor-tance, both economically and from a public healthstandpoint. Taking into account the large differences

18CH_Hui_277065 8/22/05 9:37 AM Page 414


in the market value of different fish species and theincreased prevalence of processed product on themarket, it is perhaps not surprising that speciesauthentication is fast becoming an issue of supremecommercial importance. Along with other moleculartechniques, such as DNA-based species identifica-tion (Mackie et al. 1999, Martinez et al. 2001b,Sotelo et al. 1993) and isotope distribution tech-niques for determining geographical origin (Cam-pana and Thorrold 2001), proteomics are proving tobe a powerful tool in this area, particularly foraddressing questions on the health status of theorganism, stresses or contamination levels at theplace of breeding, and postmortem treatment (Mar-tinez and Jakobsen Friis 2004). Martinez et al.

(2003) recently reviewed proteomic and other meth-ods for species authentication in foodstuffs. Since,unlike the genome, the proteome is not a static enti-ty, but changes between tissues and with environ-mental conditions, proteomics can potentially yieldmore information than genomic methods, possiblyindicating freshness and tissue information in addi-tion to species. Therefore, although it is likely thatDNA-based methods will remain the methods ofchoice for species authentication in the near term,proteomic methods are likely to develop rapidly andfind commercial uses within this field. In many cases,the proteomes of even closely related fish speciescan be easily distinguishable by eye from one anoth-er on 2D gels (Fig. 18.6), indicating that diagnostic

Figure 18.6. 2DE liver proteome maps of four salmonid fish (S. Martin and O. Vilhelmsson, unpublished). Runningconditions are as in Figure 18.2. A. Brown trout (Salmo trutta), B. Arctic charr (Salvelinus alpinus), C. rainbow trout(Oncorhynchus mykiss), D. Atlantic salmon (Salmo salar).

A

B

C D

18CH_Hui_277065 8/22/05 9:37 AM Page 415


protein spots may be used to distinguish closelyrelated species.

From early on, proteomic methods have been rec-ognized as a potential method of fish species identi-fication. During the 1960s one-dimensional electro-phoretic techniques were developed to identify theraw flesh of various species (Cowie 1968, Mackie1969, Tsuyuki et al. 1966); this was soon followedby methods to identify species in processed orcooked products (Mackie 1972, Mackie and Taylor1972). These early efforts were reviewed in 1980(Hume and Mackie 1980, Mackie 1980).

More recently, 2DE-based methods have beendeveloped to distinguish various closely relatedspecies, such as the gadoids or several flat fishes(Piñeiro et al. 1999, Piñeiro et al. 1998, Piñeiro et al.2001). Piñeiro et al. have found that Cape hake(Merluccius capensis) and European hake (Merluc-cius merluccius) can be distinguished on 2D gelsfrom other closely related species by the presence ofa particular protein spot that they identified, usingnanoelectrospray ionization mass spectrometry, asnucleoside diphosphate kinase (Piñeiro et al. 2001).Lopez et al., studying three species of Europeanmussels, Mytilus edulis, Mytilus galloprovincialisand Mytilus trossulus, found that M. trossulus couldbe distinguished from the other two species on footextract 2D gels by a difference in a tropomyosinspot. They found the difference to be due to a singleT to D amino acid substitution (Lopez et al. 2002).Recently, Martinez and Jakobsen Friis went furtherand attempted to identify not only the species pres-ent, but also their relative ratios in mixtures of sever-al fish species and muscle types (Martinez andJakobsen Friis 2004). They concluded that such astrategy would become viable once a suitable num-ber of markers have been identified, although detec-tion of species present in very different ratios isproblematic.

IDENTIFICATION ANDCHARACTERIZATION OFALLERGENS

Food safety is a matter of increasing concern to foodproducers and should be included in any considera-tion of product quality. Among issues within thisfield that are of particular concern to the seafoodproducer is that of allergenic potential. Allergic re-actions to seafood affect a significant part of the

population: about 0.5% of young adults are allergicto shrimp (Woods et al. 2002). Seafood allergies arecaused by an immunoglobulin E–mediated responseto particular proteins, including structural proteinssuch as tropomyosin (Lehrer et al. 2003). Proteo-mics provide a highly versatile toolkit to identifyand characterize allergens. As yet, these have seenlittle use in the study of seafood allergies, althoughan interesting and elegant approach has been report-ed by Yu et al. (Yu et al. 2003a) at National TaiwanUniversity. These authors, studying the cause ofshrimp allergy in humans, performed 2DE on crudeprotein extracts from the tiger prawn, Penaeus mon-odon, blotted the 2D gel onto a polyvinyl difluoride(PVDF) membrane, and probed the membranes withserum from confirmed shrimp allergic patients. Theallergens were then identified by MALDI-TOF massspectrometry of tryptic digests. The allergen wasidentified as a protein with close similarity to argi-nine kinase. The identity was further corroboratedby cloning and sequencing the relevant cDNA. Afinal proof was obtained by purifying the protein,demonstrating that it had arginine kinase activityand reacted to serum IgE from shrimp allergicpatients and, furthermore, induced skin reactions insensitized shrimp allergic patients.

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