The hemolymph proteome of the honeybee: Gel-based or gel-free?

RESEARCH ARTICLE

The hemolymph proteome of the honeybee: Gel-based

or gel-free?

Annelies Bogaerts, Geert Baggerman, Evy Vierstraete, Liliane Schoofs and Peter Verleyen

Research Group of Functional Genomics and Proteomics, K. U. Leuven, Leuven, Belgium

Received: July 17, 2008

Revised: February 12, 2009

Accepted: February 23, 2009

The honeybee has an invaluable economic impact and is a model for studying immunity,

development and social behavior. The recent sequencing and annotation of the honeybee

genome facilitates the study of its hemolymph, which reflects the physiological condition and

mediates immune responses. We aimed at making a proteomic reference map of honeybee

hemolymph and compared gel-free and gel-based techniques. One hundered and four 2-DE

spots corresponding to 62 different proteins were identified. Eight identical 2-DLC experi-

ments resulted in the identification of 32 unique proteins. One repeat was clearly not

representative for the potential of the given 2-DLC setup. Only 27% of the identified

hemolymph proteins were found by both techniques. In addition, we found proteins of three

different viruses which creates possibilities for biomarker design. Future hemolymph studies

will benefit from this work.

Keywords:

2-DE / 2-DLC / Hemolymph / Honeybee

1 Introduction

The honeybee, Apis mellifera, is so important for humans

that it is among the first insects with a sequenced and

annotated genome [1]. Besides its role as model organism

for studying social instincts and behavioral traits, the

honeybee is crucial for agriculture as a facilitator of polli-

nation. Honeybees are the primary pollinating insects in the

United States and their impact on the crop yield and quality

was estimated at 14.6 billion dollars in 2000 [2]. Honeybees

also have a medicinal value. Their honey possesses anti-

bacterial activities [3] and propolis even antioxidative, anti-

ulcer and anti-tumor activities as well [4]. Furthermore bee

venom contains a series of pharmacologically fascinating

components. Melittin, apamin and mast cell degranulated

peptide have been identified and are now successfully used

in therapeutic treatments of arthritis [5], HIV [6–8] and

other disorders. Unfortunately, bee venom can elicit sting-

induced anaphylaxis and other serious allergic diseases as

well. The response to a bee sting has historically unfolded as

a prominent model to study allergic diseases. Furthermore,

the honeybee is an interesting model for the study of innate

defense mechanisms. As the conditions within the beehive

(constant temperature, high relative humidity, high popu-

lation densities and the food sharing) are ideal for the

growth of pathogens, honeybees must have evolved a

powerful immune system to cope with these circumstances.

Moreover, the honeybee’s reaction to pathogens resembles

that of humans. For example, Apis species generate a fever

in response to a colonial infection with the fungus Asco-sphaera apis [9] and middle-aged worker bees display hygie-

nic behavior to prevent specific pathologies [10]. The

honeybee might even be useful for the study of human

diseases. Although 77% of the 929 studied human disease

genes have orthologs in Drosophila, it is expected that the

Apis genome will elicit additional orthologs [11, 12]. The

above enumeration illustrates how valuable the study of

honeybees can be. The actual list of motives for the honey-

bee genome project was much longer www.genome.gov/

pages/research/sequencing/seqproposals/honeybee_genome.

pdf.

Plasma is always among the first samples to be examined

when new separation techniques are developed. Soon afterAbbreviations: OBP, odorant binding protein; RJ, royal jelly

Correspondence: Dr. Annelies Bogaerts, Research Group of

Functional Genomics and Proteomics, K. U. Leuven, Zoological

Institute, Naamsestraat 59, 3000 Leuven, Belgium

E-mail: [email protected]

Fax: 132-16-323902

& 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2009, 9, 3201–3208 3201DOI 10.1002/pmic.200800604

the introduction of high-resolution 2-DE [13, 14] the tech-

nique was applied to study the human plasma proteins [15].

Many disease states and developmental processes have been

studied using human plasma. Like the human plasma

proteome reflects the state of the body, so does the hemo-

lymph of insects. Hemolymph is important for the recog-

nition and defense against micro-organisms; it is the major

place of resistance during infection. In order to study the

hemolymph proteome of Drosophila after infection [16, 17],

our group reported the first 2-DE database of Drosophilalarval hemolymph [18]. Meanwhile the hemolymph

proteome of the mosquito Anopheles gambiae [19] and the

silkworm Bombyx mori [20] became available. The recent

completion of the honeybee genome project allows now the

first studies on the honeybee using proteomics technologies.

So far, bee venom [21], royal jelly (RJ), pollen-bread [22] and

the hypopharyngeal gland secretome [23] have been

analyzed. Recently, Chan et al. used 1-DE in combination

with MS to study the caste differences in honeybee hemo-

lymph [24]. We, on the other hand, used both 2-DE MS and

2-DLC MS to analyze the hemolymph proteome of honeybee

workers. In this way, a first 2-DE database was established in

order to serve as a reliable reference for future physiological

studies of honeybee hemolymph.

2 Materials and methods

2.1 Animals

A. mellifera carnica were collected from a recognized

beekeeper and kept in an artificial beehive with sugars adlibitum for a maximum of 2 days before sample collection.

2.2 Sample collection and preparation

Bees were anesthetized with CO2 and kept on ice. Wings

were carefully removed on one side. Gentle squeezing

allowed collecting a droplet of hemolymph with a micro-

capillary. For every 2-DE gel or 2-DLC analysis, hemolymph

of 70 or 80 bees, respectively, was suspended in 80 or 100mL

of lysis solution containing 7 M urea, 2 M thiourea, 4%

CHAPS, 40 mM Tris, 1% DTT and Complete protease

inhibitor (Roche). The suspensions were sonicated five

times for 10 s and placed on ice during the intervals. Then

the samples were centrifuged for 12 min at 13 000 rpm

and 41C.

2.3 Gel-based technique

Supernatants were desalted using the PlusOne Mini Dialysis

Kit (GE Healthcare). Protein concentration was determined

by the method of Bradford [25]. Immobiline pH 3–10 NL

Drystrips (24 cm, GE Healthcare) were rehydrated overnight

in Destreak solution and 0.5% IPG buffer (GE Healthcare).

Samples containing 300mg of proteins were loaded in cups.

IEF was performed with the Ettan IPGphor Manifold (GE

Healthcare) at 201C and 50mA per IPGstrip; 3 h at 150 V, 3 h

at 300 V, 6 h at 1000 V and 8000 V until 50 000 Vh. Strips were

stored at �801C. Prior to SDS-PAGE, IPG strips were

immersed twice for 15 min in equilibration buffer (6 M urea,

50 mM Tris-Cl (pH 8.8), 30% glycerol and 2% SDS).

Respectively, DTT (1% w/v) and iodoacetamide 4% w/v were

added. Equilibrated strips were placed on top of a 1.5 mm

SDS-polyacrylamide gel (11.5% T; 2.6% C) and run in the

Ettan Daltsix (GE Healthcare) at 201C; 1 h at 600 V, 10 mA/gel

and 10 W; overnight at 600 V, 14 mA/gel and 15 W. After

separation, gels were stored in water containing 5% acetic

acid and 50% methanol. The 2-D gels were stained according

to Shevchenko et al. [26]. Spots were excised with a sterile

scalpel and in-gel digested with trypsin. Silver ions were

removed prior to digestion. To each spot, 25mL of 30 mM

potassium ferricyanide and 25mL of 100 mM sodium thio-

sulfate were added. The gel pieces were vortexed until the

brown color disappeared and rinsed with Milli-Q water.

Thereafter, we dehydrated the gel pieces three times with

50mL of ACN. Next, gel pieces were reswollen during 10 min

with 50mL of 100 mM ammonium bicarbonate and dehy-

drated again with ACN for 10 min. The last two steps were

repeated and spots were dried. For enzymatic digestion, gel

pieces were covered with 25mL of a digestion buffer (50 mM

ammonium bicarbonate and 5 mM CaCl2) containing 6 ng/

mL of trypsin (Promega) and incubated on ice for 45 min.

Following enzymatic digestion overnight at 371C, the resul-

tant peptides were extracted in three steps of each 30 min:

once with 80mL of 50 mM ammonium bicarbonate and twice

with 80mL of 50% ACN and 5% formic acid. The samples

were dried and prepared and analyzed by MALDI-TOF MS

(Bruker Reflex) as described by Vierstraete et al. [18]. Proteins

were identified through PMF using Mascot (Matrix Science).

One missed cleavage per peptide was allowed and an initial

mass tolerance between 0.3 and 0.1 Da was used in all sear-

ches. Carbamidomethylation was set as fixed modification,

oxidation (M) as variable. We searched three databases, the

general NCBI database and two on a local server: the Prere-

lease2 set from Beebase and all protein hits from NCBI with

[Apis] in the description. When PMF failed to identify the

protein, nanoLC-MS/MS was performed as described by

Baggerman et al. [27]. The LC system was connected in series

with the electrospray interface of the Q-TOF device. The

column eluent was directed through a stainless steel emitter

(Proteon). Needle voltage was set at 1650 V, and cone voltage

at 35 V. Nitrogen was used as nebulizing gas. Parent ions

with 2, 3 or 4 charges of sufficient abundance (threshold set

at 15 counts s�1) were automatically recognized by the soft-

ware (MassLynx 3.5, Micromass) and selected for fragmen-

tation. Argon was used as collision gas, and collision energy

was set at 25–40 eV. Fragmentation spectra were acquired

from m/z 50 to 2000. Spectra were then subjected to a Mascot

search using the two local databases mentioned.

3202 A. Bogaerts et al. Proteomics 2009, 9, 3201–3208


2.4 Gel-free technique

An aliquot of 100 mL of a 20% trichloroacetic acid solution

(41C) was added to the supernatants; incubation on ice was

for a maximum of 3 h. Next, the suspension was centrifuged

(15 min, 41C, 13 000 rpm); supernatant was removed.

Subsequently, we added 100 mL of acetone (�201C), carefully

vortexed the sample and placed it on ice for 5 min. After

centrifugation (10 min, 41C, 13 000 rpm) supernatant was

removed and pellet was left to dry at room temperature for

20 min. The pellet was resuspended in 40 mL of 200 mM

ammonium bicarbonate after which 2mg of trypsine was

added. Next, the sample was sonicated during 10 min and

incubated overnight at 371C. We filtered the sample through

a spin-down filter (Millipore) before analysis. 2-DLC MS/MS

experiments were conducted as described by Baggerman etal. [28]. PKL-files of the ten cycles were combined and

searched by Mascot using the two local databases. Para-

meters: peptide mass tolerance 0.6 Da, fragment mass

tolerance 0.3 Da, one missed cleavage, oxidation (HW) and

(M) as variable modifications. Criteria to evaluate the quality

of identifications from MS/MS data were: a protein score

greater than the significance threshold and at least two

unique peptides per protein. Significant identifications

based on one peptide were manually checked (see

Supporting Information).

3 Results and discussion

In this study, we created the first 2-DE map of hemolymph

proteins of honeybee workers (Fig. 1). The map is restricted

to proteins with a molecular mass ranging from 10 to

200 kDa and a pI from 3 to 10. The resolution of higher

molecular mass protein spots is not ideal. Note that the

hemolymph of an insect can be compared with the plasma

of vertebrates. Plasma gels typically display a bad resolution

due to the abundance of IgGs, albumins and other globu-

lins. Insect hemolymph lacks IgGs but does possess a large

amount of globulins (e.g. imaginal disc growth factor,

transferrin, arylphorins and hemolin), hampering an ideal

separation of higher MW proteins (Fig. 1 [20, 29]). The

application of clean up kits based on precipitation slightly

improved the resolution of test gels. However, as the

Figure 1. A representative 2-DE

gel image of the proteome of

worker bee hemolymph.

Proteomics 2009, 9, 3201–3208 3203


reproducibility and the number of detectable spots did not

benefit from the clean up, we opted to present an intensively

stained representative gel with a maximum number of

visible spots as a reference. A total of 104 spots, corre-

sponding to 62 different proteins, were identified with either

MALDI-MS or nanoLC-MS (Supporting Information).

In addition, we tested the potential of 2-DLC MS/MS by

running eight biologically independent experiments with

identical settings. This resulted in a total of 32 identified

proteins. Interestingly, a single 2-DLC experiment is not at

all representative. No less than 31% of these proteins were

identified in only one experiment (Fig. 2A). Figure 2B

clearly illustrates that even after eight experiments, new

proteins can still be identified. This might be due to the

limited capacity of the MS/MS device. While the mass

spectrometer selects one peak for fragmentation, co-eluting

peaks will not be fragmented. In a following experiment, the

device may randomly select other peaks, which will lead to

new identifications. In addition, peptide fragments from

abundant proteins are over-represented in the MS/MS

spectra. An important conclusion is that it is essential to

repeat a 2-DLC MS/MS experiment several times in order to

fully cover that part of the proteome that lays within the

potential of a given setup.

Comparing the data sets from the 2-DE and 2-DLC

experiments shows an overlap of only 27%. Other data

comparing gel-free and gel-based techniques are rare,

displaying a rather wide variation in overlap: 21 [30], 38 [31],

48 [32] and 54% [33] reflecting a similar variation in applied

equipment, protocols, software and samples. Interestingly,

the relative number of unique identifications differs even

more. Dumont et al. identified 45 proteins only by 2-DLC

and 114 only by 2-DE from a sample of mature rat oligo-

dendrocytes [30], whereas Wolff et al. found more unique

Bacillus subtilis proteins with 2-DLC (269) than with 2-DE

(200) [33]. These figures make clear that both techniques

potentially identify a different subset of proteins. 2-DE is

limited for the detection of extremely small, big or hydro-

phobic proteins. The main advantages of 2-DE, on the other

hand, are the simplified mass spectra and the possibility of

identifying proteins by PMF. Another positive aspect is that

the position of a 2-DE spot confines information on pI and

MW and thus a hint towards possible PTMs [34]. 2-DLC

allows identifying proteins from a much larger pI in a

completely automated way. A negative point is that some

identifications are based on just one peptide. These identi-

fications have to be manually checked to exclude all possible

false positives. We can conclude that although gel-free

techniques were developed to compensate for the drawbacks

of 2-DE, 2-DLC cannot be seen as a substitute technique for

proteomic research. Hence both techniques should be

regarded as complementary [35] and used to replenish each

other’s limitations.

The proteins in this study can be subdivided into differ-

ent categories according to their functional properties

(Fig. 3). The main protein categories are briefly discussed.

3.1 Enzymes

Enzymes are the most abundant group of hemolymph

proteins. We mainly identified proteins involved in the

carbohydrate metabolism such as alpha-glucosidase, triose-

phosphate isomerase and pyruvate kinase. The carbohy-

drate-metabolising enzymes may have particularly

interesting roles in the honeybee, as this insect primarily

feeds on nectar and pollen, which are extremely rich in

carbohydrates. Nutrition also seems to be the main factor in

Figure 2. Number of identified worker bee hemolymph proteins

throughout eight identical 2-DLC experiments. (A) The total

number of identified proteins increases with every repeat. (B)

Eight proteins were identified in every analysis, whereas ten

proteins were found in only one experiment.

enzymes

transport & storage

defense

MRJP

others

unknown

virus proteins

enzymes

transport & storage

structural proteins

defense

cell div., chrom. struct.

MRJP

others

unknown

virus proteins

34%

22%6%6%

13%

13%6%

37%

16%5%5%3%2%

11%

16%5%

2-DLC

2-DE

Figure 3. Pie charts representing the functional categories of the

hemolymph proteins identified with gel-free and gel-based

techniques. (cell div., chrom. struct. 5 cell division and chroma-

tin structure).



caste determination as larvae fed with nutrient-rich RJ

become queens, whereas others become workers. In addi-

tion, it has been shown that the expression of genes

encoding carbohydrate-metabolising enzymes is associated

with the age-dependent behavioral changes of this insect

species [36, 37]. Proteinases and peptidases are not only

involved in food digestion but also play an important role in,

for example, development and immune responses. Serine

proteases, for instance, are known to regulate several innate

defense responses such as coagulation, antimicrobial

peptide synthesis and melanisation of pathogen surfaces.

The possible involvement of serine-protease-related genes in

immunity and embryonic development in the honeybee was

described by Zou [38]. Serine protease inhibitors from the

serpin superfamily regulate protease cascades in mammals

and arthropods [39]. In insect hemolymph, serpins inhibit

activated proteases to maintain homeostasis and prevent

unregulated activation of immune responses such as mela-

nisation or Toll-mediated antimicrobial protein synthesis

[40].

3.2 Structural proteins

Structural proteins are represented only by an actin, a

tubulin and a protein similar to forked CG5424-PA, isoform

A, which is involved in actin filament bundle formation.

3.3 Transport and storage proteins

The first insect iron-binding protein was described in

Manduca sexta [41]. Later on, insect transferrin genes were

cloned from Aedes aegypti [42], B. mori [43], Drosophilamelanogaster, D. sylvestris [44], Sarcophaga peregrina [45],

Riptortus clavatus [46] and A. mellifera [47]. Besides its role as

an iron transporter, transferrin displays multiple functions

in the honeybee. The upregulation of the gene after infec-

tion with Escherichia coli suggests a role in immunity [47].

The expression of transferrin is repressed by juvenile

hormone [47, 48] and transferrin also has an additional role

in vitellogenesis [49]. Hexamerins are large storage proteins

of insects that have evolved from the copper-binding

hemocyanins. They usually consist of six identical or similar

subunits. Their presence peaks in hemolymph of late larval

and early pupal stages after which it gradually declines

during metamorphosis and adult development [50]. We

found subunit 70a, which is the sole subunit that is also

present in a large amount in adult stages [51]. Olfaction

plays a central role in the life of an insect. Food odors and

sex pheromones are transported to the sensory receptors by

odorant binding proteins (OBPs). The genome of the

honeybee carries 21 genes encoding putative OBPs [51]. This

number is less than half the repertoire of OBP genes found

in D. melanogaster, A. gambiae or Triboleum castaneum. We

identified Obp13, 14 and 15, which in honeybees belong to a

monophyletic group of OBPs called the C-minus OBPs [51].

Obp 13 and 14 are expressed during relatively narrow

developmental stages. Unlike described by Foret in 2006, we

show here that Obp15 is exclusively expressed not only in

the antennae of adult bees but also in the hemolymph and

that Obp13 is expressed not only in pupae and larvae but

also in adult worker bees. Antennal-specific protein asp3c

efficiently binds fatty acid ester components of the brood

pheromone [52]. This pheromone is involved in the regu-

lation of behavior-like feeding of the larvae, capping of the

cells and thermoregulation of the brood area in the colony.

Take-out-like protein JHBP-1 and a protein similar to

CG5867-PA, isoform 1, have juvenile hormone-binding

properties. Retinoid- and fatty-acid binding protein is

involved in lipid transport.

3.4 Immune-related proteins

Specific pattern recognition molecules identified in this

study were two forms of a protein similar to Gram-negative

binding protein 1 CG6895-PA and a protein similar to

Peptidoglycan recognition protein SA CG11709-PA. This

latter protein could be the trigger for the activation of the

prophenoloxidase cascade. This cascade results in the

production of melanine and melanisation of pathogen

surfaces. The key enzyme in this process is phenoloxidase,

which is present as an inactive precursor (prophenoloxidase)

in the plasma or hemocytes. This prophenoloxidase is

converted to phenoloxidase by endogenous trypsin-like

serine proteases.

3.5 Other identified proteins

The cephalic glands of nurse bees secrete RJ, which consists

of 90% of MRJPs [22, 53–55]; eight loci encoding MRJPs

(mrjp1– mrjp8) have been identified [55–57]. Together with

the Yellow-related proteins, they form the MRJP/Yellow

protein family. Members of this family have not only a

nutritional function. The mrjp-1 gene, for example, is

expressed in the mushroom bodies of the honeybee, impli-

cating its involvement in behavior [58]. Furthermore, it

appears that the MRJP protein subfamily evolution from the

Yellow protein family may have coincided with the evolution

of honeybee eusociality [59]. Other proteins we found in the

hemolymph were heat shock proteins, pigments, a protein

similar to imaginal disc growth factor 4 and vitellogenins.

3.6 Virus proteins

Constructing a database with all NCBI proteins having [Apis]in their description allowed to identify proteins from known

honeybee pathogens. An interesting finding was the iden-

tification of a polyprotein derived from the Kakugo virus.

Proteomics 2009, 9, 3201–3208 3205


This Picorna-like virus was previously found only in the

honeybee brain and is believed to cause aggressive behavior

in honeybee workers [60]. Similar to other Picorna viruses,

the Kakugo virus is mainly transmitted by oral infection

between colony members. We also found a polyprotein

derived from the deformed wing virus and one from the

Varroa destructor virus. These two viruses are both trans-

mitted by Varroa destructor, a parasitic mite. Varroa can

replicate only within a honeybee colony. It feeds on hemo-

lymph, spreading RNA viral agents to the bee and leaving

their victims very weakened. This can cause serious losses in

case of a significant infestation of the colony. The fact that

these viral proteins were relatively easy to identify and

clearly visible on the gel makes them potential biomarkers.

4 Concluding remarks

The hemolymph of insects plays a very important role in the

transport and storage of nutrients and is crucial for the

recognition of and defense against micro-organisms. Our

aim was to create a 2-D reference map of the honeybee

hemolymph proteome. Comparing gel-based and gel-free

techniques, we identified a total of 74 proteins belonging to

different functional categories. Both techniques are

complementary, but 2-DLC MS/MS experiments require a

sufficient number of replicates. Moreover, we have shown

the presence of several viral proteins, which makes the

hemolymph an excellent candidate subject in biomarker

research. This study provides an initial picture of the

composition of the hemolymph proteome of A. mellifera,

which undoubtedly will pave the way for future physiologi-

cal studies of the honeybee.

The data from this publication are accessible from the World-2DPAGE database http://world-2dpage.expasy.org/0006/cgi-bin/2d.cgi.

A.B. and P.V. are a PhD fellow and a postdoctoral fellow,respectively, of the Research Foundation – Flanders (FWO-Vlaanderen). We would like to acknowledge Dr. Bart Landuytand Lieve Geenen. This work has been supported by Prometa –K.U.Leuven and by the FWO grant number G041708N.

The authors have declared no conflict of interest.

5 References

[1] Weinstock, G. M., Robinson, G. E., Gibbs, R. A., Worley,

K. C. et al., Insights into social insects from the genome of

the honeybee Apis mellifera. Nature 2006, 443, 931–949.

[2] Morse, R. A., Calderone, N. W., The value of honey

bee pollination in the United States. Bee Culture 2000, 128,

1–15.

[3] Molan, P. C., The antibacterial activity of honey .1. The nature of

the antibacterial activity. Bee World 1992, 73, 5–28.

[4] Khalil, M. L., Biological activity of bee propolis in health and

disease. Asian Pac. J. Cancer Prev. 2006, 7, 22–31.

[5] Lee, J. Y., Kang, S. S., Kim, J. H., Bae, C. S., Choi, S. H.,

Inhibitory effect of whole bee venom in adjuvant-induced

arthritis. In Vivo 2005, 19, 801–805.

[6] Fenard, D., Lambeau, G., Valentin, E., Lefebvre, J. C. et al.,

Secreted phospholipases A(2), a new class of HIV inhibitors

that block virus entry into host cells. J. Clin. Invest. 1999,

104, 611–618.

[7] Fenard, D., Lambeau, G., Maurin, T., Lefebvre, J. C., Doglio,

A., A peptide derived from bee venom-secreted phospholi-

pase A(2) inhibits replication of T-cell tropic HIV-1 strains via

interaction with the CXCR4 chemokine receptor. Mol.

Pharmacol. 2001, 60, 341–347.

[8] Wachinger, M., Kleinschmidt, A., Winder, D., von Pechmann, N.

et al., Antimicrobial peptides melittin and cecropin inhibit

replication of human immunodeficiency virus 1 by suppressing

viral gene expression. J. Gen. Virol. 1998, 79, 731–740.

[9] Starks, P. T., Blackie, C. A., Seeley, T. D., Fever in honeybee

colonies. Naturwissenschaften 2000, 87, 229–231.

[10] Arathi, H. S., Burns, I., Spivak, M., Ethology of hygienic

behaviour in the honey bee Apis mellifera L-(Hymenoptera :

Apidae): Behavioural repertoire of hygienic bees. Ethology

2000, 106, 365–379.

[11] Reiter, L. T., Potocki, L., Chien, S., Gribskov, M., Bier, E., A

systematic analysis of human disease-associated gene

sequences in Drosophila melanogaster. Genome Res. 2001,

11, 1114–1125.

[12] Whitfield, C. W., Band, M. R., Bonaldo, M. F., Kumar, C. G.

et al., Annotated expressed sequence tags and cDNA

microarrays for studies of brain and behavior in the honey

bee. Genome Res. 2002, 12, 555–566

[13] Klose, J., Protein mapping by combined isoelectric focusing

and electrophoresis of mouse tissues - novel approach to

testing for induced point mutations in mammals. Human-

genetik 1975, 26, 231–243.

[14] Ofarrell, P. H., High-resolution 2-dimensional electrophor-

esis of proteins. J. Biol. Chem. 1975, 250, 4007–4021.

[15] Anderson, L., Anderson, N. G., High-resolution 2-dimen-

sional electrophoresis of human-plasma proteins. Proc.

Natl. Acad. Sci. USA 1977, 74, 5421–5425.

[16] Vierstraete, E., Verleyen, P., Sas, F., Van den Bergh, G. et al.,

The instantly released Drosophila immune proteome is

infection-specific. Biochem. Biophys. Res. Commun. 2004,

317, 1052–1060.

[17] Vierstraete, E., Verleyen, P., Baggerman, G., D’Hertog, W.

et al., A proteomic approach for the analysis of instantly

released wound and immune proteins in Drosophila mela-

nogaster hemolymph. Proc. Natl. Acad. Sci. USA 2004, 101,

470–475.

[18] Vierstraete, E., Cerstiaens, A., Baggerman, G., Van den

Bergh, G. et al., Proteomics in Drosophila melanogaster:

first 2D database of larval hemolymph proteins. Biochem.

Biophys. Res. Commun. 2003, 304, 831–838.



[19] Paskewitz, S. M., Shi, L., The hemolymph proteome of

Anopheles gambiae. Insect Biochem. Mol. Biol. 2005, 35,

815–824.

[20] Li, X. H., Wu, X. F., Yue, W. F., Liu, J. M. et al., Proteomic

analysis of the silkworm (Bombyx mori L.) hemolymph

during developmental stage. J. Proteome Res. 2006, 5,

2809–2814.

[21] Peiren, N., Vanrobaeys, F., de Graaf, D. C., Devreese, B.

et al., The protein composition of honeybee venom recon-

sidered by a proteomic approach. Biochim. Biophys. Acta

2005, 1752, 1–5.

[22] Scarselli, R., Donadio, E., Giuffrida, M. G., Fortunato, D.

et al., Towards royal jelly proteome. Proteomics 2005, 5,

769–776.

[23] Santos, K. S., dos Santos, L. D., Mendes, M. A., de Souza,

B. M. et al., Profiling the proteome complement of the

secretion from hypopharyngeal gland of Africanized nurse-

honeybees (Apis mellifera L.). Insect Biochem. Mol. Biol.

2005, 35, 85–91.

[24] Chan, Q. W. T., Howes, C. G., Foster, L. J., Quantitative

comparison of caste differences in honeybee hemolymph.

Mol. Cell Proteomics 2006, 5, 2252–2262.

[25] Bradford, M. M., Rapid and sensitive method for

quantitation of microgram quantities of protein utilizing

principle of protein-dye binding. Anal. Biochem. 1976, 72,

248–254.

[26] Shevchenko, A., Wilm, M., Vorm, O., Mann, M., Mass

spectrometric sequencing of proteins from silver stained

polyacrylamide gels. Anal. Chem. 1996, 68, 850–858.

[27] Baggerman, G., Huybrechts, J., Clynen, E., Hens, K. et al.,

New insights in adipokinetic hormone (AKH) precursor

processing in Locusta migratoria obtained by capillary

liquid chromatography-tandem mass spectrometry.

Peptides 2002, 23, 635–644.

[28] Baggerman, G., Boonen, K., Verleyen, P., De Loof, A.,

Schoofs, L., Peptidomic analysis of the larval Drosophila

melanogaster central nervous system by two-dimensional

capillary liquid chromatography quadrupole time-of-

flight mass spectrometry. J. Mass Spectrom. 2005, 40,

250–260.

[29] Furusawa, T., Rakwal, R., Nam, H. W., Hirano, M. et al.,

Systematic investigation of the hemolymph proteome of

Manduca sexta at the fifth instar larvae stage using one-

and two-dimensional proteomics platforms. J. Proteome

Res. 2008, 7, 938–959.

[30] Dumont, D., Noben, J. P., Moreels, M., Vanderlocht, J. et al.,

Characterization of mature rat oligodendrocytes: a proteo-

mic approach. J. Neurochem. 2007, 102, 562–576.

[31] Kohler, C., Wolff, S., Albrecht, D., Fuchs, S. et al., Proteome

analyses of Staphylococcus aureus in growing and non-

growing cells: a physiological approach. Int. J. Med.

Microbiol. 2005, 295, 547–565.

[32] Salzano, A. M., Arena, S., Renzone, G., Ambrosio, C. D.

et al., A widespread picture of the Streptococcus

thermophilus proteome by cell lysate fractionation

and gel-based/gel-free approaches. Proteomics 2007, 7,

1420–1433.

[33] Wolff, S., Otto, A., Albrecht, D., Zeng, J. S. et al., Gel-free

and gel-based proteomics in Bacillus subtilis – a compara-

tive study. Mol. Cell Proteomics 2006, 5, 1183–1192.

[34] Gorg, A., Weiss, W., Dunn, M. J., Current two dimensional

electrophoresis technology for proteomics (vol 4, pg 3665,

2004). Proteomics 2005, 5, 826–827.

[35] Baggerman, G., Vierstraete, E., De Loof, A., Schoofs, L., Gel-

based versus gel-free proteomics: a review. Comb. Chem.

High Throughput Screen. 2005, 8, 669–677.

[36] Ohashi, K., Natori, S., Kubo, T., Expression of amylase

and glucose oxidase in the hypopharyngeal gland

with an age-dependent role change of the worker

honeybee (Apis mellifera L.). Eur. J. Biochem. 1999, 265,

127–133.

[37] Whitfield, C. W., Cziko, A. M., Robinson, G. E., Gene

expression profiles in the brain predict behavior in indivi-

dual honey bees. Science 2003, 302, 296–299.

[38] Zou, Z., Lopez, D. L., Kanost, M. R., Evans, J. D., Jiang, H. B.,

Comparative analysis of serine protease-related genes in

the honey bee genome: possible involvement in embryonic

development and innate immunity. Insect Mol. Biol. 2006,

15, 603–614.

[39] Reichhart, J. M., Tip of another iceberg: Drosophila serpins.

Trends Cell Biol. 2005, 15, 659–665.

[40] Kanost, M. R., Clarke, T., Proteases. Comprehensive Mole-

cular Insect Science (Gilbert, L. I., Iatrou, K., Gill, S., eds)

2005, 4, 247–266.

[41] Huebers, H. A., Huebers, E., Finch, C. A., Webb, B. A. et al.,

Iron-binding proteins and their roles in the tobacco horn-

worm, Manduca-sexta (L). J. Comp. Physiol. B Biochem.

Syst. Environ. Physiol. 1988, 158, 291–300.

[42] Yoshiga, T., Hernandez, V. P., Fallon, A. M., Law, J. H.,

Mosquito transferrin, an acute-phase protein that is up-

regulated upon infection. Proc. Natl. Acad. Sci. USA 1997,

94, 12337–12342.

[43] Yun, E. Y., Kang, S. W., Hwang, J. S., Goo, T. W. et al.,

Molecular cloning and characterization of a cDNA encoding

a transferrin homolog from Bombyx mori. Biol. Chem. 1999,

380, 1455–1459.

[44] Yoshiga, T., Georgieva, T., Dunkov, B. C., Harizanova, N.

et al., Drosophila melanogaster transferrin – cloning,

deduced protein sequence, expression during the life cycle,

gene localization and up-regulation on bacterial infection.

Eur. J. Biochem. 1999, 260, 414–420.

[45] Kurama, T., Kurata, S., Natori, S., Molecular characterization

of an insect transferrin and its selective incorporation into

eggs during oogenesis. Eur. J. Biochem. 1995, 228, 229–235.

[46] Hirai, M., Watanabe, D., Chinzei, Y., A juvenile hormone-

repressible transferrin-like protein from the bean bug,

Riptortus clavatus: cDNA sequence analysis and protein

identification during diapause and vitellogenesis. Arch.

Insect Biochem. Physiol. 2000, 44, 17–26.

[47] Kucharski, R., Maleszka, R., Transcriptional profiling reveals

multifunctional roles for transferrin in the honeybee, Apis

mellifera. J. Insect Sci. 2003, 3, 27–34.

[48] Nascimento, A. M., Cuvillier-Hot, V., Barchuk, A. R., Simoes,

Z. L. P., Hartfelder, K., Honey bee (Apis mellifera) transfer-

Proteomics 2009, 9, 3201–3208 3207


rin-gene structure and the role of ecdysteroids in the

developmental regulation of its expression. Insect Biochem.

Mol. Biol. 2004, 34, 415–424.

[49] Nichol, H., Law, J. H., Winzerling, J. J., Iron metabolism in

insects. Annu. Rev. Entomol. 2002, 47, 535–559.

[50] Danty, E., Arnold, G., Burmester, T., Huet, J. C. et al.,

Identification and developmental profiles of hexamerins in

antenna and hemolymph of the honeybee, Apis mellifera.

Insect Biochem. Mol. Biol. 1998, 28, 387–397.

[51] Foret, S., Maleszka, R., Function and evolution of a gene

family encoding odorant binding-like proteins in a social

insect, the honey bee (Apis mellifera). Genome Res. 2006,

16, 1404–1413.

[52] Briand, L., Swasdipan, N., Nespoulous, C., Bezirard, V. et al.,

Characterization of a chemosensory protein (ASP3c) from

honeybee (Apis mellifera L.) as a brood pheromone carrier.

Eur. J. Biochem. 2002, 269, 4586–4596.

[53] Sano, O., Kunikata, T., Kohno, K., Iwaki, K. et al., Char-

acterization of royal jelly proteins in both Africanized and

European honeybees (Apis mellifera) by two-dimensional

gel electrophoresis. J. Agric. Food Chem. 2004, 52, 15–20.

[54] Santos, K. S., dos Santos, L. D., Mendes, M. A., de Souza,

B. M. et al., Profiling the proteome complement of the

secretion from hypopharyngeal gland of Africanized nurse-

honeybees (Apis mellifera L.). Insect Biochem. Mol. Biol.

2005, 35, 85–91.

[55] Schmitzova, J., Klaudiny, J., Albert, S., Schroder, W.

et al., A family of major royal jelly proteins of the

honeybee Apis mellifera L. Cell. Mol. Life Sci. 1998, 54,

1020–1030.

[56] Albert, T., Klaudiny, J., The MRJP/YELLOW protein family of

Apis mellifera: identification of new members in the EST

library. J. Insect Physiol. 2004, 50, 51–59.

[57] Klaudiny, J., Hanes, J., Kulifajova, J., Albert, S., Simuth, J.,

Molecular-cloning of 2 cdnas from the head of the nurse

honey-bee (Apis-mellifera L) for coding related proteins of

royal jell‘‘y. J. Apic. Res. 1994, 33, 105–111.

[58] Kucharski, R., Maleszka, R., Hayward, D. C., Ball, E. E., A

royal jelly protein is expressed in a subset of Kenyon cells in

the mushroom bodies of the honey bee brain. Naturwis-

senschaften 1998, 85, 343–346.

[59] Drapeau, M. D., Albert, S., Kucharski, R., Prusko, C.,

Maleszka, R., Evolution of the Yellow/Major Royal Jelly

Protein family and the emergence of social behavior in

honey bees. Genome Res. 2006, 16, 1385–1394.

[60] Fujiyuki, T., Takeuchi, H., Ono, M., Ohka, S. et al., Novel

insect picorna-like virus identified in the brains of aggres-

sive worker honeybees. J. Virol. 2004, 78, 1093–1100.



Documents

The hemolymph proteome of the honeybee: Gel-based or gel-free?