Larvae of the cotton bollworm (H. armigera) were reared on an artificial diet described (made from powder of wheat germs and soybeans with various vitamins as well as inorganic salts) by Zhao et al.  at 28°C with 60-70% relative humidity in our laboratory. The light:dark schedule was 14:10 h.
Preparation of protein samples
For 2-DE analysis, we used samples from fifth-instar feeding larvae (5th-F) and sixth-instar metamorphically committed larvae (6th-M). The 5th-F larvae were defined as the larvae that had shed their old fourth instar cuticle and had grown for 24 h. The 6th-M larvae were defined as the larvae at 72 h, 84 h, 96 h, 108 h, and 120 h after molting into the 6th instar.
The epidermis was dissected from the 5th-F larvae or the 6th-M larvae at the indicated time points, homogenized by glass homogenizer in 1 ml sample buffer [40 mM Tris, 3 mM EDTA, 1 mM phenylmethanesulfonyl fluoride], and centrifuged at 12,000 rpm (24,300 g) for 10 min at 4°C. The supernatants were passed through 0.45 μm microporous filters (Xing Ya, Shanghai, China). Total proteins in the samples were determined by the Bradford method 30. The metamorphosis sample was a mixture of 1 mg protein sample from each of the five stages of the sixth instar larvae described above.
Each protein was precipitated with two volumes of acetone containing 10% (w/v) trichloroacetic acid and 20 mM dithiothreitol (DTT) at -20°C for 2 h. After being centrifuged at 12,000 rpm (24,300 g) for 15 min, the precipitate was washed twice with two volumes of pre-cooled acetone containing 20 mM DTT for 15 min and centrifuged to collect the proteins. The proteins were then dried at room temperature.d
Two-dimensional gel electrophoresis (2-DE)
The dried protein samples were redissolved in a rehydration solution containing 8 M urea, 2 M thiourea, 4% 3-[(3-Cholamidopropyl)dimethylammonio]propanesulfonic acid, 65 mM DTT and 0.2% (w/v) Ampholyte pH 3.0-10.0 (GE Healthcare, United States). Eight hundred micrograms of protein were loaded on an 18-cm nonlinear Immobiline IPG Drystrip (pH 3-10) (GE Healthcare) in a rehydration tray for 14 h. An Ettan IPGphor3 system (GE Healthcare) was used for the first-dimensional isoelectric focusing (IEF) at 250 V for 1 h, 500 V for 1 h, 1,000 V for 5 h, followed by linearly ramping to 10,000 V over 3 h and then holding at 10,000 V until 60,000 V-h had been accumulated. After IEF, the IPG strip was first equilibrated with an equilibration buffer (0.375 M Tris-HCl, pH 8.8, 6 M urea, 20% glycerol, 2% SDS) containing 65 mM DTT for 10 min followed by 135 mM iodoacetamide for 10 min with constant shaking. After equilibration of the focused IPG strip, the strip was transferred to the top of a 12% SDS polyacrylamide gel and sealed with 1% low melting agarose. The second-dimensional electrophoresis was performed on a Protean II xi system (Bio-Rad) at 10 mA per gel for 30 min, and then 25 mA for approximately 6 h. Three independent repeats were performed. The detailed protocol for 2-DE is described in the instruction manual from GE Healthcare.
Protein staining and imaging
After electrophoresis, the gels were first stained with the Pro-Q Diamond Phosphoprotein Stain (Molecular Probes, United States) following the manufacturer's instructions. Briefly, the gels were fixed with 50% (v/v) methanol and 10% (v/v) acetic acid for 60 min twice. After washed with ultrapure water, the gels were incubated in the dark in Pro-Q Diamond Phosphoprotein Stain with gentle agitation for 1.5-2 h. The gels were destained with a destaining solution [20% (v/v) acetonitrile, 50 mM sodium acetate, pH 4.0] 3 times, 30 min each. The gels were imaged with a Typhoon Trio+ System (GE Healthcare, United States) and the images were optimized using phosphoprotein molecular weight standards (Invitrogen, United States). Then the gels were stained with colloidal Coomassie Brilliant Blue G-250 [10% (w/v) ammonium sulfate, 1% (w/w) phosphoric acid, 0.1% Coomassie Blue G-250] and destained with ultrapure water. The gel images were analyzed using ImageMaster 2D Platinum 6.0 software (GE Healthcare) to identify differentially expressed and phosphorylated proteins.
The differentially expressed and phosphorylated protein spots were manually picked from the gel and placed individually into methanol-treated tubes. Each gel piece was washed 3 times with distilled water. Then 200 μL of 200 mM ammonium bicarbonate with 40% acetonitrile was added to each tube and incubated at 37°C for 30 minutes. After that, the solution was removed from the tube. The gel piece in each tube was suspended in 100 μL acetonitrile and dehydrated for 5 min before the excess acetonitrile was discarded. The gel piece was dried in vacuum for 15 min and then treated with 5 μL of a trypsin solution [20 μg/mL trypsin (Proteomics Grade, Sigma, United States), 40 mM ammonium bicarbonate, 9% acetonitrile]. The tube was incubated on ice for 45 min and then excess trypsin solution was removed. To cover the gel piece, 5 μL of 40 mM ammonium bicarbonate in a 9% acetonitrile solution was added and incubated overnight at 37°C. After the incubation, the liquid from the gel piece was transferred to a new labeled tube. Then 5 μL of 0.1% trifluoroacetic acid in a 50% acetonitrile solution was added to the gel piece and incubated for 30 min at 37°C. After that, the solution was collected and combined with the liquid from the previous step. The combined sample solution was used for MALDI-TOF-MS (matrix assisted laser desorption/ionization time-of-flight mass spectrometry) analysis.
MALDI-TOF-MS and MS/MS analysis
The trypsin-digested peptides were mixed with a MALDI matrix [7 mg/mL α-cyano-4-hydroxycinnamic acid, 0.1% trifluoroacetic acid and 50% acetonitrile] and spotted on the MALDI target plates. MS and MS/MS spectra were obtained with an ABI 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, United States) operating in a result-dependent acquisition mode. Peptide mass maps were acquired in a reflectron mode (1000 V accelerating voltage) with 1,000 laser shots per spectrum. Six external standards (mass standard kit for the 4700 Proteomics Analyzer calibration mixture, Part Number 4333604, Applied Biosystems) were used to calibrate each spectrum to a mass accuracy within 50 ppm. The MS/MS data were acquired with stop conditions and 3,000-6,000 laser shots were accumulated for each spectrum (Additional files 1). MS/MS analysis were performed at collision energy of 1 kV and the collision gas pressure of 2.0 × 10-7 to 3 × 10-8 Torr.
The MASCOT search engine (version 1.9, Matrix Science, http://www.matrixscience.com/search_form_select.html) was used to search all of the tandem mass spectra against NCBInr_Metazoa Database. Carbamidomethyl (C) (cysteine carbamidomethylation) and Oxidation (M) (methionine oxidation) were selected as fixed and variable modifications. One missing trypsin cleavage was allowed. Peptide mass tolerance and fragment mass tolerance were set to 100 ppm or 0.6 Da. High confidence identifications had statistically significant search scores (greater than 95% confidence, equivalent to MASCOT expect value p < 0.05), were consistent with the protein's experimental pI and MW, and accounted for the majority of ions present in the mass spectra.
Reverse Transcription PCR
Total RNA was isolated from the epidermis of the 5th-F, 6th-F and 6th-M larvae. In 20E-induction experiment, total RNA was extracted from epidermis of larvae injected with 20E for different time periods. Five micrograms of RNA was used to reverse transcribe the first strand cDNA (First Strand cDNA Synthesis Kit, Sangon, China), which was then used as a template (0.5 ng) in PCR reactions with gene-specific primers. Specific PCR primers for the genes of the proteins identified in MALDI-TOF-MS were designed from expression sequence tags of H. armigera (Table 2). PCR cycles were as follows: one cycle (94°C, 2 min); 27 cycles (94°C, 30 s; 55°C, 40 s; 72°C, 30 s), followed by a last cycle (72°C, 10 min). A β-actin gene fragment from H. armigera was also amplified as a control. Each RT-PCR assay was replicated three times.
To confirm expression changes for some of the interesting proteins identified, the epidermis of fifth feeding larvae (5th-F), sixth feeding larvae (6th-F) and sixth-metamorphically committed larvae (6th-M) were dissected and homogenized for Western blotting. The protein extracted from each tissue was quantified by the Bradford method. Equal amounts of protein (50 μg) were subjected to 12.5% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then electro-transferred onto nitrocellulose membranes. The membranes were incubated in a blocking buffer (10 mM Tris-buffered saline) containing 2% milk power at room temperature for 1 h, then incubated with primary antibodies: diluted 1:100 for anti-calponin polyclonal antibodies (generated in our lab) and 1:500 for anti-hexamerin monoclonal antibody (a gift from Dr. Gang Ma, University of Adelaide, Australia) at 4°C overnight, respectively. Goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) diluted 1:10000 was adopted as a secondary antibody. 4-chloro-1-naphthol was used as a HRP substrate for visualizing the peroxidase activity. The quantity of protein loaded was controlled by SDS-PAGE with two gels simultaneously, one for transferring and the other for Coomassie Brilliant Blue staining.
Induction with 20E was performed as follows. A 10 mg/mL stock solution of 20E in dimethyl sulfoxide (DMSO) was diluted 1:100 in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 1.8 mM KH2PO4). The 6th instar feeding larvae (6th-6 h, 6th-12 h, 6th-24 h and 6th-48 h) were each injected with 500 ng 20E in 5 μl of the dilution, and then incubated for 12 h. An equal volume of DMSO diluted in PBS was injected into control larvae.
To verify the identified phosphoprotein, λ protein phosphatase (λPPase) was added to the epidermis homogenate of 20E-injected larvae at a final concentration of 4 μg/100 μl reaction buffer [500 mM Hepes (N-2-hydroxyethylpiperazine-N-ethane-sulphonicacid), pH 7.5, 1 mM EDTA, and 20 mM MnCl2] and incubated at 37°C for 10 min before subjected to Western blot analysis. To reveal whether phosphorylation of the identified protein was regulated by protein kinase C (PKC), a PKC specific inhibitor, CC (chelerythrine chloride), was injected into the larvae together with 20E. The epidermis proteins of each sample were extracted 12 h after injection and subjected to SDS-PAGE and Western blotting.