Systematic Investigation of the Hemolymph Proteome of Manduca

Systematic Investigation of the Hemolymph Proteome of Manduca sexta at the Fifth Instar Larvae Stage Using One- and Two-Dimensional Proteomics Platforms Takako Furusawa,† Randeep Rakwal,‡,§ Hyung Wook Nam,4 Misato Hirano,‡ Junko Shibato,‡ Yu Sam Kim,4 Yoko Ogawa,⊥ Yasukazu Yoshida,⊥ Karl J. Kramer,# Yoshiaki Kouzuma,† Ganesh Kumar Agrawal,§,¶ and Masami Yonekura*,† Food Function Laboratory, School of Agriculture, Ibaraki University, Ami 300-0393, Japan, Human Stress Signal Research Center (HSS), National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba West, 16-1 Onogawa, Tsukuba 305-8569, Japan, Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB), Kathmandu, Nepal, Protein Network Research Center, Yonsei University, Seoul 120-749, Korea, HSS, AIST Kansai Center, 1-8-31 Midorigaoka, Ikeda 563-8577, Japan, and Grain Marketing and Production Research Center, ARS-USDA, 1515 College Avenue, Manhattan, Kansas 66502 Received July 1, 2007

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. Keywords: Manduca sexta • hemolymph • proteomic analysis • gel-based approach • mass spectrometry • proteins

1. Introduction The leaf-chewing solanaceous specialist Manduca sexta (holometabolous; lepidopteran) is a well-known insect pest of tobacco plants. It is interesting to note that tobacco is the * To whom correspondence should be addressed. E-mail: yonekura@ mx.ibaraki.ac.jp. Fax: +81-29-888-8683. † Ibaraki University. ‡ AIST. § RLABB. 4 Yonsei University. ⊥ AIST Kansai Center. # ARS-USDA. ¶ Present address: University of Missouri-Columbia, Biochemistry Department, 204 Life Sciences Center, Columbia, MO 65211.

938 Journal of Proteome Research 2008, 7, 938–959 Published on Web 01/17/2008

world’s most widely grown crop plant, with America, China, Brazil, and India involved in its cultivation. The larvae or caterpillar of M. sexta (also called the tobacco hornworn) are voracious leaf feeders that reduce the yield and quality of the crop and are theoretically the most potentially damaging insect pests of tobacco, with a heavy infestation causing total crop destruction. Thus, M. sexta can be considered as the world’s most destructive pest and has been widely studied in the last 40 years. These four decades have resulted in 2144 scientific publications (as of April, 2007) on PubMed with the keyword “Manduca sexta”. M. sexta has been an excellent insect model organism/system for understanding the physiological control of growth, develop10.1021/pr070405j CCC: $40.75

 2008 American Chemical Society

Investigation of the Hemolymph Proteome of Manduca sexta ment, and metamorphosis of insects for half a century. This is mainly due to easy availability of its life stages, rapid growth and life cycle, easy culture, and, maybe the most important, the presence of a large quantity of hemolymph (insect blood). Current research on M. sexta is wide ranging and includes flight mechanisms in the moth, larval nicotine resistance, hormonal regulation of development, and hemolymph physiology, to name a few hot areas of interest. Due to these reasons and interests, M. sexta has been aptly referred to as the laboratory star in the world of insect physiologists.1 Hemolymph, an open circulatory system that circulates among all organs, takes on metabolism and regulation functions, such as nutrient and hormone transport, response to injury, and immunity. The hemolymph proteins and their functions are being actively researched, and most of the information on hemolymph proteins has come from the studies of the Lepidoptera.2 Several studies have shown that the mechanisms to defend against invading pathogens and parasites,3,4 system for transport and metabolism,5,6 and regulation of insect metamorphosis7,8 can be attributed to components residing in the hemolymph. Although the hemolymph is readily obtainable from various stages such as larva, pupa, to adult, the fifth instar larvae is the stage of choice for extracting hemolymph.9 This is due to the fact that the fifth instar is (i) bigger in size, (ii) rich in hemolymph, and (iii) a transition period of biosynthesis, melanization, and metamorphosis from larva to pupa. For these reasons, this stage has been the focus of research for a long time, resulting in the publication of 379 scientific papers with the keywords “Manduca sexta” and “hemolymph” on PubMed. Studies on hemolymph have mainly been conducted on purification and identification of proteinaceous components. However, a large-scale proteomics analysis study on the fifth instar hemolymph or any other stages of the life cycle of M. sexta has not been reported to date. Proteomics is a prominent technology that has being widely applied to address the biological questions in prokaryotes,10–12 animals,13–15 and plants.16–21 Here we apply two complementary proteomics approaches, one- and two-dimensional gel electrophoresis (1and 2-DGE) coupled with Edman sequencing of the N-terminal amino acids and tandem mass spectrometry (MS), to investigate the hemolymph proteome at the fifth instar larval stage of M. sexta. This study reports the identification of several novel proteins in hemolymph along with a number of proteins previously reported during the four decades of independent and targeted studies. Use of both 1-DGE and 2-DGE based approaches helped in identification of 123 proteins. A highresolution 2-D gel reference map was also established for future comparative proteomics analysis at other stages of the M. sexta life cycle.

2. Materials and Methods 2.1. Hemolymph. Manduca sexta larvae were reared using an artificial diet at 27 °C with a photoperiod of 16/8 h light/ dark.22 The extracted and dried hemolymph from fifth instar larvae was stored at -80 °C until extraction of total protein. 2.2. Extraction of Total Protein. Extraction of total protein was performed by precipitating the total proteins in finely ground (in a prechilled mortar and pestle with liquid nitrogen) hemolymph powder (ca. 150 mg) with cold trichloroacetic acid (TCA)/acetone extraction buffer [TCAAEB, (acetone containing 10% (w/v) TCA, and 0.07% mercaptoethanol (ME)] followed by solubilization of proteins in LB.20,23,24 Briefly, the proteins were

research articles

precipitated for 1 h at -20 °C, followed by centrifugation at 15 000 rpm for 15 min at 4 °C. The pellet was washed twice with chilled wash buffer [acetone containing 0.07% ME, 2 mM EDTA, and EDTA-free proteinase inhibitor cocktail tablets (Roche Diagnostics GmbH, Mannheim, Germany) in a final volume of 100 mL buffer] followed by removal of residual acetone. The pellet was subsequently solubilized in lysis buffer [7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 18 mM Tris-HCl (pH 8.0), 14 mM trizma base, two EDTA-free proteinase inhibitor cocktail tablets in a final volume of 100 mL buffer, 0.2% (v/v) Triton X-100 (R), containing 50 mM dithiothreitol (DTT); hereafter called LB-TT], incubated for 20 min at 4 °C with occasional vortexing, and centrifuged at 15 000 rpm for 15 min at 10 °C. In this experiment, a further purification/ clean-up of the solubilized protein samples was performed using the 2-D Clean-Up Kit (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) as per the instructions provided along with the kit. The pellet was resolubilized in LB-TT to give the total soluble protein. The supernatant was used for protein quantification by a Coomassie Plus protein assay kit (PIERCE, Rockford, IL) and stored in aliquots at -80 °C. 2.3. One-Dimensional Gel Electrophoresis and Mass Spectrometry Analysis. The total soluble protein obtained in the previous step was precipitated using a Protein Precipitation Kit (Calbiochem, Darmstadt, Germany), and the pellet was resolubilized in homogenization buffer [HB, 0.2 M Tris-HCl buffer, pH 7.8, containing 5 mM EDTA.2Na, 14 mM 2-ME, 10% (v/v) glycerol, and 2 EDTA-free proteinase inhibitor tablets (Roche Diagnostics GmbH, Mannheim, Germany) per 100 mL of buffer solution in MQ water]. Sodium dodecyl sulfate (SDS) sample buffer [2.5X, 62 mM Tris (pH 6.8) containing 10% (v/v) glycerol, 2.5% (w/v) SDS, and 5% (v/v) 2-ME, pH 6.8] was added to maximize solubilization of the protein pellet. The supernatant after centrifugation was used for protein quantification by a Coomassie Plus protein assay kit. Just before electrophoresis, a drop of bromophenol blue (BPB) was added to the protein samples and boiled for 1 min at 95 °C. An amount of 50 µg of protein was loaded per well for 1-DGE. SDS-polyacrylamide gel electrophoresis (SDS-PAGE, 4% T, 2.6% C stacking gels, pH 6.8 and 12.5% T, 2.6% C separating gels, pH 8.8) was carried out using polyacrylamide gels on a Nihon Eido (Tokyo, Japan) vertical electrophoresis unit at a constant current of 35 mA for approximately 3 h. The running buffer was composed of 0.025 M Tris, 0.192 M glycine, and 0.2% (w/v) SDS. A portion of 5 µL of the commercially available “ready-to-use” molecular mass standards (Precision Plus Protein Standards, Dual Color, BioRad, Hercules, CA, USA) was loaded in the well adjacent to the samples. The gel was stained with Coomassie brilliant blue (CBB) R-250. Each lane was sliced into six pieces of gel matrix and digested with 1 µg of trypsin at 37 °C for 18 h. The tryptic peptide samples were separated by a C18 reversed-phase column and analyzed on a nanoelectrospray ionization mass spectrometer (nESI-LC-MS/MS). Ultimate nanoLC systems, combined with the FAMOS autosampler and Switchos column switching valve (LC-Packings, Amsterdam, Netherlands), were used. The samples were loaded onto the precolumn (2 cm × 200 µM i.d.; Zorbax 300SB-C18, 5 µM, Agilent, CA) and washed with the loading solvent (H2O/0.1% formic acid; flow rate of 4 µL/min) for 10 min to remove salts. Subsequently, a Switchos II column switching device transferred flow paths to the analytical column (15 cm × 75 µM i.d.; Zorbax 300SB-C18, 5 µM, Agilent). The nanoflow eluted at a flow rate of 200 nL/min using a 110 min Journal of Proteome Research • Vol. 7, No. 3, 2008 939

research articles gradient elution from 0% solvent A to 32% solvent B, where solvent A was 0.1% formic acid with 5% acetonitrile and solvent B was 0.1% formic acid with 90% acetonitrile in water. The column outlet was coupled directly to the high-voltage ESI source, which was interfaced to the QSTAR mass spectrometer (Applied Biosystems, Foster City, CA). The nanospray voltage was typically 2.3 kV in the nESI-LC-MS/MS mode. The nESILC-MS/MS running on the QSTAR instrument was acquired in the “Information Dependent Acquisition” mode, which allows the user to acquire MS/MS spectra based on an inclusion mass list and dynamic assessment of relative ion intensity. The data acquisition time was set to 3 s per spectrum over a mass/ charge (m/z) range of 400–1500 Da. AnalystQS software (version 1.0; Applied Biosystems, Foster City, CA) was used for generating a peaklist. Acquired data were searched against the National Center for Biotechnology Information (NCBI) nonredundant protein database (nrdb90) for an animal sequence using the MASCOT software package (version 2.1, Matrix Sciences, UK; www.matrixscience.com). The NCBInr database contained 2 481 719 sequences and 841 197 525 residues, and the taxonomy was Metazoa (animal; 719 806 sequences). Parameters used were peptide mass tolerance (1.0 Da), MS/MS ion mass tolerance (0.8 Da), allowance of two tryptic miscleavages, differential modification (methionine as oxidation), and default selection of charge state ions (only +2, +3). The cutoff score/expectation value for accepting individual MS/MS spectra was 42, which indicates identity or extensive homology of probability lower than 0.05 (p < 0.05). The threshold employed was -10* log(P), where P is the probability that the observed match is a random event; protein scores are derived from ion scores as a nonprobabilistic basis for ranking protein hits. If peptides matched to multiple members of a protein family, we focused on the unique peptide of the identified proteins. The unique peptide of each protein was manually selected to reduce the multimatching peptide mistake, and then the MSQuant 1.4 (www.cebi.sud.dk) program was used to regenerate the protein identification list. The MSQuant 1.4 program eliminates peptides having scores lower than the applied cutoff score of 42, thereby helping in the unambiguous identification of isoforms/individual members of a protein family. Multiple protein IDs having the same accession number were considered as a single nonredundant protein for the protein list provided in this study. The peptide sequences were listed along with the corresponding proteins (Table 1). 2.4. Two-Dimensional Gel Electrophoresis. 2-DGE was carried out using precast IPG strip gels on an IPGphor unit (GE Healthcare) followed by the second dimension using ExcelGel XL SDS gels on a Multiphor II horizontal electrophoresis unit (GE Healthcare). The volume carrying 750 µg of total soluble protein was mixed with LB-TT containing 0.5% (v/v) pH 4–7 IPG buffer to bring it to a final volume of 340 µL. A trace of BPB was added and centrifuged at 15 000 rpm for 15 min followed by pipetting into an 18 cm strip holder tray placed into the IPGphor unit. IPG strips (pH 4–7; 18 cm, GE Healthcare) were carefully placed onto the protein samples avoiding air bubbles between the sample and the gel. The IPG strips were allowed to passively rehydrate with the protein samples for 1.5 h, followed by overlaying the IPG strips with cover fluid (mineral oil), and this was directly linked to a five-step active rehydration and focusing protocol as described previously.23 The whole procedure was controlled at 20 °C, and a total of 68 902 Vh was used for the 18 cm strip. The strip gels were 940

Journal of Proteome Research • Vol. 7, No. 3, 2008

Furusawa et al. incubated in equilibration buffer (50 mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS) containing 2% (w/ v) DTT for 10 min (twice) with gentle agitation, followed by incubation in the same equilibration buffer supplemented with 2.5% (w/v) iodoacetamide for the same time periods as above at room temperature. The IPG strip was placed onto the gradient (12–14%) gels. To visualize the protein spots, the polyacrylamide gels were stained with colloidal CBB G-250. Protein patterns in the gels were recorded as digitalized images using a digital scanner (CanoScan 8000F, resolution 300 dpi, 16 bit-grayscale pixel depth) and saved as TIFF files. ImageMaster 2D Platinum software version 5.0 (GE Healthcare) was used to determine the total number of spots (those having % volume greater than 0.01 were accepted) on the 2-D gels. Moreover, spots present in at least three gel replications were selected for further analysis. 2.5. In-Gel Digestion. The colloidal CBB stained protein spots were excised from the 2-D gels using a gel picker (One Touch Spot Picker, P2D1.5, The Gel Company, San Francisco, CA) and transferred to sterile 1.5 mL microcentrifuge tubes. The gels were first destained with 50 mM ammonium bicarbonate in 50% acetonitrile followed by 100% acetonitrile until they were completely destained with minor modifications. Briefly, protein spots were washed twice with 100 mM ammonium bicarbonate (pH 8.5; hereafter called AMBIC) and then dehydrated with acetonitrile. The gel pieces were reduced with 10 mM DTT at 56 °C for 45 min and alkylated with 50 mM iodoacetamide for 45 min at room temperature in the dark in AMBIC solution. Gel pieces were washed with 20 mM AMBIC, dehydrated with acetonitrile, and air-dried. Gel pieces were subjected to in-gel trypsin digestion with 20 µL of 20 mM AMBIC containing 15 ng/µL of sequence grade modified trypsin (17 000 U/mg; Promega, Madison, WI, USA) at 37 °C for 18 h. The peptides were extracted from the gel pieces two times with 20 µL of 20 mM AMBIC and three times with 20 µL of 0.5% trifluoroacetic acid (TFA) in 50% acetonitrile. The peptides extracted in the five steps were combined together and concentrated to 20 µL by using a centrifugal concentrator (CC105; TOMY, Tokyo, Japan). The resulting tryptic peptides were analyzed on either the nLC Linear ion-trap time-of-flight MS (nLC-IT-TOF-MS/MS, Hitachi High-Technologies Corporation, Hitachi, Japan) or the LCQ Deca linear ion trap MS (Thermo Electron, USA). 2.6. nLD-IT-TOF-MS/MS and Protein Identification. The HPLC/ESI mass spectrometer instrument used was a Nano Frontier system (Hitachi High-Technologies) consisting of a capillary HPLC system based on the AT10PV nanoGR generator and an ESI-TOF-MS. The analytical column was MonoCap for nanoflow (0.05 mm i.d., 150 mm length, GL-Science, Tokyo, Japan). The trap column was monolith (0.2 mm i.d., 5 mm length, Kyoto Monotech, Kyoto, Japan), which was cut off to an arbitrary length and was custom made. A silica tip (tip diameter of 10 µM; New Objective, Woburn, MA, USA) was used as an electrosprayer. The HPLC conditions were as follows: The flow rate of the nanoflow pump was set at 100 nL/min. Solvent A was 2% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid, and solvent B was 98% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid. The composition of solvent B was linearly increased from 2% at 0 min to 45% at 60 min, kept at 98% until 80 min, and then returned to the initial condition of 2%. The ESI-IT-TOF-MS conditions were as

score

1204

1145

116 692

67 72

196

166 65 137 46

171 155 183 120

366

51 257

96 160

peptide

17

16

2 10

1 1

3

3 1 2 2

3 2 3 2

6

1 5

1 2

gi|5326830 gi|29469967

gi|4262357 gi|27733411

gi|84831

gi|7262927 gi|511297 gi|9651929 gi|27733409

gi|2654518 gi|2654518 gi|21630233 gi|21630231

gi|857370

gi|134436 gi|134436

gi|27733417 gi|134436

gi|1378128

gi|431347

acc. no.

3 4

serpin 3bb alaserpin precursor (serpin 1)b

plasmatocyte-spreading peptide precursorb antimicrobial peptide gloverinb

scolexin Ab leureptinb

lysozyme (EC 3.2.1.17)b

β-1,3-glucan recognition proteinb hemolinb immulectin-2b peptidoglycan recognition protein 1Bb

pro-phenol oxidase subunit 1; proPO-p1b pro-phenol oxidase subunit 1; proPO-p1 serine proteinase-like protein 2b serine proteinase-like protein 1b

pro-phenol oxidaseb

MW

5.07 5.07

5.26 5.07

5.25

43510.13 43510.13

51128.25 43510.13

43457.91

5 6

8.41 9.39

14004.78 18444.55

Other Defense Proteins (4) 4 6.98 30372.7 3 6.99 46537.05

Lysozyme (1) 8.94 13983.8

sexta sexta sexta sexta

sexta sexta sexta sexta

M. sexta M. sexta

M. sexta M. sexta

M. sexta

M. M. M. M.

Pattern Recognition (4) 3 5.27 54567.17 3 7.91 45665.21 4 6.1 37423.55 6 6.29 21530.35 6

M. M. M. M.

7.01 7.01 5.83 5.16

M. sexta

M. sexta M. sexta

M. sexta M. sexta

M. sexta

Manduca sexta

species

78965.66 78965.66 43488.52 45322.22

2 3 3 3

Pro-phenol Oxidase (5) 2 5.68 80060.7

5 6

3

alaserpin precursor (serpin 1) alaserpin precursor (serpin 1)

pI

Serine Protease Inhibitor (6) 3 5.56 43683.28

fraction

serpin 1b

serine protease inhibitorb

protein name

Defense (20)

Table 1. List of Hemolymph Proteins Separated by 1-DGE and Identified by nESI-LC-MS/MSa

NGNLAVFSR VGVFDGIPK, LFLQTNQIR, NVYNLEEIDLSR, NLLSTLPLGLFDNLPK, VSVNAFQSPSLELLDLSR IVFPDDDQDVAPR TGLTASGSGVWQLGK, VFGTLGQNDQGLFGK

CELVHELR, QGFPENLMR, FQAWYGWR, DYGLFQINDK, CSDLLIDDITK, DWVCLVENESSR

DPSDAIIVPPIVTAK, TLVGGPIMSEAEPYR, VSIPDDGYTLFAFHGK QLGEDESIADSILAR, SPAIDGDVPLVGYTIK GDFFSVEGIPLKK, QSVFTGIHATFSR, LHEIPANWHEAR EPNSQQLNAIK, RWPEWTNDVSEYKD

VQNYAEIFPAK, NIEEAIATGNVILPDK, QSTESSLTIPFEQTFR, NIEEAIATGNVILPDKSTK DQFLEAIQK, FVDDVFNIYKEK, GEDKVVFEIPDHYYPDKYK DQFLEAIQK DVPAVYVNVPAFR, AGEWDTQTIKEELEHQER KIEVPVVDR, FGEFPWMVAILK

TIKVPTMIGK, YADVPELDAK, WVEDQTNNR, FKIETTTDLK, ESLYVDAAIQK, ITTYSFHFVPK, GLELNDDFAAVSR, NLVDPDALDETTR, TKESLYVDAAIQK, VEINKPFFFSLK, LYNTEVEIYLPK, DVFGSEVQNVDFVK, IKNLVDPDALDETTR, ESNDQFTAQMFSEVVK, IETTTDLKEVLSNMNIK, FKIETTTDLKEVLSNMNIK, ANPGQNVVLSAFSVLPPLGQLALASVGESHDELLR SVEAAGAINK, TIKVPTMIGK, YADVPELDAK, WVEDQTNNR, FKIETTTDLK, ESLYVDAAIQK, GLELNDDFAAVSR, NLVDPDALDETTR, TKESLYVDAAIQK, LYNTEVEIYLPK, DVFGSEVQNVDFVK, IKNLVDPDALDETTR, ESNDQFTAQMFSEVVK, IETTTDLKEVLSNMNIK, FKIETTTDLKEVLSNMNIK, ANPGQNVVLSAFSVLPPLGQLALASVGESHDELLR SELASALGFGLDR, VASTSNANFLLSPLGLK KLFTPGAAR, SVLVNAIYFK, FKIETTTDLK, ESLYVDAAIQK, GLELNDDFAAVSR, TKESLYVDAAIQK, LYNTEVEIYLPK, DVFGSEVQNVDFVK, ESNDQFTAQMFSEVVK, FKIETTTDLKEVLSNMNIK TKESLYVDAAIQK GLELNDDFAAVSR

matched peptide(s)

Investigation of the Hemolymph Proteome of Manduca sexta

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Journal of Proteome Research • Vol. 7, No. 3, 2008 941

942


60

1

2 6

4

3

4

4

3

3

acc. no.

protein name

fraction

pI

MW

Transport and Metabolism (19) species

matched peptide(s)

4180 gi|7511788 apolipophorin precursor proteinb

Lipid Transport and Metabolism (6) 1 8.66 366946.52 M. sexta

ITFSQILK, SEIDVFDK, IDSSIVLSK, VFLVGHTSK, TIIEGSGSVK, VVEHFLGPK, GDIIDYLLK, HLTFPGTCR, LDVATNIDIR, YELSGFVLHK, TVVDFLNYIK, GNGLIIIDFKK, GKLDDIEYSGK, NVLSGSTSFISK, LGFSVQGNEHR, YLDDYTLTVR, LGVAPELGLQMR, AAAGLLDMPNLPK, SFELLLDTEGAK, ENFNYFLNALK, ILPTDELKEFAK, ANLNNANLEAFSR, AHTVAELVLPTER, GVETFFNVALGER, FLVDYSNSGSEDK, ENFNYFLNALKK, FYQEIADDKVFK, TEDPQALYDNLVK, KIDIDIEGQLQDK, QFIQTAAHNIIQR, AVQLVSQAYEAFSK, GSDVVLAFNNQLNPK, LFGTDAVFLSFGDDK, MLDQILGGCNSGINK, TKPNDVNVGFIGHLK, NIAHGALFLQDNLVK, AVVVNGQHIFTFDGR, VITNLHSSTGVHVNAK, NMVVPLVSQLVDMLK, AIDVEGSFNVNQQQR, SSHEGEKNNNYVELK, GSYGSDIGFELAGVGTIK, EISHVFNEIVQYIAK, AHPVLDIQYHSPSSDK, DYQYYEFTTEESNR, DGLYNGHMDMTISDAPK, VLVPHVEPFVLDYNYK, SASMVVDSSYAPHYSTLK, AESAPYNNELDLDIYVK, DYQYYEFTTEESNRK, IDMSTPGMIVNVINAGLDLR, VDNINDICLDAVSEANVQK, AVYKPIFEYSLPDGSSPGSK, INIGHIETPAVFSSHATISGSR, VSNSNLDQEIIDYEGQINFK, SHLLFMDAELAYPTSVGLPLR, IELGITNIEASQGQIFDLPLRPGAVK

Protein (Amino acid) Transport and Metabolism (9) 201 gi|136429 trypsin precursorb 1 7 24409.48 Sus scrofa VATVSLPR, LGEHNIDVLEGNEQFINAAK, IITHPNFNGNTLDNDIMLIK 199 gi|136429 trypsin precursor 2 7 24409.48 S. scrofa VATVSLPR, LGEHNIDVLEGNEQFINAAK, IITHPNFNGNTLDNDIMLIK 262 gi|136429 trypsin precursor 5 7 24409.48 S. scrofa VATVSLPR, LSSPATLNSR, LGEHNIDVLEGNEQFINAAK, IITHPNFNGNTLDNDIMLIK 268 gi|136429 trypsin precursor 6 7 24409.48 S. scrofa VATVSLPR, LSSPATLNSR, LGEHNIDVLEGNEQFINAAK, IITHPNFNGNTLDNDIMLIK 185 gi|3318722 chain E, leech-derived tryptase inhibitor trypsin complexb 3 8.26 23472.78 S. scrofa LSSPATLDSR, LGEHNIDVLEGNEQFINAAK, IITHPNFNGNTLDNDIMLIK 236 gi|3318722 chain E, leech-derived tryptase inhibitor trypsin complex 4 8.26 23472.78 S. scrofa VATVSLPR, LSSPATLDSR, LGEHNIDVLEGNEQFINAAK, IITHPNFNGNTLDNDIMLIK 95 gi|400673 ommochrome-binding protein precursorb 4 5.89 30614.72 M. sexta MGYLNLATK, VIDLGDYNVNAFTK 398 gi|400673 ommochrome-binding protein precursor 5 5.89 30614.72 M. sexta MGYLNLATK, DGIYTYDYATK, VIDLGDYNVNAFTK, DGMATAVDTTNHIVYLGGK, SFGEISGVKDGMATAVDTTNHIVYLGGK, LISETGEDSIWGAAFDKDDNIVYSNEDNIVK 51 gi|400673 ommochrome-binding protein precursor 6 5.89 30614.72 M. sexta MGYLNLATK

peptide score

Table 1. Continued

research articles Furusawa et al.

121 gi|136206 1038 gi|136206

2 16

transferrin precursorb transferrin precursor

494

2717

39

score

gi|1168527

gi|1168527

acc. no. b

arylphorin β chain precursor


protein name

162 gi|136206 transferrin precursor 62 gi|7272336 ferritinb

9

peptide

3 1

173 gi|9718

3

6

3 6 5

2

fraction

pI

MW

species

matched peptide(s)

8.66 366946.52 M. sexta VYLDALLR, RVYLDALLR, GSSSPSFAAGQK, AAAGLLDMPNLPK, SFELLLDTEGAK, NKPDEDVVVAILK, AGTGESIEASIQILK, TEDPQALYDNLVKR, NLLDSEPVHQVGNFITSSLR, VSAIEEYLYKPFSVGENGAR, SKDLSQLEQHLVFLSLGNAR, SIIFDVPHGTSSASGNLNSVISAVK, NDFVNSIANPDAGIKDLQVLQSMLNVESK 8.66 366946.52 M. sexta AAAGLLDMPNLPK, NDFVNSIANPDAGIK 6.98 20793.37 M. sexta DAPAGGNAFEEMEK, TFSEQFNSLVNSK 6.29 23182.27 M. sexta TFSHLIDASK, LPLENENQGK, AHSIHAWILSK, CTIAEYKYDGK, ASVYNSFVVNGVK, CTIAEYKYDGKK, KASVYNSFVVNGVK, EYMEGDLEIAPDAK, VVNLVPWVLATDYK, NYAINYNCNYHPDKK, FISNDFSEAACQYSTTYSLTGPDRH 6.29 23182.27 M. sexta TFSHLIDASK, LPLENENQGK, ASVYNSFVVNGVK

2

1

fraction

5.95

5.95

pI

83848.83

83848.83

MW

Storage (18)

M. sexta

M. sexta

species

NSNDFLIFK, AVEEFLQLYR, DVFVYHDGEYFPYK, MQDGVVSADLAAQHGIVK, LSNGLGDIPEFSWYSPVK, VLDLFQDVDQVNPNDEYYK, NSNDFLIFKDDSVPMTELYK, LLEQNKVPHDMSEDYGYLPK, AINFVGNYWQDNADLYGEEVTK SYEIIAR, SSYPHDFK, QVLGAAPKPFDK, DPMFYQLYNR, YYEFSPFYDR, FFELDWFTYK, KAVEEFLQLYR, LNHKPFSVSIDIK, AFDQKIDFHDFK, VPHDMSEDYGYLPK, EYNIEANIDNYSNK, FNVPSHVMHSNVVPK, DVFVYHDGEYFPYK, EYNIEANIDNYSNKK, MQDGVVSADLAAQHGIVK, GEIYYYFYQQLMAR, LRDEAIGVFHLFYYAK, YDDNGFPLKLENNWNK, LSNGLGDIPEFSWYSPVK, TKMQDGVVSADLAAQHGIVK, IGKEYNIEANIDNYSNKK, VLDLFQDVDQVNPNDEYYK, VYLNEGQFLYAYYIAVIQR, FLDTYEMSFLQFLQNGHFK, NSNDFLIFKDDSVPMTELYK, LLEQNKVPHDMSEDYGYLPK, KVLDLFQDVDQVNPNDEYYK, GTEGGFPFQFFVFVYPFNADSK, AINFVGNYWQDNADLYGEEVTK, DTQGFVVPAPYEVYPQFFANLNTMLK, AINFVGNYWQDNADLYGEEVTKDYQR, KDTQGFVVPAPYEVYPQFFANLNTMLK,

matched peptide(s)

Iron Transport and Metabolism (4) 1 6.98 75222.87 M. sexta AMSVFAFSR, IPNQDFVVFQEYR, 2 6.98 75222.87 M. sexta ALSTFFAK, IPLTMLMK, AMSVFAFSR, GHGAPELVVR, DLPINNLDQLK, IPNQDFVVFQEYR, SVQDNGSDLASVDDMR, QLADSGAADKPEWFTK, CDYPDNYSGYEGALR, KYNLHPVFHEVYGELK, IHHVADNIPIKPIDYLNK, DIRPILDCVQENSEDACLK, ACSWAARPWQGLIGHNDVLAK, KFFGLPVGTTPASPSNENPEEFR, NLGEFFSGGSCLPGVDKPENNPSGDDVSK, DFLSDVSIAHTPLSLAQMLATRPDLFNIYGEFLK 3 6.98 75222.87 M. sexta ALSTFFAK, AMSVFAFSR, IHHVADNIPIKPIDYLNK 5 5.44 26507.66 M. sexta NLAGHTTDLKR

insecticyanin B form precursor (blue biliprotein) (INS-b)

101 gi|7511788 apolipophorin precursor protein 198 gi|84819 apolipophorin III precursorb 728 gi|9718 insecticyanin B form precursor (blue biliprotein) (INS-b)b

protein name

2 2 11

acc. no.

830 gi|7511788 apolipophorin precursor protein

score

Transport and Metabolism (19)

13

peptide

Table 1. Continued


research articles


944

score

1077

394

236

115 412

2082

550

248

53 220

peptide

17

6

4

2 6

31

10

4

1 4

Table 1. Continued


gi|114240 gi|228382

gi|114240

gi|114240

gi|114240

gi|1168527 gi|114240

gi|1168527

gi|1168527

gi|1168527

acc. no.

6 1 2

3

4 6 1

arylphorin R chain precursor



arylphorin R chain precursor Met-rich storage protein SP1Ab

5

4

3

fraction

arylphorin β chain precursor arylphorin R chain precursorb




protein name

6.1 9.13

6.1

6.1

6.1

5.95 6.1

5.95

5.95

5.95

pI

83866.31 29547.46

83866.31

83866.31

83866.31

83848.83 83866.31

83848.83

83848.83

83848.83

MW

Storage (18)

M. sexta M. sexta

M. sexta

M. sexta

M. sexta

M. sexta M. sexta

M. sexta

M. sexta

M. sexta

species

matched peptide(s)

VVDVVVDKLVTFFEYYDFDASNSVFWSK, DLAPFEAFIQDNKPLGYPFDRPVVDAYFK, LSYFTEDIGLNSYYYYFHSHLPFWWNSER, VVDVVVDKLVTFFEYYDFDASNSVFWSKEEVK IDFHDFK, NSNDFLIFK, AVEEFLQLYR, FFELDWFTYK, DVFVYHDGEYFPYK, MQDGVVSADLAAQHGIVK, LSNGLGDIPEFSWYSPVK, VLDLFQDVDQVNPNDEYYK, FLDTYEMSFLQFLQNGHFK, LLEQNKVPHDMSEDYGYLPK, NSNDFLIFKDDSVPMTELYK, AINFVGNYWQDNADLYGEEVTK, AINFVGNYWQDNADLYGEEVTKDYQR, KDTQGFVVPAPYEVYPQFFANLNTMLK, VVDVVVDKLVTFFEYYDFDASNSVFWSK, DLAPFEAFIQDNKPLGYPFDRPVVDAYFK, VVDVVVDKLVTFFEYYDFDASNSVFWSKEEVK VKDVDAAFVER, AVEEFLQLYR, AFDQKIDFHDFK, EYNIEANIDNYSNKK, IGKEYNIEANIDNYSNKK, AINFVGNYWQDNADLYGEEVTK SSYPHDFK, AVEEFLQLYR, EYNIEANIDNYSNKK, MQDGVVSADLAAQHGIVK AVEEFLQLYR, EYNIEANIDNYSNKK VVEDFLLLYR, HVLGAAPKPFNK, FYELDWFVHK, AFDKEINFHDVK, NVNQLDYEAEYYK, QSSDFLFFK SYEINAR, KVFSLFK, YGPFKER, DFETFYK, MQDGFLNK, IINYINEFK, IKMQDGFLNK, TSPVDAIFVEK, FNVPFYVPQK, DIKNFPYGYK, VVEDFLLLYR, HVLGAAPKPFNK, GFEFSIFYER, DPVFYQLYDR, FYELDWFVHK, KVVEDFLLLYR, LNHKPFSVSIGVK, AFDKEINFHDVK, DYDVEANIDNYSNKK, YSFIPSALDFYQTSLR, FLDMFEMTFLQYLQK, IGKDYDVEANIDNYSNK, FLDMFEMTFLQYLQK, QYLQPYNQNDLHFVGVK, IGKDYDVEANIDNYSNKK, DGYPFQLFVFVYPYQAVPK, QSSDFLFFKEDSLPMSEIYK, AVNFVGNYWQANADLYNEEVTK, QPNMHFEDVHVYHEGEQFPYK, SIVPDSKPFGYPFDRPVHPEYFK, YSFIPSALDFYQTSLRDPVFYQLYDR QSSDFLFFK, VVEDFLLLYR, GFEFSIFYER, YDSNGFPIPLAK, FYELDWFVHK, KVVEDFLLLYR, NVNQLDYEAEYYK, FLDMFEMTFLQYLQK, AVNFVGNYWQANADLYNEEVTK, SIVPDSKPFGYPFDRPVHPEYFK SDVAVDAVFK, VVEDFLLLYR, NVNQLDYEAEYYK, AVNFVGNYWQANADLYNEEVTK NVNQLDYEAEYYK VTLDVLSDK, MPLGFPLDR, NSYDMHNLVR, LVDNVDMSILDTTR


127

2

acc. no.

score

108

2

62

1

peptide

49

score

protein name

6

3

3 1 2

2

fraction

gi|32423714

3

Actin

fraction

b

5.3

pI

3 3 4

fraction

88377.23

88377.23

29547.46 88377.23 88377.23

8.1 7.64 5.39

pI

3

1

41824.76

MW

species

pI

Haemaphysalis longicornis

5.31

6.97

48086.76 48144.25 25410.99

MW

M. sexta

M. sexta

M. sexta M. sexta M. sexta

M. sexta

species

fraction

Cell Structure (1)

proteasome 26S subunit, ATPase 3, interacting proteinb CG12214-PA, isoform Ab

protein name

gi|19921964

gi|25092696

protein name

MW

29547.46

Metamorphosis (3)

8.71

8.71

9.13 8.71 8.71

9.13

pI

Storage (18)

Protein-Protein Interaction (2)

imaginal disk growth factor IDGF-like proteinb juvenile hormone-binding protein precursorb

b

protein name

methionine-rich storage protein 1

methionine-rich storage protein 1

Met-rich storage protein SP1A methionine-rich storage protein 1b methionine-rich storage protein 1

Met-rich storage protein SP1A

acc. no.

acc. no.

gi|47607477 gi|10801564 gi|400079

acc. no.

gi|159526

gi|159526

gi|228382 gi|159526 gi|159526

gi|228382

1

peptide

222 147 134

432

7

3 2 2

59 168 1605

1 3 24

score

755

11

peptide

score

peptide

Table 1. Continued

matched peptide(s)

matched peptide(s)

Drosophila melanogaster

Mus musculus

species

LEKLVGLAPNK

AEAAAGAPGIILR

matched peptide(s)

STWGSIWHGIK, YNLLLESPQAR, MVPLNENLDVDR EGFTALVR, STWGSLWHGIK YSYDLQDDSK, IVELCYVDVVHNIR

matched peptide(s)

SYELPDGQVITNGNER, TTGIVLDSGDGVSHTVPIYEGYALPHAILR

53142.67

24748.09

MW

Pieris rapae Bombyx mori M. sexta

species

LIDINVNR, VTLDVLSDK, MPLGFPLDR, FVDVVIFHK, FVDVVIFHKK, LNFVEMDTFIYK, HIDVSTFYTPNMK, LVDNVDMSILDTTR, KVDTITDMRDMLIK, LVDNVDMSILDTTRK, STVLLDKMPLGFPLDR FVDVVIFHK YNNMIVVTDENLK, QIDMTYFFTNNMK, QGGLPLQLYVIVSPVR DGTVIHLK, VNIDVVSDK, VTNTFVMFK, HSDMIYVAR, NIDTSNMVMK, YTHEELDFPGVK, VTDKNLVVIDWR, TDVVKEFINMFK, LDMVEIDSFLYK, AVTDPVVANYYGIK, TGMVLPTIDMNTMK, YNNMIVVTDENLK, RLDMVEIDSFLYK, EYVLEENTDKYLK, QIDMTYFFTNNMK, NIWDKTFDNSGSGFK, DDLTYLDSDMLMHR, QGGLPLQLYVIVSPVR, TYKDVMMMSSDNMLR, LLNHILQPTIYEDIR, KDDLTYLDSDMLMHR, FTVCFDTMPLGFPFDR, LDMVEIDSFLYKLETGK, LLNHILQPTIYEDIREVAR LDMVEIDSFLYK, AVTDPVVANYYGIK, YNNMIVVTDENLK, RLDMVEIDSFLYK, QIDMTYFFTNNMK, QGGLPLQLYVIVSPVR, NSLEMHGVIEQRPWIR DDLTYLDSDMLMHR, KDDLTYLDSDMLMHR


research articles


946

164 49 48 56

3 1 1 1

gi|25090512 gi|54640661 gi|24585053 gi|7511167

acc. no. b

hemolymph glycoprotein precursor GA10602-PAb CG10600-PAb hypothetical protein ZK484.4b

protein name

5 3 2 5

fraction

5.88 7.87 8.99 8.93

pI

Unclassified (4)

25941.08 37588.38 150552.97 98458.7

MW

M. sexta D. melanogaster D. melanogaster Caenorhabditis elegans

species

NSFPSVEAAK, GELDEVFKK, GIVDPQAIKK QKSCTLEDIACLK GGENNVELANGGK ENLNKLLEK

matched peptide(s)


insecticyanin B form precursor (blue biliprotein) (INS-b) apolipophorin-3 precursor (apolipophorinIII) (ApoLp-III) hypothetical protein lysozyme 50S ribosomal protein L24 inducible serine protease inhibitor 1 (ISPI-1) (fragment)

13

16

15

14

5

4

3

arylphorin subunit R precursor arylphorin subunit β precursor lipoprotein-releasing system transmembrane protein lolC serpin 1

protein name

2

band no.

eekyteenddldieqvikda khfsrxllvhelrwggfpen dlvpgsnyvkqhprgspiaq dlvxgsnyxkqhpxgspiaq

dapaggnafeemekhakefq

gdifypgyxpdvkpvddfdl

qetdlqkilresndqftaqm

tpasv-mrxlliqyas

skvpvkhsfkvkdydanfye

savpvkhyqhhykvs

amino acid sequence

4.93/13847.87 8.91/16087.31 10.28/11404.26 cannot be computed

6.98/20793.37

6.29/23182.27

5.01/44380.96

7.04/45234.25

5.95/83848.83

6.10/83866.31

pI/MW (Da) theoretical

Q3LB46 Q26363 Q2L282 P81905

P13276

Q00630

Q25494

Q87EF5

P14297

P14296

acc. no.

121 138 106 50

189

206

398

413

703

702

length

95 80 50 57.895

100

95

100

50

80

73.333

identity %

95 80 77.778 78.947

100

95

100

91.667

85

86.667

similarity %

Table 2. M. sexta Hemolymph Proteins Separated by 1-DGE and Their Identification with N-Terminal Amino Acid Sequencing

unclassified lysozyme ribosomal protein serine protease inhibitor

lipid transport and metabolism

serine protease inhibitor lipid transport and metabolism

lipid transport and metabolism

storage

storage

functional category

M. sexta M. sexta Bordetella avium (strain 197N) Galleria mellonella (Wax moth)

M. sexta

M. sexta

M. sexta

Xylella fastidiosa (strain Temecula1/ATCC 700964)

M. sexta

Manduca sexta

species

a Identified proteins from hemolymph of M. sexta were classified into functional classes. The table lists peptide, score, accession no., protein names, fraction no., theoritical pI, theoritical mass (kDa), species, and matched peptides. b Nonredundant.

score

peptide

Table 1. Continued


5.1/45

5.2/45

5.3/45

5.4/45

serine protease inhibitora

serine protease inhibitora

serpin 1a

serpin 1a

serine protease inhibitor



18

19

20

21

22

23

24

5.8/45

5.9/45


serpin 1a

25

26

5.8/45

5.6/45

5.5/45

pI/MW (kDa) exptl

protein name

spot no.

5.88/44032.88

5.56/43683.28

5.56/43683.28

5.56/43683.28

5.56/43683.28

5.25/43457.91

5.19/43941.50

5.56/43683.28

6.15/35117.36


queries matched score

gi|1378125

gi|431347

gi|431347

gi|431347

gi|431347

gi|1378128

gi|1378127

gi|431347

26

32

26

18

6

23

21

20

144

293

349

170

124

209

632

234

45

50

46

31

17

37

38

35

30

sequence coverage (%)

Serine Protease Inhibitor (11) gi|431335 11 401

acc. no.

Defense (27)

matched peptide(s)

DVFADLNR, GLELNDDFAAVSR, DVFGSEVQNVDFVK, SVEAAGAINK, NLVDPDALDETTR, SVLVNAIYFK, VPTMIGKK, YADVPELDAK, KLFTPGAAR ESNDQFTAQMFSEVVK, GVDLK, GLELNDDFAAVSR, DVFGSEVQNVDFVK, SVEAAGAINK, IKNLVDPDALDETTR, SVLVNAIYFK, DKFVK, TMDRDFHVSK, TIKVPTMIGK, YADVPELDAK, LYNTEVEIYLPK, KLFTPGAAR ESNDQFTAQMFSEVVK, DVFADLNR, GVDLK, DVFGSEVQNVDFVK, SVEAAGAINK, NLVDPDALDETTR, SVLVNAIYFK, DKFVK, TMDRDFHVSK, TIKVPTMIGK, YADVPELDAK, IETTTDLK, EVLSNMNIK, KLFTPGAAR, SNGQHLFNGICFQP ESNDQFTAQMFSEVVK, DVFADLNR, GVDLK, SVEAAGAINK, IKNLVDPDALDETTR, NLVDPDALDETTR, SVLVNAIYFK, DKFVK, TMDRDFHVSK, VPTMIGK, YADVPELDAK, LYNTEVEIYLPK, IETTTDLK, EVLSNMNIK, KLFTPGAAR, QDKTTLFSGVFQS, TTLFSGVFQS YADVPELDAK, NLVDPDALDETTR, LYNTEVEIYLPK, DVFGSEVQNVDFVK, ESNDQFTAQMFSEVVK ESNDQFTAQMFSEVVK, GLELNDDFAAVSR, DVFGSEVQNVDFVK, NLVDPDALDETTR, SVLVNAIYFK, TIKVPTMIGK, YADVPELDAK, LYNTEVEIYLPK, TKESLYVDAAIQK, ITTYSFHFVPK DVFADLNR, GVDLK, DVFGSEVQNVDFVK, IKNLVDPDALDETTR, NLVDPDALDETTR, SVLVNAIYFK, DKFVK, TMDRDFHVSK, YADVPELDAK, LYNTEVEIYLPK, IETTTDLK, IETTTDLKEVLSNMNIK, EVLSNMNIK, KLFTPGAAR, TKESLYVDAAIQK, AFIEVNEEGAEAAAANAFK, ITTYSFHFVPK, VEINKPFFFSLK, NSMFSGVCVQP ESNDQFTAQMFSEVVK, DVFADLNR, GVDLK, DVFGSEVQNVDFVK, SVEAAGAINK, IKNLVDPDALDETTR, NLVDPDALDETTR, SVLVNAIYFK, DKFVK, TMDRDFHVSK, TIKVPTMIGK, VPTMIGK, YADVPELDAK, LYNTEVEIYLPK, FKIETTTDLK, IETTTDLKEVLSNMNIK, EVLSNMNIK, EVLSNMNIKK, KLFTPGAAR, LFTPGAAR, TKESLYVDAAIQK, AFIEVNEEGAEAAAANAFK, ITTYSFHFVPK ESNDQFTAQMFSEVVK, DVFADLNR, AVKGVDLK, GVDLK, SVEAAGAINK, WVEDQTNNR, IKNLVDPDALDETTR, SVLVNAIYFK, TMDRDFHVSK, TIKVPTMIGK, YADVPELDAK, LYNTEVEIYLPK, FKIETTTDLK, EVLSNMNIK, KLFTPGAAR, TKESLYVDAAIQK, VIPPVLK, ANDQSLFNGICLQP

Table 3. M. sexta Hemolymph Proteins Separated by 2-DGE and Their Identification with nLD-IT-TOF-MS/MS and nESI-LC-MS/MS


research articles


948

Journal of Proteome Research • Vol. 7, No. 3, 2008 6.7/49

hemolina

hemolin immulectin-2 [M. sexta]a immulectin-3 [M. sexta]a

immulectin-3 [M. sexta]

immulectin-3 [M. sexta]

peptidoglycan recognition protein 1B [M. sexta]a peptidoglycan recognition protein 1B [M. sexta]

15

30 31 32

33

34

46

47

5.4/55

β-1,3-glucan recognition protein [M. sexta]

9

6.7/20

6.3/20

6.6/33

6.4/33

5.9/39 6.0/39 5.7/32

6.29/21530.35

6.29/21530.35

5.86/33867.46

5.86/33867.46

7.91/45665.21 6.10/37423.55 5.86/33867.46

7.91/45665.21

5.27/54567.17

5.27/54567.17

5.3/55

β-1,3-glucan recognition protein [Manduca sexta]a

8

5.56/43683.28

5.88/44032.88


5.3/42


28

6.1/45

pI/MW (kDa) exptl

serpin 1

protein name

27

spot no.

Table 3. Continued

21

35

queries matched

235

217

score

gi|27733409

gi|27733409

gi|55139125

gi|55139125

gi|511297 gi|9651929 gi|55139125

gi|511297

gi|7262927

14

1

12

9

3 2 12

10

16

129

64

191

110

79 88 130

174

178

Pattern Recognition (10) gi|7262927 13 112

gi|431347

gi|1378125

acc. no.

Defense (27)

37

10

30

24

8 9 34

23

25

18

42

44

sequence coverage (%) matched peptide(s)

LVIIQHTDTPGCDTDDACAAR, ITFIGSYNSK, ITFIGSYNSKEPNSQQLNAIK, EPNSQQLNAIK, CGVDNGHLSSDYK, CGVDNGHLSSDYKVVGHR, QLLDTDSPGR, QLLDTDSPGRK

LEAIYPK, CTGLLGTAQCK, DPSDAIIVPPIVTAK, TFAFK, VEISAK, NYGIR, NYVSGLLR, TLVGGPIMSEAEPYR, VTAPAGGFYK, ALLVDYVR LEAIYPK, VSIPDDGYTLFAFHGK, GQMLFEDNFNKPLADGR, RDPSDAIIVPPIVTAK, DPSDAIIVPPIVTAK, KTFAFK, TFAFK, VEISAK, NYGIR, VACVKGNTEYIK, GNTEYIK., TLVGGPIMSEAEPYR, VTAPAGGFYK, VTAPAGGFYKEANEQNVEAAAR, EANEQNVEAAAR DQPAEVLFR, KDEGSLVFLKPEAK, DEGSLVFLKPEAK, KPVEGSWLK, ITQSPEGDLYFTSVEK, YVCAAK, SPAIDGDVPLVGYTIK, NGELVPMYVSNDMIAK, VVASPSGLTIK ESQATVLECVTENGDKDVK, QLGEDESIADSILAR CHLEGAVLASPLNSNLK, GDFFSVEGIPLKK TRDMFTEEYSSGPHCAR, DMFTEEYSSGPHCAR, LIPQEGLVAGSCSDALPYICYK, TAELSMTECGTVDK, TAELSMTECGTVDKGYQLSAK, TGHCYK, ELLAR, YPTGLIK, SGQLDDIGCAK, VPFICEKHPNNIMPVPNNV, HPNNIMPVPNNV TAELSMTECGTVDK, CIAEGGQLAVINSAVEANVLK, ELLAR, YPTGLIK, SGQLDDIGCAK, VPFICEK, HPNNIMPVPNNV TRDMFTEEYSSGPHCAR, DMFTEEYSSGPHCAR, TAELSMTECGTVDKGYQLSAK, CIAEGGQLAVINSAVEANVLK, ELLAR, SGQLDDIGCAK, VPFICEKHPNNIMPVPNNV LVIIQHTDTPGCDTDDACAAR

DVFADLNR, GVDLK, DVFGSEVQNVDFVK, SVEAAGAINK, IKNLVDPDALDETTR, NLVDPDALDETTR, SVLVNAIYFK, DKFVK, TMDRDFHVSK, TIKVPTMIGK, VPTMIGK, YADVPELDAK, LYNTEVEIYLPK, FKIETTTDLK, IETTTDLK, IETTTDLKEVLSNMNIK, KLFTPGAAR, LFTPGAAR, LENLLK, TKESLYVDAAIQK, ESLYVDAAIQK, VIPPVLK, ANDQSLFNGICLQP ESNDQFTAQMFSEVVK, DVFADLNR, GLELNDDFAAVSR, DVFGSEVQNVDFVK, IKNLVDPDALDETTR, NLVDPDALDETTR, SVLVNAIYFK, TMDRDFHVSK, YADVPELDAK, LYNTEVEIYLPK, FKIETTTDLK, IETTTDLKEVLSNMNIK, LFTPGAAR, TKESLYVDAAIQK, ESLYVDAAIQK, AFIEVNEEGAEAAAANAFK


ommochrome-binding protein precursor (OBP) (YCP)a

35

multicystatin [M. sexta]a

53

protein name

6.7/13

lysozyme [M. sexta, peptide partial, 120 aa]a

49

spot no.

6.8/16

serine protease-like protein [M. sexta]a

29

5.8/30

pI/MW (kDA) exptl

6.4/41

6.2/49

serine proteinase-like protein 2 [M. sexta]a

14

acc. no.

queries matched

Defense (27)

score

135

300

56

Lysozyme (1) 16 123

11

25

12

queries matched

score

matched peptide(s)

MGYLNLATK, SFGEISGVK, DGMATAVDTTNHIVYLGGK, DGIYTYDYATK, LYFSSPVGFYAVNEADR, LYFSSPVGFYAVNEADRK

matched peptide(s)

YLQSIGSTKPHK, EITVDCK, EITVDCKINNQK

KHFSR, CELVHELR, CELVHELRR, QGFPENLMR, DWVCLVENESSR, YTDKVGR, NGSRDYGLFQINDK, CSDLLIDDITK, CSDLLIDDITKASTCAK, NHCQGSLPDISSC

IFIAPK, WQDLK, LAYWR, FSDWR, IPEFPK, EAAAVIPK, GLDFSDR, TPIIIPR, IEQPDGR, DPFFYR, LDSLTSAR, LCNSLKR, MFIEMDR, FGDEEEVSR, KMFIEMDR, LSDVTEPNPR, FGDEEEVSRK, NLPWALSDQR, NLDKIPEFPK, EPIPEAYYPK, DLSIQGSDPRR, VQNYAEIFPAK, FLDSQVFTQAR, FTHLNHRPFR, EAPHNVRPYSR, NLPWALSDQRK, GLDFSDRGPVYAR, DYTATDLEEEHR, FVVPLSAGENTITR, VHAWVDDIFQSFK, ELSCVEASMFCGLK, NIEEAIATGNVILPDK, RPVDGLNVTIDDMER, YLESFGVIADEATTMR, QSTESSLTIPFEQTFR, ELSCVEASMFCGLKDK, FSDWREPIPEAYYPK, NIEEAIATGNVILPDKSTK, KLDIDMLGNMMEASVLSPNR, INLNPQLFNYCYTVAIMHR, MFIEMDRFVVPLSAGENTITR, SELAAFNYCGCGWPQHMLVPK, YLESFGVIADEATTMRDPFFYR, GEDKVLFELTEQFLTPEYANNGLELNNR CFASGWGK, YQVILKK, KIEVPVVDR, IEVPVVDR, DTCKGDGGSPLVCPSEYEK, GDGGSPLVCPSEYEK, GDGGSPLVCPSEYEKDR, HGEYCQCVPYYLCK, DGLTINDPTLDGNGLLDIR, FGEDENNDRLCQESVER, LCQESVER, VLKDPSEVVKPKPDPSK, AGEWDTQTIKEELEHQER, DVQEILIHK, DFKPLSLK, NDIALLR, NCVANGWGK, NVFGVQGLYAVILK, KVEQDMVPHSR, VEQDMVPHSR, CNTQLQK, DTCQGDGGAPLACPIGNNR, HWVDENMNK, WGYGSSTYSV DNYAVALGK, SDVKEIHIPPQFK, LQLKDGIMGK, DGIMGK, ETLKLSK, VVENPYIDAATCISESPASFR, GDGGAGVAFPSQEQGVQR


Protein (Amino Acid) Transport and Metabolism (5) 5.89/30614.72 gi|400673 12 205 24

acc. no.


3

71

25

47

11

71


Cystatin Protease Inhibitor (1) gi|110225182 3 54

gi|233964

gi|27733421

gi|21630233

gi|56418466

Pro-phenol Oxidase (4) gi|75038472 66 2212

pI/MW (kDA) theoretical

8.81/70612.02

8.94/13983.80

6.30/33065.59

5.83/43488.52

6.10/43149.26

5.2/51

serine proteinase-like protein 4 [M. sexta]a

12

5.68/80060.70


5.5/68

pI/MW (kDa) exptl

phenoloxidase subunit 2 (proPO-p2)a,b

protein name

3

spot no.

Table 3. Continued


research articles


950

ommochrome-binding protein precursor (OBP) (YCP)

ommochrome-binding protein precursor (OBP) (YCP) ommochrome-binding protein precursor (OBP) (YCP) ommochrome-binding protein precursor (OBP) (YCP)

36

37

Journal of Proteome Research • Vol. 7, No. 3, 2008 6.7/84

6.7/68

transferrin precursora,b

transferrin precursorb

4

5.7/27

5.5/27

6.3/30

6.1/30

pI/MW (kDa) exptl

2

39

38

protein name

spot no.

Table 3. Continued

6.98/75222.87

6.98/75222.87

5.89/30614.72

5.89/30614.72

5.89/30614.72

5.89/30614.72

pI/MW (kDa) theoretical

2

6

22

15

queries matched

107

133

221

178

score

12

18

22

31


gi|136206

98

3656

74

Iron Transport and Metabolism (3) gi|136206 46 1773 60

gi|400673

gi|400673

gi|400673

gi|400673

acc. no.

Transport and Metabolism (8))

matched peptide(s)

TWVAAK, WSPDPK, VLGLSEK, ALSTFFAK, YLCVDGSK, IPLTMLMK, IPLTMLMK, VALECVPAR, AMSVFAFSR, MNDHSISPK, GHGAPELVVR, LCVPAAYMK, YEAVIVVHK, TPNYAVAVVK, ANYTEVIER, QCGSDSSAWK, TDEEPDAPFR, LCVTSNVALSK, NNNVIFNNAAK, TIHDVISSCGLA, DLPINNLDQLK, DCEQMLEVPTK, GLATTEKLDFEK, CLAHNNGEVAFTK, DRVECLSFVQQR, IPNQDFVVFQEYR, SVQDNGSDLASVDDMR, QLADSGAADKPEWFTK, CDYPDNYSGYEGALR, DVLSSFATCNVAMAPSR, QADFVPVDPEDMYVASK, DIRPILDCVQENSEDACLK, FFGLPVGTTPASPSNENPEEFR, KFFGLPVGTTPASPSNENPEEFR, NLGEFFSGGSCLPGVDKPENNPSGDDVSK IDDLR, LDFEK, TWVAAK, RAVFPK, WSPDPK, VLGLSEK, LAPLREK, ALSTFFAK, IPLTMLMK, VALECVPAR, AMSVFAFSR, MNDHSISPK, GHGAPELVVR, LCVPAAYMK, AIQSDHCVK, YEAVIVVHK, TPNYAVAVVK, QCGSDSSAWK, TDEEPDAPFR, TPNYAVAVVKK, LCVTSNVALSK, NNNVIFNNAAK, SKVALECVPAR, KQCGSDSSAWK, VECLSFVQQR, GTAYNKIDDLR, DLPINNLDQLK, VALECVPARDR, DCEQMLEVPTK, GLATTEKLDFEK, CLAHNNGEVAFTK, YLCVDGSKAPITGK, DRVECLSFVQQR, FKTIHDVISSCGLA, MNDHSISPKENELK, IPNQDFVVFQEYR, SVQDNGSDLASVDDMR, QLADSGAADKPEWFTK, CDYPDNYSGYEGALR, DVLSSFATCNVAMAPSR, YNLHPVFHEVYGELK, QADFVPVDPEDMYVASK, SQYSHLCSMCEHPER, VKQLADSGAADKPEWFTK, TDEEPDAPFRYEAVIVVHK, SVQDNGSDLASVDDMRVAAAAK, DIRPILDCVQENSEDACLK, DAGGRDVLSSFATCNVAMAPSR, ACSWAARPWQGLIGHNDVLAK, FFGLPVGTTPASPSNENPEEFR, LCVPAAYMKDCEQMLEVPTK, KFFGLPVGTTPASPSNENPEEFR, SCHSSYSTFSGLHAPLFYLINK, IPNQDFVVFQEYRTDEEPDAPFR, NLGEFFSGGSCLPGVDKPENNPSGDDVSK

DGMATAVDTTNHIVYLGGK, VIDLGDYNVNAFTK

TVLKMGYLNLATK, MGYLNLATK, SFGEISGVK, DGMATAVDTTNHIVYLGGK, DGIYTYDYATK, GKLYFSSPVGFYAVNEADR, LYFSSPVGFYAVNEADR, DDNIVYSNEDNIVK MGYLNLATK, SFGEISGVK, DGIYTYDYATK, VIDLGDYNVNAFTK, GKLYFSSPVGFYAVNEADR, LYFSSPVGFYAVNEADR, LYFSSPVGFYAVNEADRK DGMATAVDTTNHIVYLGGK, VIDLGDYNVNAFTK, LYFSSPVGFYAVNEADR, LYFSSPVGFYAVNEADRK




44

45

5

HSC70a

protein name

arylphorin β subunit precursor

51

spot no.

arylphorin β subunit precursor

arylphorin subunit R precursora,b arylphorin β subunit precursora

17

16

1

protein name

insecticyanin B form precursor (blue biliprotein) (INS-b)a

42

spot no.

transferrin precursor

protein name

6

spot no.

Table 3. Continued

5.50/71863.38


pI/MW (kDa) exptl

6.0/65

5.95/83830.79

5.95/83830.79

5.95/83830.79

5.4/15

6.3/47

6.2/47

6.10/83866.31


pI/MW (kDa) exptl

5.6/114

6.29/23182.27

6.29/23182.27

6.29/23182.27

6.98/75222.87


6.7/21

6.6/23

6.3/24

6.7/61

pI/MW (kDa) exptl

8

queries matched

225

score

8


gi|1495233

acc. no.

gi|159491

gi|159491

gi|159491

gi|114240

acc. no.

gi|9718

gi|9718

18

queries matched

87

264

349

score

95

537

343

65

score

Chaperone (1)

5

19

11

4

queries matched

Storage (4)

11

41

26


5

19

12

4


33

64

Lipid Transport and Metabolism (3) gi|9718 16 123 45

gi|136206

acc. no.


matched peptide(s)

NTTIPTK, VAYKGEDK, STMEPVEK, MVNHFVQEFK, NALESYCFNMK, SQIHDIVLVGGSTR, TTPSYVAFTDTER, TFFPEEVSSMVLTK, QKELEGICNPIITK, IINEPTAAAIAYGLDK, NQVAMNPNNTIFDAK, IINEPTAAAIAYGLDKK, NQVAMNPNNTIFDAKR, TVQNAVITVPAYFNDSQR, GEDKTFFPEEVSSMVLTK, QTQTFTTYSDNQPGVLIQVFEGER

matched peptide(s)

AFDQKIDFHDFK, SYEIIAR, QVLGAAPKPFDK, YIYEYK, QYLQPYSSEK, VVDVVVDK, SSYPHDFK, IFMAPK, FVAGDNK, NSNDFLIFK SYEIIAR, QVLGAAPKPFDK, QYLQPYSSEK, VVDVVVDK, SSYPHDFK, SEAAVDAVVK, IFMAPK, YDDNGFPLK, FVAGDNK, FVAGDNKIVR, NSNDFLIFK, LLEQNKVPHDMSEDYGYLPK, LMLPR, QHNMFFK, FNVPSHVMHSNVVPK DVDAAFVER, EYNIEANIDNYSNK, EYNIEANIDNYSNKK, AVEEFLQLYR, TGFLPK

IFLGPK, TGFMPK, SYEINAR, DFETFYK

matched peptide(s)

LPLENENQGK, CTIAEYK, CTIAEYKYDGK, EYMEGDLEIAPDAK, YVMTFK, NYAINYNCNYHPDK, NYAINYNCNYHPDKK, AHSIHAWILSK, SKVLEGNTK, VLEGNTKEVVDNVLK, EVVDNVLK, TFSHLIDASK LPLENENQGK, CTIAEYK, CTIAEYKYDGK, ASVYNSFVVNGVK, EYMEGDLEIAPDAK, YVMTFK, VVNLVPWVLATDYK, NYAINYNCNYHPDK, NYAINYNCNYHPDKK, VLEGNTK, EVVDNVLK, TFSHLIDASK, FISNDFSEAACQYSTTYSLTGPDR, FISNDFSEAACQYSTTYSLTGPDRH LPLENENQGK, CTIAEYK, EYMEGDLEIAPDAK, YVMTFK, NYAINYNCNYHPDK, EVVDNVLK, TFSHLIDASK

ALSTFFAK, CLAHNNGEVAFTK, YLCVDGSK, VLGLSEK, TPNYAVAVVK, AIQSDHCVK, TWVAAK


research articles


952


a

Nonredundant.

b

N.D.c

7.08/47827.53


gi|85726208

acc. no.

4

43

score


Unclassified (9)

5

queries matched

Metamorphosis (1)

gi|62241290

129

score

queries matched

Cell Fate (1)

22

queries matched

Cell Structure (1)

acc. no.

gi|576368

acc. no.

pI/MW (kDa) exptl

5.25/55103.26


5.29/41828.20


protein name

6.5/51

pI/MW (kDa) exptl

5.6/54

pI/MW (kDa) exptl

5.5/45

pI/MW (kDa) exptl

NanoLD/Linear-Trap-TOF-MS. c N.D. ) not determined.

7, 11, 40, 41, 43, 48, 50, 52, 54

spot no.

imaginal disk growth factor-like protein [Mamestra brassicae]a

protein name

spot no.

13

protein disulfide-isomerase like protein ERp57 [Bombyx mori]a

protein name

chain A, β-Actin-profilin complexa

protein name

10

spot no.

22

spot no.

Table 3. Continued

77

9

acc. no.

11

matched peptide(s)

queries matched

score


matched peptide(s)

MVPLNENLDVDR, AITNFK, YPGLK, VLLSVGGDVDTEDADK, GLCTGDKYPILR

matched peptide(s)

STCEQFSVSGYPTLK, KGELSSEYNGPR, ESDLKGEFLK, ENYHGLVGVR

matched peptide(s)

GDDDIAALVVDNGSGMCK, AGFAGDDAPR, AVFPSIVGRPR, HQGVMVGMGQK, DSYVGDEAQSKR, LDLAGR, DLTDYLMK, GYSFTTTAER, MQKEITALAPSTMK, EITALAPSTMK, QEYDESGPSIVHR



score

30



research articles

Investigation of the Hemolymph Proteome of Manduca sexta follows: ESI voltage, +1.3 kV; curtain (nitrogen) gas flow rate, 1.0 L/min without heating; scan range, m/z 200–2000. Nano Frontier Data Processing P/N:3807051-01 Hitachi HighTechnologies Corp. software was used for generating a peaklist. Acquired data were searched against the NCBInr 20061227-1 (4 318 227 sequences; 1 487 489 705 residues) protein database (Lepidoptera, M. sexta; 452 sequences) using the MASCOT software package (version 2.1, Matrix Sciences). Parameters used were peptide mass tolerance (0.5 Da), MS/MS ion mass tolerance (0.5 Da), allowance of one tryptic miscleavage, differential modification (methionine as oxidation), and default selection of charge states ions (+1, +2, and +3). The cutoff score/expectation value for accepting individual MS/MS spectra was 60, which indicates identity or extensive homology of probability lower than 0.05 (p < 0.05). The threshold employed was -10* log(P), where P is the probability that the observed match is a random event; protein scores are derived from ion scores as a nonprobabilistic basis for ranking protein hits. For peptides matching to multiple members of a protein family, we focused on the unique peptide of the identified proteins. The unique peptide of each protein was manually selected to reduce the multimatching peptide mistake to regenerate the protein identification list. By eliminating peptides having scores lower than the applied cutoff score of 42, it was possible to have an unambiguous identification of isoforms/individual members of a protein family. Multiple protein IDs having the same accession number were considered as a single nonredundant protein for the protein list provided in this study. The peptide sequences were listed along with the corresponding proteins (Table 3). 2.7. nESI-LC-MS/MS and Protein Identification. The peptides in 20 µL were used for mass spectral analysis by LC-MS/ MS through an nESI source. Briefly, online capillary LC included a monolithic reverse-phase trap column (0.2 mm × 5 cm, MonoCap for fast-flow, GL Science, Tokyo, Japan) and a fast-equilibrating C18 capillary column (monolith-type column; i.d, 0.1 mm; length, 50 mm; GL Science). The sample was loaded onto peptide traps for concentration and desalting prior to final separation by the C18 column using a linear acetonitrile gradient ranging from 5% to 65% solvent B [H2O/acetonitrile/ formic acid, 10:90:0.1 (v/v)] in solvent A [H2O/acetonitrile/ formic acid, 98:2:0.1 (v/v)] for a duration of 40 min. The m/z ratios of eluted peptides and fragmented ions from a fusedsilica Fortis Tip emitter (150 µM o.d., 20 µM i.d.; AMR Inc., Tokyo, Japan) were analyzed in the data-dependent positive acquisition mode on LC-MS/MS. Following each full scan (400–2000 m/z), a data-dependent triggered MS/MS scan for the most intense parent ion was acquired. The heated fusedsilica Fortis Tip emitter was held at ion sprays of 1.8 kV and a flow rate of 300 nL/min. Xcalibur version 1.1 (Thermo Fisher Scientific K.K.) software was used for generating a peaklist. Acquired data were searched against the NCBInr protein database (nrdb90) for an animal sequence using the MASCOT software package (version 2.1, Matrix Sciences). The NCBInr database contained 3 947 950 sequences and 1 358 419 857 residues, and the taxonomy was Metazoa (animal; 967 093 sequences). Parameters used were peptide mass tolerance (2.0 Da), MS/MS ion mass tolerance (0.8 Da), allowance of one tryptic miscleavage, differential modification (methionine as oxidation) and static modification (cysteine as carbamidomethylation), default selection of charge state ions (only +2, +3), and a minimum of two nonredundant peptides. The cutoff score/expectation value for accepting

individual MS/MS spectra was 39, which indicates identity or extensive homology of probability lower than 0.05 (p < 0.05). The threshold employed was -10* log(P), where P is the probability that the observed match is a random event; protein scores are derived from ion scores as a nonprobabilistic basis for ranking protein hits. When peptides matched to multiple members of a protein family, the unique peptide of the identified proteins was considered. The unique peptide of each protein was manually selected to reduce the multimatching peptide mistake to regenerate the protein identification list. For unambiguous identification of isoforms/individual members of a protein family, the experimental molecular mass and pI of the protein spot on 2-D gel was taken into account. Single nonredundant proteins were assigned for multiple protein IDs with the same accession number. The peptide sequences were listed along with the corresponding proteins (Table 3).

3. Results and Discussion Studies on hemolymph of M. sexta have dealt mostly with the purification and identification of proteins of interest.25–30 Although some insect hemolymph proteomics studies have been performed, such as those for Bombyx mori L and Drosophila melanogaster,9,31,32 a proteome-scale investigation aimed at creating a hemolymph proteome of M. sexta is entirely lacking. Our own interest in hemolymph proteins, especially in the mechanism for insect defense and metamorphosis, led us to embark on the present study. 3.1. Experimental Strategy. The experimental strategy for a systematic analysis of the hemolymph proteome is presented in Figure 1. The life cycle of M. sexta begins with an egg. Briefly, eggs are spherical (approximately 1.4 mm in diameter) and translucent blue-green to yellow-green in color and typically hatch 2–4 days after they are laid. The larvae feed only on solanaceous plants and normally undergo development from the first to fifth instar larvae stages. Larval development time is on average about 20 days. The fifth instar larval body length and weight, respectively, is about 11 times (an average of 7.0-80.0 mm) and 1000 times (an average of 1-1000 mg) that of the first instar larval. The pupal (brown or reddish brown, elongate-oval measuring 45.0-60.0 mm in length) stage is maintained for about 20 days. During the pupal stage, structures of the adult moth form within the pupal case that is shed during eclosion. The adult males and females are sexually dimorphic. The whole cycle can be completed within 30-50 days, but this varies due to environment.33 Hemolymph from fifth instar larvae was used for total protein extraction using TCA/acetone followed by solubilization in LB-TT to give the total soluble protein. The total protein was then used for 1-DGE and 2-DGE analyses. The tryptic peptides either from 1-DGE or 2-DGE were analyzed by LCMS/MS (nESI-LC-MS/MS or nLC-IT-TOF-MS/MS). Proteins were identified using the SwissProt/NCBI nonredundant protein database and MASCOT search engine. N-Terminal amino acid sequencing of protein bands blotted onto PVDF membranes from the SDS-PAGE gels was also performed to get additional information on separated protein bands. 1- and 2-DGE together identified 123 proteins, representing 58 nonredundant proteins (based on accession number, Figure 3). 1-DGE and 2-DGE separately identified 33 and 25 proteins, respectively, where only 12 proteins were common in both data sets. From these results, it is quite clear that different proteins are present and/or identified only by 1-DGE but not by 2-DGE. In all, there are 33 such proteins from 1-DGE. Twenty of the Journal of Proteome Research • Vol. 7, No. 3, 2008 953

research articles

Furusawa et al.

Figure 1. Experimental strategy and proteomics workflow. Starting with an egg, the emerged larvae normally undergo development from the first to the fifth instar larvae stages, followed by the pupal stage culminating in the adult moth (imago). The hemolymph from the fifth instar larvae was extracted and lyophilized before being processed for protein extraction (by grinding in liquid nitrogen and precipitating total proteins by TCA/acetone). The pellet was solubilized in LB-TT and used for 1-DGE and 2-DGE. The separated proteins on gel were stained with CBB R-250 (1-DGE) and colloidal CBB G-250 (2-DGE), excised from gels, in-gel tryptic digested for analyses by LC-MS/MS (nLD-IT-TOF-MS/MS or nESI-LC-MS/MS), and identified using the MASCOT search engine and SwissProt/NCBI nonredundant protein databases. We are grateful to Colin L. Miller ([email protected], Sierra Vista, AZ) for kind permission to use the M. sexta life cycle photographs presented in this figure.

33 proteins have a pI value of over 7.0, which are basic in nature, and thus may not be represented on the 2-D gel. Some other proteins were membrane proteins, which are not well 954


represented on 2-D gel, suggesting that 1-DGE and 2-DGE are complementary approaches whose applications result in a more comprehensive proteome investigation. This finding is


Figure 2. 1-D gel profile of Manduca sexta hemolymph proteins. (A) Proteins were extracted from distilled water diluted hemolymph powder by solubilization with SDS-sample buffer and separated by 12.5% SDS-PAGE. (B) Proteins were visualized with CBB R-250. Identified proteins are indicated on the right-hand side and low molecular weight markers on the left-hand side. The electroblotted proteins on the PVDF membrane were sequenced on an Applied Biosystems 494 protein sequencer (Perkin-Elmer-ABI, Foster City, CA) as described previously.67 The obtained sequences were used to interrogate databases with Web accessible search programs like FastaA and Fasta3 (available online from EMBL Outstation of the European Bioinformatics Institute), to identify homology to proteins already present in the protein and nucleic acid databases.

Figure 3. Distribution of 58 identified nonredundant proteins. Numbers in parentheses indicate the total number of proteins found by 1-DGE coupled with either N-terminal amino acid sequencing or MS and by 2-DGE in conjunction with MS and show a distribution of 58 nonredundant identified proteins. Fortyfour nonoverlapping proteins are in black and white, and 14 overlapping proteins are shaded in gray.

in line with previous reports that not one but multiple proteomics approaches are needed to dig deeper into the proteome.17,20,34–36 The proteomics data, including a 2-D map, have also been posted to the Web site http://foodfunc.agr.ibaraki.ac.jp/ mansehemoprot.html, which we hope will serve as a proteomics resource for the scientific community, especially the M. sexta (insect) researchers. 3.2. 1-DGE Analyses Identified 49 Hemolymph Proteins. For 1-DGE, two different experiments were carried out. In one case, total solubilized proteins separated by SDS-PAGE were analyzed by LC-MS/MS, and in the second case, blotted proteins onto the PVDF membrane were analyzed by Nterminal amino acid sequencing. N-Terminal amino acid

research articles

sequencing is a low-throughput analysis but provides good sequence information on the N-terminal amino acid sequence (which is usually not conserved) of a protein, and thus an additional protein assignment is possible when used in parallel to MS analysis. By using two protein identification methods, Edman sequencing and tandem MS, we could identify more proteins compared to that with MS/MS only. A total of 67 proteins were identified from the six excised fractions, representing 39 nonredundant proteins (Table 1). The fractions 1, 2, 3, 4, 5, and 6 gave protein assignments of 8, 10, 21, 8, 9, and 11 proteins, respectively. Out of these 39 proteins, 10 proteins were identified for the first time from the hemolymph. Single peptide matched protein data were also presented in Table 1; their corresponding MS/MS spectra are shown in Supplementary Figure 1. On the other hand, N-terminal amino acid sequencing of the 15 major and minor polypeptide bands by CBB staining (Figure 2) resulted in the identification of 10 nonredundant proteins (Table 2). Out of these 10 proteins, 6 were previously reported from M. sexta, whereas 4 are novel proteins. These results indicate that the hemolymph is rich in proteins related to defense, transport and metabolism, storage, and metamorphosis. 3.3. Establishment of a 2-D Gel Reference Map. To establish a high-resolution 2-D gel reference map for hemolymph, we used a narrow range (pH 4–7) IPG strip to minimize spot overlapping. Recently, it has been shown that a wide range IPG strip carries spot overlaps, and the density of spots is much higher in the region between pH 4 and 7.37 The 2-D gel profile shows that most of the proteins are in the pH range of 5-7 and between approximate molecular masses 20 and 100 kDa (Figure 4). The ImageMaster software analysis of gel detected 235 protein spots. Fifty-four intensely stained colloidal CBB spots were randomly selected covering the high to low molecular mass and acidic to basic pI range. The excised protein spots were identified by LC-MS/MS (nESI-LD-MS/MS or nLCIT-TOF-MS/MS) analysis. The protein IDs obtained after the MASCOT search are presented in Table 3. A total of 46 proteins representing 25 nonredundant proteins were identified. A spectrum for spot number 3 is given in Supplementary Figure 2 (A-C) as a reference. Out of 54 proteins analyzed, only one protein spot (number 22) gave two confident IDs (serine protease inhibitor, score 124, and chain A, β-Actin-profilin complex, score 129) indicating that use of narrow range IPG strips dramatically reduces the protein overlap.21 Single peptide matched protein data were also presented in Table 3; the corresponding MS/MS spectra is shown in Supplementary Figure 3. These data were used to create a 2-D gel reference map (Figure 4). One of the important features of 2-DGE is the finding of isoelectric species of a given protein. For example, a proteomics study of seed filling in rapeseed has identified multiple isoelectric species of enzymes involved in the carbon assimilation pathway.38 In this study, multiple isoelectric species for the β-1,3-glucan recognition protein (spot numbers 8 and 9), serine protease inhibitor superfamily (spot numbers 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, and 28), immulectin-3 (spot numbers 32, 33, and 34), ommochrome-binding protein precursor (spot numbers 35, 36, 37, 38, and 39), insecticyanin B form precursor (spot numbers 42, 44, and 45), and peptidoglycan recognition protein 1B (spot numbers 46 and 47) were identified. These isoelectric species might be due to alternative splicing of the same gene or posttranslational modification (PTM). For example, the serpin 1 (serine proteinase inhibitor) Journal of Proteome Research • Vol. 7, No. 3, 2008 955

research articles

Furusawa et al.

Figure 4. Development of a 2-D gel map of the fifth instar hemolymph of M. sexta. The proteins were extracted by the TCA/acetone protocol, and the pellet was solubilized in LB-TT. The total soluble proteins were separated on precast IPG strips (18 cm, pH 4–7) in the first dimension followed by precast gradient (12–14%) ExcelGel SDS-PAGE in the second dimension. Proteins were visualized by colloidal CBB G-250 staining. Molecular masses were determined by running standard protein markers (2.5 µL/gel; Bio-Rad). Total spot numbers (ImageMaster 2D platinum software 5.0) detected on gel are given at the bottom right-hand corner. Marked protein spots were analyzed: spots numbers 1, 2, 3, and 4 were analyzed by nLD-IT-TOF-MS/MS and the rest of the proteins by nESI-LC-MS/MS. Proteins were identified using the MASCOT search engine and SwissProt/NCBI nonredundant protein databases.

gene of M. sexta encodes for a family of plasma serpins produced through alternate exon splicing.39 Therefore, the finding of many serpins/serine protease inhibitors is not surprising. Moreover, the serpin 1 protein also carries potential phosphorylation sites with high score [9 (Ser), 3 (Thr), and 4 (Tyr) high score (NetPhos 2.0)], suggesting that the protein may be phosphorylated. Similarly, the immulectin-3 protein has a high score prediction for a signal peptide (SignalP 3.0) between amino acid positions 18 and 19 (VQG-SN). The cleaved protein has a predicted pI/MW of 5.85/31.973.11 compared to the uncleaved protein pI/MW of 5.86/33867.46. Although the observed pI of the immunolectin-3 protein spots on 2-D gel are slightly different from the theoretical pIs, the predicted molecular masses of spot 32 (most probably a cleaved product) are lower than the spots 33 and 34, which may represent the uncleaved immunolectin-3 proteins. The immunolectin-3 protein also has 4 (Ser), 3 (Thr), and 5 (Tyr) high score predicted phosphorylation sites (NetPhos 2.0) that might account for the closeness of pIs between spots 33 and 34. 3.4. Functional Categorization of the Identified Hemolymph Proteins Reveals Defense-Related Proteins as the Predominant Proteins. The identified 58 nonredundant proteins were classified into 10 functional categories (Figure 5), out of which defense (49%), transport and metabolism (15%), storage (9%), and metamorphosis (7%) were the most highly represented. Together, these four categories accounted for 80% of the identified proteins in the hemolymph. These and other proteins are briefly discussed below. 3.4.1. Defense-Related Proteins. Our current data from SDS-PAGE with N-terminal amino acid sequencing and 1-/2DGE in conjunction with MS have revealed that defensive proteins form the largest category of the identified proteins. We identified three serine protease inhibitors including six 956


serpins. The serine protease inhibitors of the serpin superfamily are essential regulators of serine protease cascades that mediate the host defense responses.39 These proteins are present in the hemolymph and work to remove excess proteases and maintain homeostasis. Serpins, irreversible covalent “suicide” protease inhibitors,40 regulate hemolymph coagulation, melanization, and antimicrobial protein synthesis in arthropods.27,40 Serpins from human plasma have been studied intensively because they are important regulators of serine proteases involved in inflammation, blood coagulation, fibrinolysis, and complement activation.39 In M. sexta, the serpin-1 gene encodes 12 variants through alternative exon usage. These serpins differ in their reactive center loops near the carboxyl terminus, which are encoded by variants of exon 9. In this study, we could identify a total of five serpin-1’s and an alaserpin precursor. Four serine protease-like proteins, nonproteolytic serine proteinase homologues, thought to be involved in regulating a variety of processes in the hemolymph39 were also identified along with two prophenoloxidases (proPO4). It has been reported that for generating an active pheonoxidase, serine proteinase homologues are required as a cofactor. This reaction has been implicated in melanotic encapsulation, wound healing, and protein cross-linking.4 Also identified were the hemolin, peptidoglycan recognition protein 1B, and β-1,3-glucan recognition protein, which serve as a surveillance mechanism upon binding to the microbial surface molecules triggering phagocystosis, nodule formation, encapsulation, melanization, synthesis of antimicrobial peptides/proteins, etc.4 Two proPO were identified, which are proteolytically activated by serine proteinase homologues. This reaction is implicated in melanotic encapsulation, wound healing, and protein cross-linking.4 Finally, an immulectin-2 previously reported to be present at a constitutively low level in the hemolymph but induced after


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Figure 5. Hemolymph proteins representing 10 functional categories. Out of 123 identified proteins, 58 proteins were nonredundant. The pie chart shows the distribution of these nonredundant proteins into their functional classes by percentage.

injection of Gram-negative bacteria or lipopolysaccharide41 was also found close to the immulectin-3 protein spot (Figure 4). This protein was shown to protect the larvae from Gramnegative bacterial attack41 and may serve as an important marker protein for bacterial infection. The identification of many defensive proteins in the hemolymph lies in the nature of the insect immune system. Insects have first-line defense barriers in the outer exoskeleton, the peritrophic matrix of the midgut epithelium, and the chitinous lining of the trachea. But, these physical barriers cannot prevent penetrations by pathogenic microbes. Once microbes have entered the hemocoel, they are exposed to humoral and cellular responses. Humoral defenses include the production of antimicrobial peptides, the induction of lectin synthesis, and the activation of the proPO system, whereas cellular defenses involve hemocyte-mediated immune responses,4 which include phagocytosis and encapsulation.42 3.4.2. Transport and Metabolism. The second largest category is transport and metabolism. These proteins are needed for insect homeostasis and metamorphosis in the fifth instar larvae. For example, lipid transport processes via the circulatory system of animals are a vital function that utilizes highly specialized lipoprotein complexes. These complexes of protein and lipid impart solubility to otherwise insoluble lipids. The apoprotein components of lipoprotein complexes serve to stabilize the lipid components, modulate particle metabolism, and function as ligands for receptor-mediated endocytosis of lipoproteins,5 and the major hemolymph lipoprotein (lipophorin, M. sexta) transports carotenes and thus has an intense yellow color.43 The mix of yellow-colored lipoprotein and bluecolored biliprotein provides the larvae with camouflage: the green coloration of larvae closely matches that of chlorophyll in the host plant.43 The ommochrome-binding protein precursor (OBP, previously designated YCP), which is different from the lipoprotein, binds to an ommochrome (visual pigment), ommatin D, which is a yellow chromophore and is not observed prior to the wandering stage.26 Iron must be acquired to provide catalysis for oxidative metabolism, but it must be controlled to avoid destructive oxidative reactions. The transferrin precursor and ferritin balance two properties of ionic iron.44 Thus, these proteins are needed and exist in the hemolymph. 3.4.3. Storage. Insect larval storage proteins (LSPs), mainly synthesized during larval development, are stored in the hemolymph and sequestered in the fat body where they serve

as sources of nitrogen and amino acids for utilization by pupae and adults during metamorphosis and reproduction.2,45 LSPs are important for insect development, and therefore numerous studies concerning their structure, biosynthesis, regulation, and evolution have been conducted during recent years.46–48 Storage proteins are synthesized in the fat body, secreted into the larval hemolymph, and taken up by the fat body shortly before pupation.46 Two classes of LSPs in insects are methionine-rich LSPs and aromatic amino acid rich arylphorins.46,47 The methionine-rich SP-1 mRNA in the fat body appears in the early last instar larvae stage and accumulates to a maximum level at the end of the last instar larvae stage.49 Arylphorins are present throughout larval development, though their concentration in the hemolymph increases greatly in the last instar larvae stage.50 3.4.4. Others. The other 16 identified proteins were divided into seven functional categories, namely, metamorphosis, cell structure, protein–protein interaction, cell fate, chaperone, ribosomal protein, and unclassified. The insect hemolymph juvenile hormone (JH) binding protein (hJHBP) regulates peripheral titers of its ligands, the JHs, and prevents the JH from being hydrolyzed by general esterases by combining with it specifically. Its appearance in both the larval and pupal epidermis after ecdysis depended on the presence of JHs during the molt.51 So far, the JHBP has been purified from the hemolymph of the fourth or early fifth instar larvae M. sexta, and the protein is present at relatively high levels in both the larval stages.52 It is expected that further research on this protein will be a key step in the elucidation of JH action leading to new insights into the mechanism of insect metamorphosis. The hemolymph glycoprotein precursor is a low-abundance hemolymph protein synthesized in the fat body and present in both males and females during all stages of development.53 However, the function of the protein remains to be clarified in future studies. 3.5. Identification of 18 Novel Hemolymph Proteins. The novel hemolymph proteins account for almost 30% (18 proteins) of the total identified proteins. These proteins were previously not identified in the hemolymph of M. sexta. These proteins are lipoprotein-releasing system transmembrane protein lolC, 50S ribosomal protein L24, inducible serine protease inhibitor 1 (ISPI-1), trypsin precursor, chain E, leech-derived tryptase inhibitor trypsin complex, CG12214-PA, isoform, proteasome 26S subunit ATPase 3 interacting protein, Actin, GA10602-PA, CG10600-PA, three imaginal disk growth factors Journal of Proteome Research • Vol. 7, No. 3, 2008 957

research articles (IDGFs), HSC70, ERp57, Chain A, β-Actin-profilin complex protein, and two hypothetical proteins. The lipoprotein-releasing system transmembrane protein lolC is homologous to permease proteins of the ABC transporter.54 The protein recognizes and releases lipoproteins anchored to the periplasmic leaflet of the inner membrane.55 As lipid transport is an important function for hemolymph, it is natural for this protein to be present therein. The 50S ribosomal protein L24 is one of two assembly initiator proteins, and it binds directly to the 5′ end of the 23S rRNA where it nucleates assembly of the 50S subunit and/or one of the proteins that surrounds the polypeptide exit tunnel on the outside of the subunit.56 Inducible serine protease inhibitor 1 (ISPI-1), previously identified from the hemolymph of the last instar larvae of the wax moth, inhibits trypsin and the toxin protease PR2 of fungus Metarhizium anisopliae but does not inhibit chymotrypsin, subtilisin, proteinase K, porcine pancreatic elastase, and the toxin protease PR1 of M. anisopliae.57 Thus, its presence in M. sexta hemolymph is not surprising, and it forms a new member of the serine proteinase inhibitor family. The trypsin precursor and chain E, leech-derived tryptase inhibitor trypsin complex proteins are synthesized as inactive precursor zymogens that are cleaved during limited proteolysis to generate their active forms. The CG12214-PA, isoform A, belongs to the leucine-rich repeats (LRRs) ribonuclease inhibitor (RI)-like subfamily. LRRs are 20–29 residue sequence motifs present in many proteins that participate in protein–protein interactions and have different functions and cellular locations. The proteasome 26S subunit ATPase 3 interacting protein belongs to the 26S proteasome subfamily and is responsible for the bulk of protein turnover as well as for the degradation of regulatory short-halflife proteins such as transcription factors, cyclins, and for the production of antigenic peptides presented by the class I major histocompatibility complex.58 The Actin protein identified here showed high homology (97% identity) to an M. sexta Actin described from intersegmental muscles.59 Actin is a highly conserved protein involved in various types of cell motility and is ubiquitously expressed in all eukaryotic cells and thus was identified from the hemolymph. The GA10602-PA protein has an amiloride-sensitive sodium channel domain, but function of this protein has not been determined. Three proteins, CG10600-PA, a ZK484.4, and a hypothetical protein, remain as unknown proteins. Three proteins (including spot number 13, Figure 4) were identified as IDGFs from different species, Pieris rapae, Bombyx mori, and Mamestra brassicae. These three proteins are highly homologous: the B. mori and M. brassicae IDGF have 82.7% homology; the M. brassicae and P. rapae IDGF have 89.1% homology; B. mori and P. rapae have 85.7% homology. The IDGF family plays a role in regulating the cell line of insects. The IDGF derived from P. rapae is produced in fat body cells and hemocytes.60 The IDGF protein of B. mori is a 20hydroxyecdysone, which induces programmed cell death (PCD) and tissue degeneration, during pupal metamorphosis.61 The IDGF of M. brassicae has been shown to work without insulin.62 It stimulated the growth of cultured imaginal disk cells and has been named MbIDGF, a new member of the IDGF family that functions as a growth factor. The first identified IDGF from Drosophilia requires insulin, whereas the IDGF of M. brassicae works without insulin. This suggests the presence of IDGF proteins with different requirements for the hormone insulin, 958


Furusawa et al. and it remains to be seen how the M. sexta IDGF functions during growth stimulation. HSC70 (spot number 5, Figure 4) showed homology to a heat shock protein from Chilo suppressalis Walker. HSC70 protects other proteins from harmful conditions, such as oxidative stress, extreme temperature changes, and toxic drugs. This chaperone also controls protein degradation, directing selected, misfolded, or covalently modified proteins toward the proteasome system.63 Interestingly, a BLAST search revealed its high (98%) homology with a previously identified HSC70 from the prothoracic gland of M. sexta.64 Spot number 10 was a protein disulfide-isomerase (PDI) like protein ERp57. The ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the PDI family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. ERp57 functions as a subunit of specific complexes formed with the ER lectins calreticulin and calnexin.65 The chain A, β-Actinprofilin complex protein was identified from spot number 22 (Figure 4). Once the Actin/profilin complex has formed, phosphoinositides can break the complex apart, releasing the ATPbound monomer and inducing polymerization.66 It should be noted that this protein was identified with high score from the same spot containing the serine protease inhibitor. As spot overlaps do occur on 2-D gels,21,37 the possibility that one spot includes more than one protein cannot be ruled out.

4. Conclusions M. sexta belonging to Lepidoptera, the second most numerous order of insects that includes many species important for agriculture and forestry, is a good model organism to study insect physiology and biochemistry. Advances in M. sexta research will not only facilitate the development of new strategies for pest control but also help in the elucidation of insect biology. This investigation represents a systematic proteomics study of hemolymph proteins expressed during the fifth instar larvae stage in M. sexta using two widely used complementary approaches, 1-D and 2-D gel-based proteomics coupled with N-terminal amino acid sequencing and LC-MS/ MS. Proteins belonging to 10 functional categories were identified, including 18 novel proteins. However, proteins involved in defense, transport and metabolism, storage, and metamorphosis were the most highly represented. These identified proteins with numerous known functional proteins correlate well with the fifth instar larvae stage and physiology, which is most prone to attack by natural pathogens and pests, and as voracious leaf feeders, they are under constant threat from any changing environmental factor. Another important outcome of the study is the establishment of a high-resolution 2-D gel reference map, which could be used for comparative functional proteomics. Application of this established proteomics approach to other stages of the life cycle will help in understanding how these protein expressions change in time and space during growth and development of M. sexta and under the constantly changing environment.

Acknowledgment. We are grateful to Colin. L. Miller, [email protected], Sierra Vista, AZ, USA, for kind permission for the use of the M. sexta life cycle photographs. During her study at HSS, AIST, T.F. greatly appreciates the support and encouragement from Drs. Hitoshi Iwahashi and Yoshinori Masuo. This work was partly supported by a grant from the Korea Science and Engineering Foundation through Protein Network Research

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Investigation of the Hemolymph Proteome of Manduca sexta Center at Yonsei University (Grant No. R112000078010010). We also appreciate the expert technical support of Kimiyoshi Kohda of Hitachi High-Technologies Corporation (Hitachi-Naka 312-0057, Japan). R.R. and G.K.A. greatly appreciate the support from Prof. V.P. Agrawal (Founding Director, RLABB, Nepal) in establishing the collaboration with Prof. Yu-Sam Kim at Yonsei University, Korea.

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Systematic Investigation of the Hemolymph Proteome of Manduca

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