Functional expression and mutant analysis of thioredoxin-fused CEL-III, a hemolytic lectin from the marine invertebrate Cucumaria echinata.

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. New expression system of CEL-III was constructed, and the recombinant thioredoxin-fused CEL-III (Trx-CEL-III) showed strong hemolytic and carbohydrate-binding activity as same as authentic CEL-III. Mutation analysis of Trx-CEL-III suggested that carbohydrate binding to subdomain 1α and 2ß of CEL-III might be important for the hemolytic activity.

Purification, cDNA cloning and characterization of Kunitz-type protease inhibitors from Apios americana tubers.

Two kinds of Kunitz-type protease inhibitors, AKPI1 and AKPI2, were purified from Apios americana tubers by four steps of column chromatographies and their cDNA cloning was performed. AKPI1 cDNA consist of 809 nucleotides, and the matured protein had 190 amino acids with 20,594 Da. AKPI2 cDNA consist of 794 nucleotides, and the matured protein had 177 amino acids with 19,336 Da. P1 site of AKPI2 was Leu88, suggested the target enzyme was chymotrypsin. On the other hand, Gly85-Ile86-Ser87 was positioned around P1 site of AKTI1. Sequence analysis suggested that two forms (single-chain and two-chain form) of AKPI2 protein were present in the tubers. Recombinant AKPI2 expressed by E.coli system showed inhibitory activity toward serine proteases and heat stability. The Ki values toward chymotrypsin and trypsin were 4 × 10-7 M and 6 × 10-6 M, respectively.Abbreviations: AAL: Apios americana lectin; AATI: Apios americana Bowman-Birk type trypsin inhibitor; ACE: angiotensin-converting enzyme; IPTG: isopropyl-ß-D-thio-galactopyranoside; Ki: inhibition constant; KPIs: Kunitz-type protease inhibitors; L-BAPA: Benzoyl-L-arginine p-nitroanilide monohydrochloride; L-BTPA: Benzoyl-L-tyrosine p-nitroanilide; PFLNA: Pyr-Phe-Leu-p-nitroanilide; RP-HPLC: reverse-phase high-performance liquid chromatography; RT-PCR: reverse transcription-polymerase chain reaction; SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SLIC: sequence and ligation independent cloning; STANA: N-Succinyl-Ala-Ala-Ala-p-nitroanilide; SHR: spontaneously hypertensive rats; TFA: trifluoroacetic acid; UTR: untranslated region.

Unraveling the seed endosperm proteome of the lotus (Nelumbo nucifera Gaertn.) utilizing 1DE and 2DE separation in conjunction with tandem mass spectrometry.

Nelumbo nucifera (Gaertn.) or lotus, is an aquatic plant native to India, and presently consumed as food mainly in China and Japan. Lotus is also widely used in Indian and Chinese traditional medicine. Extracts from different parts of the lotus plant have been reported to show diverse biological activities-antioxidant, free radical scavenging, anti-inflammatory, and immunomodulatory. Despite this, little work has been done in isolating and identifying proteins responsible for these activities, or yet importantly to establish a lotus proteome. The aim of our group is to develop a proteome catalog of the lotus plant, starting with its seed, the nutrient rich food source. In this present study, the seed endosperm-most abundant in proteins, and main nutrient storage tissue-was targeted for protein extraction by testing five different extraction protocols, followed by their proteomic analyses using complementary 1DE and 2DE approaches in conjunction with MS/MS. The inventory of 66 nonredundant proteins obtained by 1DE-MS and the 30 obtained by 2DE-MS provides the first catalog of the lotus seed endosperm, where the most abundant protein functions were in categories of metabolic activities related to carbohydrate metabolism and nutrient storage.

Purification and cDNA cloning of a lectin and a lectin-like protein from Apios americana Medikus tubers.

An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono- or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved.

Molecular cloning, functional expression, and characterization of isolectin genes of hemolytic lectin CEL-III from the marine invertebrate Cucumaria echinata.

CEL-III is a hemolytic lectin purified from the marine invertebrate Cucumaria echinata. Previous research has indictated that CEL-III is composed of several isoforms. Here we identified five CEL-III isolectin genes, designated CEL-III-L1, CEL-III-L2, CEL-III-S1, CEL-III-S2, and CEL-III-LS1, by cDNA cloning. The deduced amino acid sequences suggested they shared 94.0-99.8% identical residues. Among the amino acid residues involved in carbohydrate binding, the His residue, which contributes to stacking with sugar, in subdomain 1α was replaced by Tyr in CEL-III-L2. The recombinant proteins were expressed in Escherichia coli or insect cells. rCEL-III-L2 showed higher hemolytic activity than those of the other isolectins. Furthermore, an apparent oligomer band of rCEL-III-L2 was detected on erythrocyte membranes, although the other isolectins showed smear bands. These results suggest that Tyr36 of CEL-III-L2 is important for the expression of hemolytic activity and oligomerization.

Identification of a royal jelly glycoprotein that carries unique complex-type N-glycans harboring the T-antigen (Galß1-3GalNAc) unit.

In this study, we identified a royal jelly glycoprotein (RJG) that carries a unique complex-type N-glycans harboring the T-antigen (Galß1-3GalNAc) unit. The amino acid sequence of the tryptic glycopeptide harboring the T-antigen unit was G-E-S-L-X-K (X might be glycosylated Asn), confirmed in the major royal jelly glycoprotein 1 (MRJP1), which is also expressed in the mushroom body of the honeybee brain.

Purification, characterization, and cDNA cloning of a Bowman-Birk type trypsin inhibitor from Apios americana Medikus tubers.

An Apios americana trypsin inhibitor, AATI, was purified from Apios tubers by chromatography on DEAE Cellulofine A-500 and Sephadex G-50. The molecular mass of AATI was determined to be 6,437 Da by matrix-assisted laser desorption and ionization time-of-flight mass spectrometer (MALDI-TOF-MS). It showed strong inhibitory activity toward serine proteases, and the inhibition constants toward trypsin and chymotrypsin were 3.0 x 10(-9) M and 1.0 x 10(-6) M respectively. The inhibitory activity was not affected by heating at 80 degrees C for 2 h or by incubation at a wide range of pH values, suggesting that AATI has remarkable heat-stability and pH-stability. AATI cDNA consists of 552 nucleotides, and includes an open reading frame encoding a protein of 116 amino acids. The results of N-terminal amino acid sequencing of AATI and MALDI-TOF-MS analysis suggested that the deduced amino acid sequence had 50 and seven extra amino acids at the N- and C-termini respectively. Thus the mature AATI protein consists of 59 amino acid residues. Comparison of the amino acid sequence with those of the trypsin inhibitors from plants suggests that AATI belongs to the Bowman-Birk family and that it contains two possible reactive sites toward trypsin at Lys62 and Arg88.

Royal jelly peptides inhibit lipid peroxidation in vitro and in vivo.

Royal jelly peptides (RJPx) isolated from hydrolysates of water-soluble royal jelly proteins prepared with protease P exhibited significantly stronger hydroxyl radical-scavenging activity (p<0.001), and antioxidant activity against lipid peroxidation (LPO, p<0.001), than did water-soluble royal jelly protein (WSRJP) in vitro. We also investigated the in vivo antioxidant activity of RJPx against ferric nitrilotriacetate (Fe-NTA)-induced LPO. Male Wistar rats were divided into a control group (Group C), an Fe-NTA group (Group Fe), and an Fe-NTA with RJPx group (Group Fe+R). Rats in Group Fe+R were fed RJPx (2 g/kg body weight) daily for 5 wk. Fe-NTA (8 mg Fe/kg body weight) was then intraperitoneally injected, and serum lipid levels were examined 2 h later. Serum total cholesterol (TC) levels were lower (p<0.05) while low-density lipoprotein (LDL) and LPO were significantly higher (p<0.01) in Group Fe than in Group C. TC (p<0.05) and LPO levels (p<0.01) were lower in Group Fe+R than in Group Fe. Our data suggest that RJPx may inhibit LPO both in vitro and in vivo.

Purification of a cysteine protease inhibitor from larval hemolymph of the tobacco hornworm (Manduca sexta) and functional expression of the recombinant protein.

A cysteine protease inhibitor (CPI) with an apparent molecular mass of 11.5kDa was purified from larval hemolymph of the tobacco hornworm (Manduca sexta) by gel filtration on Sephadex G-50 followed by hydrophobic and ion-exchange column chromatographies. The purified cysteine proteinase inhibitor, denoted as MsCPI, strongly inhibited the plant cysteine protease, papain, with a K(i) value of 5.5 x 10(-9)M. Nucleotide sequence analysis of a partial cDNA encoding MsCPI indicated that MsCPI consists of 105 amino acid residues in a sequence that is similar to sarcocystatin A from Sarcophaga peregrina. However, northern blotting and PCR analyses using the specific primers of MsCPI suggested that the mRNA encoding MsCPI had a size of more than 12 kilobases, which included at least six tandemly repeated MsCPI segments. MsCPI was expressed in Escherichia coli and the recombinant protein effectively inhibited cysteine proteases from plants as well as from animals such as cathepsins B (K(i), 6.8 nM), H (3.0 nM), and L (0.87 nM). There was no inhibition exhibited toward trypsin, chymotrypsin, subtilisin, pepsin or themolysin.

Search for novel stress-responsive protein components using a yeast mutant lacking two cytosolic Hsp70 genes, SSA1 and SSA2.

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heat-shocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.

Role of defense/stress-related marker genes, proteins and secondary metabolites in defining rice self-defense mechanisms.

Rice, a first cereal crop whose draft genome sequence from two subspecies (japonica-type cv. Nipponbare and indica-type 93-11) was available in 2002, along with its almost complete genome sequence in 2005, has drawn the attention of researchers worldwide because of its immense impact on human existence. One of the most critical research areas in rice is to discern the self-defense mechanism(s), an innate property of all living organisms. The last few decades have seen scattered research into rice responses to diverse environmental stimuli and stress factors. Our understanding on rice self-defense mechanism has increased considerably with accelerated research during recent years mainly due to identification and characterization of several defense/stress-related components, genes, proteins and secondary metabolites. As these identified components have been used to study the defense/stress pathways, their compilation in this review will undoubtedly help rice (and others) researchers to effectively use them as a potential marker for better understanding, and ultimately, in defining rice (and plant) self-defense response pathways.

System, trends and perspectives of proteomics in dicot plants Part I: Technologies in proteome establishment.

The first 3 years of the 21st century have seen the impact of plant proteomics on functional genomics that has enhanced our understanding, not only on the plant genome(s), but also more importantly, on the functional aspect of proteins. This is mainly due to availability of the complete genome sequence of the Arabidopsis thaliana-a dicotyledoneous (dicot) model plant-and technological advancements in proteomics. Proteomic analyses of a variety of dicot plants, including both Arabidopsis and the model legume species, barrel medic (Medicago truncatula), have greatly helped in an efficient separation, identification and cataloguing of a large number of proteins, and thereby defining their proteomes. Therefore, we have composed an inclusive review on dicot plant materials, as of February 2004, that provides system, trends and perspectives of proteomics in growth and development and the environment. The review is summarized and discussed as three individual, but interlinked, entities: Part I, technologies in proteome establishment (this review), Part II, proteomes of the complex developmental stages [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)], and Part III, unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)]. This review deals with the diverse proteomic technologies being used in proteome development of different dicot plants.

System, trends and perspectives of proteomics in dicot plants Part II: Proteomes of the complex developmental stages.

This review is devoted to the proteomes of the complex developmental stages of dicotyledoneous (dicot) plant materials. The two core technologies, two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (MS), independently or in combination with each other, are propelling dicot plant proteomics to new discoveries and functions, with the establishment of tissue-specific and organelle proteomes, mostly in Arabidopsis thaliana and Medicago truncatula, revealing their complexity and specificity. These experimental proteomes have provided a good start towards the establishment of high-density 2-DGE reference maps and peptide mass fingerprint databases, for not only the model dicot plants, A. thaliana and M. truncatula, but also other important dicot plants, which will serve as a basis for proteomes of many other dicot plants and plant materials.

System, trends and perspectives of proteomics in dicot plants. Part III: Unraveling the proteomes influenced by the environment, and at the levels of function and genetic relationships.

This review is devoted to the proteomics studies in dicotyledoneous (dicot) plants, such as Arabidopsis, Medicago, potato, soybean, and tomato, under the influence of the environment and at the functional and genetic relationship levels, where the two core technologies, two-dimensional gel electrophoresis (2-DGE) and mass spectrometry (MS) have been instrumental in unraveling the proteomes affected therein. Abiotic and biotic stress responses, including the affect of allergens, the symbiotic interaction between the members of the Leguminoseae family and genera of nitrogen fixing bacteria, phosphoproteomics, and proteomics in revealing the genetic relationships between species and genera have been the subject of many proteomics studies, and these are discussed in this review. In all, these studies have complemented and extended the studies of developmental proteomics [G.K. Agrawal, M. Yonekura, Y. Iwahashi, H. Iwahashi, R. Rakwal, J. Chromatogr. B (2004)].

Stability of A+U-rich element binding factor 1 (AUF1)-binding messenger ribonucleic acid correlates with the subcellular relocalization of AUF1 in the rat uterus upon estrogen treatment.

The nucleocytoplasmic shuttling protein, A+U-rich element binding factor 1 (AUF1), is one of the RNA-binding proteins that specifically bind adenylate-uridylate rich elements (AREs) in mRNA 3'-untranslated regions (UTRs), and acts as a regulator of ARE-mediated mRNA degradation in the cytoplasm. We previously reported that in the female rat uterus, the levels of specific AUF1 isoform mRNAs (p40/p45) were increased by 17 beta-estradiol (E2) treatment. Therefore, we examined the role of AUF1 in the regulation of E2-mediated mRNA turnover in the rat uterus. We identified ABIN2 and Ier2/pip92 mRNAs as candidate targets of AUF1 in the rat uterus. We found that AUF1-binding elements were present in the 3'-UTR of both mRNAs and that the 3'-UTRs functioned as mRNA turnover regulatory elements. In the ovariectomized rat uterus, the nucleocytoplasmic localization of AUF1p40/p37 isoform proteins was regulated by E2. We also found that cytoplasmic AUF1-bound mRNA levels changed coincidentally with the cytoplasmic levels of AUF1p40/p37. Finally, we confirmed that the subcellular localization of AUF1p40 controlled the stability of target mRNAs in vitro, such that cytoplasmically localized AUF1p40 led to marked mRNA stabilization, whereas nuclear-localized AUF1p40 stabilized target mRNA only slightly. These results suggested that E2-inducible ARE-containing gene transcripts are regulated, at least in part, via mRNA stabilization through the nucleocytoplasmic relocalization of AUF1.

Immature Seed Endosperm and Embryo Proteomics of the Lotus (Nelumbo Nucifera Gaertn.) by One-Dimensional Gel-Based Tandem Mass Spectrometry and a Comparison with the Mature Endosperm Proteome.

Lotus (Nelumbo nucifera Gaertn.) seed proteome has been the focus of our studies, and we have recently established the first proteome dataset for its mature seed endosperm. The current study unravels the immature endosperm, as well as the embryo proteome, to provide a comprehensive dataset of the lotus seed proteins and a comparison between the mature and immature endosperm tissues across the seed's development. One-dimensional gel electrophoresis (SDS-PAGE) linked with tandem mass spectrometry provided a protein inventory of the immature endosperm (122 non-redundant proteins) and embryo (141 non-redundant proteins) tissues. Comparing with the previous mature endosperm dataset (66 non-redundant proteins), a total of 206 non-redundant proteins were identified across all three tissues of the lotus seed. Results revealed some significant differences in proteome composition between the three lotus seed tissues, most notably between the mature endosperm and its immature developmental stage shifting the proteins from nutrient production to nutrient storage.

Molecular cloning of a multidomain cysteine protease and protease inhibitor precursor gene from the tobacco hornworm (Manduca sexta) and functional expression of the cathepsin F-like cysteine protease domain.

A Manduca sexta (tobacco hornworm) cysteine protease inhibitor, MsCPI, purified from larval hemolymph has an apparent molecular mass of 11.5 kDa, whereas the size of the mRNA is very large (∼9 kilobases). MsCPI cDNA consists of a 9,273 nucleotides that encode a polypeptide of 2,676 amino acids, which includes nine tandemly repeated MsCPI domains, four cystatin-like domains and one procathepsin F-like domain. The procathepsin F-like domain protein was expressed in Escherichia coli and processed to its active mature form by incubation with pepsin. The mature enzyme hydrolyzed Z-Leu-Arg-MCA, Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA rapidly, whereas hydrolysis of Suc-Leu-Tyr-MCA and Z-Arg-Arg-MCA was very slow. The protease was strongly inhibited by MsCPI, egg-white cystatin and sunflower cystatin with K(i) values in the nanomolar range. When the MsCPI tandem protein linked to two MsCPI domains was treated with proteases, it was degraded by the cathepsin F-like protease. However, tryptic digestion converted the MsCPI tandem protein to an active inhibitory form. These data support the hypothesis that the mature MsCPI protein is produced from the MsCPI precursor protein by trypsin-like proteases. The resulting mature MsCPI protein probably plays a role in the regulation of the activity of endogenous cysteine proteases.

Proteomics of two cultivated mushrooms Sparassis crispa and Hericium erinaceum provides insight into their numerous functional protein components and diversity.

Mushroom can be defined as a macrofungus with a distinctive fruiting body. Mushrooms of class Basidiomycete are primarily wood degradation fungi, but serve as food and a part of traditional medicine used by humans. Although their life cycle is fairly well-established, the information on the molecular components, especially proteins are very limited. Here, we report proteomics analysis of two edible mushrooms (fruiting bodies) Sparassis crispa and Hericium erinaceum using one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) based complementary proteomics approaches. 1-DGE coupled with liquid chromatography and mass spectrometry identified 77 (60 nonredundant proteins) and 121 (88 nonredundant proteins) proteins from S. crispa and H. erinaceum, respectively. 2-DGE analysis revealed 480 and 570 protein spots stained with colloidal coomassie brilliant blue in S. crispa and H. erinaceum, respectively. Of the 71 and 115 selected protein spots from S. crispa and H. erinaceum 2D gel blots on polyvinyldifluoride (PVDF) membranes, respectively, 29 and 35 nonredundant proteins were identified by N-terminal amino acid sequencing. Identified nonredundant proteins from 1- or 2-DGE belonged to 19 functional categories. Twenty-one proteins were found common in both S. crispa and H. erinaceum proteomes, including 14-3-3 protein and septin. Together this study provides evidence for the presence of a large number of functionally diverse proteins, expressed in the fruiting body of two economically important mushrooms, S. crispa and H. erinaceum. Data obtained from 1-DGE and 2-DGE analyses is accessible through the mushroom proteomics portal http://foodfunc.agr.ibaraki.ac.jp/mushprot.html.

Comprehensive royal jelly (RJ) proteomics using one- and two-dimensional proteomics platforms reveals novel RJ proteins and potential phospho/glycoproteins.

Royal jelly (RJ) is an exclusive food for queen honey bee (Apis mellifera L.) that is synthesized and secreted by young worker bees. RJ is also widely used in medical products, cosmetics, and as health foods. However, little is known about RJ functionality and the total protein components, although recent research is attempting to unravel the RJ proteome. We have embarked on a detailed investigation of the RJ proteome, using a modified protein extraction protocol and two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with tandem mass spectrometry. Simultaneously, we examined total soluble protein from RJ collected at 24, 48, and 72 h after honey bee larvae deposition twice (in two flower blooming seasons), to check differences, if any, in RJ proteome therein. Both 1- and 2-D gels stained with silver nitrate revealed similar protein profiles among these three time points. However, we observed a clear difference in two bands (ca. MW of 55 and 75 kDa) on 1-D gel between the first and the second collection of RJ. A similar difference was also observed in the 2-D gel. Except for this difference, the protein profiles were similar at the 3 time points. As the RJ from 48 (or sometimes 72) is commercially used, we selected the RJ sample at 48 h for detailed analysis with the first collection. 1-DGE identified 90 and 15 proteins from the first and second selection, respectively; in total, 47 nonredundant proteins were identified. 2-DGE identified 105 proteins comprising 14 nonredundant proteins. In total, 52 nonredundant proteins were identified in this study, and other than the major royal jelly protein family and some other previously identified proteins, 42 novel proteins were identified. Furthermore, we also report potentially post-translationally modified (phosphorylation and glycosylation) RJ proteins based on the Pro-Q diamond/emerald phosphoprotein/glycoprotein gel stains; MRJP 2p and 7p were suggested as potential phosphoproteins. The 2-DGE data were integrated to develop a 2-D gel reference map, and all data are accessible through RJ proteomics portal (http://foodfunc.agr.ibaraki.ac.jp/RJP.html).

Systematic investigation of the hemolymph proteome of Manduca sexta at the fifth instar larvae stage using one- and two-dimensional proteomics platforms.

Manduca sexta is an excellent insect model for studying insect physiology, including hemolymph proteins. Larvae stages of this insect are highly damaging to tobacco leaves causing a drastic decrease in crop yield. Investigation on the larval biology should help in controlling its destructive potential, thus increasing crop yields. The hemolymph is the source of its immunity to disease and environmental factors, which invariably involves protein components. To better understand the physiology of M. sexta and the protein components expressed during its life cycle, two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1-DGE and 2-DGE) in conjunction with N-terminal amino acid sequencing and liquid chromatography-mass spectrometry, were employed to analyze the fifth instar larvae hemolymph proteins. These proteomics approaches together identified 123 proteins, which constituted a total of 58 nonredundant proteins and belonged to 10 functional categories. Defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) categories were highly represented accounting for 80% of the identified proteins. Besides identification of previously reported proteins, 18 novel proteins were identified, which include the lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1, imaginal disk growth factor, protein disulfide-isomerase-like protein ERp57, etc. The 2-DGE data were integrated to develop a 2-D gel reference map. Data obtained from 1-DGE and 2-DGE analyses are accessible through the M. sexta proteomics portal ( http://foodfunc.agr.ibaraki.ac.jp/mansehemoprot.html). Together, this study provides evidence for the presence of a large number of functionally diverse protein families in the hemolymph of M. sexta. These proteins correlate well with the fifth instar stage, the transition from larvae to pupae.