iTRAQ-Based Quantitative Proteomic Analysis of Digestive Juice across the First 48 Hours of the Fifth Instar in Silkworm Larvae
Abstract
:1. Introduction
2. Results
2.1. Protein Profiling
2.2. Identification of Differentially Expressed Proteins
2.3. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes Pathway Enrichment Analysis of DEPs
2.4. Protein–Protein Interaction Network Analysis of DEPs
2.5. Spatial Expression Profile of Genes of the Identified Proteins
2.6. Expression Analysis of Genes Corresponding to DEPs
3. Discussion
4. Materials and Methods
4.1. Collection of Samples and Protein Preparation
4.2. Protein Digestion and iTRAQ Labeling
4.3. Strong Cation Exchange Fractionation and NanoLC-MS/MS Analysis
4.4. Protein Identification and Annotation
4.5. Tissue Expression Patterns Based on Microarray Data
4.6. RT-qPCR Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Acknowledgments
Conflicts of Interest
References
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Time (h) | Number | Accession | SilkDB Accession | Fold Change | Function |
---|---|---|---|---|---|
DJ24/0 h | 1 | H9J8I4 | BGIBMGA005826 | 2.11 | Alcohol dehydrogenase activity |
2 | H9IW77 | BGIBMGA001508 | 2.19 | Carboxylic ester hydrolase activity | |
3 | Q58I81 | — | 4.18 | Ester bonds hydrolase activity | |
4 | H9JTK9 | BGIBMGA012871 | 2.02 | Ester bonds hydrolase activity | |
5 | H9ISX5 | BGIBMGA000353 | 3.24 | Innate immune | |
6 | Q86D78 | BGIBMGA003512 | 2.72 | Beta-glucosidase | |
7 | H9JCF2 | BGIBMGA007153 | 2.73 | Glycosyl hydrolase activity | |
8 | H9JL39 | BGIBMGA010240 | 3.96 | Chitinase activity | |
9 | H9JSN7 | BGIBMGA012548 | 3.21 | Sphingomyelin phosphodiesterase activity | |
10 | B1Q138 | — | 2.37 | Carboxylesterase activity | |
11 | H9JTY2 | BGIBMGA012994 | 3.01 | CN_hydrolase activity | |
12 | B1Q137 | — | 3.08 | Carboxylesterase activity | |
13 | H9IWD7 | BGIBMGA001568 | 2.57 | Maltase-glucoamylase activity | |
14 | B2ZZX0 | BGIBMGA008818 | 4.87 | Phosphatase activity | |
15 | H9JT78 | BGIBMGA012740 | 2.82 | Peroxidase activity | |
16 | H9JBZ7 | BGIBMGA007042 | 2.92 | Peroxidase activity | |
17 | P10831 | — | 3.33 | Peptidase regulator | |
18 | Q03383 | — | 2.02 | Antichymotrypsin | |
19 | C0J8G5 | BGIBMGA003292 | 3.63 | Serine protease inhibitor | |
20 | H9IXK0 | BGIBMGA001983 | 10.24 | Serine protease inhibitor | |
21 | H9JH30 | BGIBMGA008827 | 13.22 | Serine protease inhibitor | |
22 | I3VR74 | BGIBMGA008061 | 2.53 | AMP deaminase activity | |
23 | B5TZ28 | BGIBMGA007915 | 4.92 | Metallopeptidase activity | |
24 | H9J232 | BGIBMGA003569 | 2.96 | Serine protease activity | |
25 | H9J267 | BGIBMGA003604 | 3.19 | Serine protease activity | |
26 | H9JL75 | BGIBMGA010276 | 3.15 | Serine protease activity | |
27 | H9JJ25 | BGIBMGA009526 | 4.40 | Serine protease activity | |
28 | H9JJ26 | BGIBMGA009527 | 3.72 | Serine protease activity | |
29 | H9IWJ8 | BGIBMGA001630 | 2.32 | Cholinesterase activity | |
30 | H9JDE9 | BGIBMGA007546 | 2.19 | Carboxylesterase activity | |
31 | H9J067 | BGIBMGA002902 | 2.84 | Cholinesterase activity | |
32 | H9IWK8 | BGIBMGA001640 | 2.89 | Aminopeptidase activity | |
33 | H9JJ19 | BGIBMGA009520 | 6.95 | WD repeat domain phosphoinositide-interacting | |
34 | Q1HPY8 | — | 4.25 | Guanine nucleotide binding | |
35 | H9J1D5 | BGIBMGA003322 | 2.28 | Juvenile hormone binding | |
36 | H9JK60 | BGIBMGA009911 | 2.05 | Ubiquitin-activating | |
37 | H9IVY4 | BGIBMGA001415 | 2.08 | Ubiquitin-mediated protein binding | |
38 | C1K001 | BGIBMGA004287 | 5.34 | N/A | |
39 | H9JN76 | BGIBMGA010979 | 2.20 | N/A | |
40 | H9J1D8 | BGIBMGA003325 | 3.80 | N/A | |
41 | H9JXD9 | BGIBMGA014204 | 2.36 | N/A | |
42 | H9JPZ3 | BGIBMGA011598 | 2.39 | N/A | |
43 | H9J1D9 | BGIBMGA003326 | 2.33 | N/A | |
DJ48/0 h | 1 | H9IW76 | BGIBMGA001507 | 5.27 | Lipase |
2 | C7EPE2 | BGIBMGA000158 | 5.42 | Glucose-methanol-choline (GMC) oxidoreductase activity | |
3 | H9JFH1 | BGIBMGA008268 | 3.06 | Aldehyde oxidase activity | |
4 | H9J8I4 | BGIBMGA005826 | 3.90 | Alcohol dehydrogenase activity | |
5 | H9JTY7 | BGIBMGA012999 | 2.94 | Glucose dehydrogenase activity | |
6 | H9ISL2 | BGIBMGA000239 | 3.87 | Peroxidase activity | |
7 | H9ISL1 | BGIBMGA000238 | 7.03 | Peroxidase activity | |
8 | H9JT78 | BGIBMGA012740 | 3.46 | Peroxidase activity | |
9 | H9IX93 | BGIBMGA001876 | 2.78 | Alpha-amylase activity | |
10 | Q86D78 | BGIBMGA003512 | 3.44 | Beta-glucosidase | |
11 | B2DD57 | BGIBMGA005696 | 2.81 | Glycosyl hydrolase activity | |
12 | H9JCF2 | BGIBMGA007153 | 3.18 | Glycosyl hydrolase activity | |
13 | A0A077JI83 | BGIBMGA006066 | 3.90 | O-Glycosyl hydrolase activity | |
14 | H9ISX5 | BGIBMGA000353 | 8.99 | Innate immune | |
15 | H9JPS6 | BGIBMGA011531 | 2.85 | Phospholipase C activity | |
16 | H9IVS0 | BGIBMGA001351 | 3.11 | Oxidoreductase activity | |
17 | B2ZZX0 | BGIBMGA008818 | 2.74 | Phosphatase activity | |
18 | Q9NGS0 | — | 3.05 | N/A | |
19 | P81902 | — | 3.16 | Trypsin inhibitor | |
20 | Q03383 | — | 3.14 | Antichymotrypsin | |
21 | C0J8G5 | BGIBMGA003292 | 7.33 | Serine protease inhibitor | |
22 | C0J8H1 | — | 12.35 | N/A | |
23 | C4B489 | BGIBMGA004445 | 2.19 | Serine protease activity | |
24 | H9JFI3 | BGIBMGA008280 | 3.17 | Serine protease activity | |
25 | H9J229 | BGIBMGA003566 | 2.04 | Serine protease activity | |
26 | H9JKH3 | BGIBMGA010024 | 2.37 | Serine protease activity | |
27 | H9JG67 | BGIBMGA008514 | 2.02 | Serine protease activity | |
28 | H9JY09 | BGIBMGA014427 | 2.44 | Serine protease activity | |
29 | H9J231 | BGIBMGA003568 | 2.72 | Serine protease activity | |
30 | H9JJ26 | BGIBMGA009527 | 2.64 | Serine protease activity | |
31 | H9JIY5 | BGIBMGA009486 | 2.33 | Carboxypeptidase activity | |
32 | H9JES0 | BGIBMGA008017 | 2.37 | AMP deaminase activity | |
33 | H9JEW2 | BGIBMGA008059 | 4.47 | Aminopeptidase activity | |
34 | H9JDE9 | BGIBMGA007546 | 3.86 | Carboxylesterase activity | |
35 | H9JE19 | BGIBMGA007766 | 2.35 | Phosphoric diester hydrolase activity | |
36 | B5TZ28 | BGIBMGA007915 | 3.13 | Metallopeptidase activity | |
37 | H9IWJ8 | BGIBMGA001630 | 2.01 | Cholinesterase activity | |
38 | H9JSJ8 | BGIBMGA012509 | 3.51 | Glucosinolate sulphatase activity | |
39 | H9J064 | BGIBMGA002899 | 2.18 | Carboxylesterase activity | |
40 | H9J067 | BGIBMGA002902 | 3.26 | Cholinesterase activity | |
41 | B2ZDZ0 | BGIBMGA009544 | 2.04 | Carboxylesterase activity | |
42 | C0SQ80 | BGIBMGA008354 | 2.00 | Odorant binding | |
43 | A1YQ87 | BGIBMGA005493 | 3.08 | Phosphopyruvate hydratase activity | |
44 | H9JP12 | BGIBMGA011266 | 3.08 | Insect hexamerins | |
45 | H9J128 | BGIBMGA003215 | 4.77 | RNA binding | |
46 | H9JLC5 | BGIBMGA010326 | 3.09 | Mitochondrial carriers | |
47 | H9IVY4 | BGIBMGA001415 | 3.89 | Ubiquitin-mediated protein binding | |
48 | H9IT95 | BGIBMGA000475 | 5.80 | Cation binding | |
49 | Q69FX2 | BGIBMGA008221 | 2.34 | Innate immunity and lipid metabolism | |
50 | S5M110 | BGIBMGA005577 | 2.25 | Carbohydrate derivative binding | |
51 | C1K001 | BGIBMGA004287 | 3.16 | N/A | |
52 | H9JYG4 | BGIBMGA009648 | 5.55 | DNA binding | |
53 | H9IYN2 | BGIBMGA002366 | 2.16 | N/A | |
54 | Q2F645 | BGIBMGA014211 | 2.03 | Transketolase activity | |
55 | H9J1D5 | BGIBMGA003322 | 4.40 | Juvenile hormone binding | |
56 | H9J5L9 | BGIBMGA004809 | 3.95 | Lyase activity | |
57 | H9J1D8 | BGIBMGA003325 | 4.85 | N/A | |
58 | H9JT75 | BGIBMGA012737 | 2.58 | Peroxidase activity | |
59 | H9JXD9 | BGIBMGA014204 | 2.02 | N/A | |
60 | H9JPZ3 | BGIBMGA011598 | 3.59 | N/A | |
61 | H9J3M9 | BGIBMGA004116 | 2.02 | Transferase activity | |
62 | H9J1D9 | BGIBMGA003326 | 8.57 | N/A | |
63 | H9JXN1 | BGIBMGA014298 | 12.23 | N/A | |
DJ48/24 h | |||||
1 | H9IW76 | BGIBMGA001507 | 2.87 | Lipase | |
2 | C7EPE2 | BGIBMGA000158 | 4.05 | Glucose-methanol-choline (GMC) oxidoreductase activity | |
3 | H9JFH1 | BGIBMGA008268 | 2.12 | Aldehyde oxidase activity | |
4 | H9ISL2 | BGIBMGA000239 | 2.30 | Peroxidase activity | |
5 | H9ISL1 | BGIBMGA000238 | 4.67 | Peroxidase activity | |
6 | H9IX93 | BGIBMGA001876 | 2.52 | Alpha-amylase activity | |
7 | B2DD57 | BGIBMGA005696 | 2.01 | Glycosyl hydrolase activity | |
8 | A0A077JI83 | BGIBMGA006066 | 3.67 | O-Glycosyl hydrolase activity | |
9 | A1YQ87 | BGIBMGA005493 | 3.06 | Phosphopyruvate hydratase activity | |
10 | P81902 | — | 2.03 | Trypsin inhibitor | |
11 | C0J8H1 | — | 2.02 | N/A | |
12 | H9JKL1 | BGIBMGA010062 | 3.09 | Serine-type endopeptidase activity | |
13 | H9J5K7 | BGIBMGA004797 | 2.14 | Metallocarboxypeptidase activity | |
14 | H9JG67 | BGIBMGA008514 | 2.28 | Serine protease activity | |
15 | H9J5P0 | BGIBMGA004830 | 2.12 | Metallocarboxypeptidase activity | |
16 | H9JIY5 | BGIBMGA009486 | 2.41 | Carboxypeptidase activity | |
17 | H9JG68 | BGIBMGA008515 | 4.20 | Serine protease activity | |
18 | H9JHZ0 | BGIBMGA009138 | 2.00 | Aminopeptidase activity | |
19 | A7LIK7 | BGIBMGA004403 | 2.14 | 30K protein | |
20 | H9J128 | BGIBMGA003215 | 2.72 | RNA binding | |
21 | H9JLC5 | BGIBMGA010326 | 3.06 | Mitochondrial carriers | |
22 | H9IVY4 | BGIBMGA001415 | 2.24 | Ubiquitin-mediated protein binding | |
23 | H9JUE4 | BGIBMGA013157 | 3.54 | Ribosomal protein | |
24 | Q8N0P2 | BGIBMGA002381 | 3.43 | 70 kilodalton heat shock protein | |
25 | H9J5L9 | BGIBMGA004809 | 2.80 | Lyase activity | |
26 | H9J1D9 | BGIBMGA003326 | 3.69 | N/A | |
27 | H9JXN1 | BGIBMGA014298 | 6.68 | N/A |
Number | Map_Name | Map_ID | Protein_ID | Definition | Fold Change |
---|---|---|---|---|---|
1 | Galactose metabolism | map00480 | F8V3L0 | gamma-glutamyltranspeptidase [24] | 0.47 |
2 | Lysosome | map04142 | H9JSN7 | sphingomyelin phosphodiesterase [25] | 3.21 |
3 | Sphingolipid metabolism | map00600 | H9JSN7 | sphingomyelin phosphodiesterase [26] | 3.21 |
4 | Arachidonic acid metabolism | map00590 | F8V3L0 | gamma-glutamyltranspeptidase [24] | 0.47 |
5 | Taurine and hypotaurine metabolism | map00430 | F8V3L0 | gamma-glutamyltranspeptidase [24] | 0.47 |
6 | Neuroactive ligand-receptor interaction | map04080 | H9JL75 | trypsin [27] | 3.15 |
7 | Folate biosynthesis | map00790 | B2ZZX0 | alkaline phosphatase [28] | 4.18 |
8 | Thiamine metabolism | map00730 | B2ZZX0 | alkaline phosphatase [28] | 4.18 |
9 | Ubiquitin mediated proteolysis | map04120 | H9JK60 | ubiquitin-activating enzyme E1 [29] | 2.05 |
10 | Caffeine metabolism | map00232 | H9JFX4 | xanthine dehydrogenase/oxidase [30] | 0.28 |
Number | Map_Name | Map_ID | Protein_ID | Definition | Fold Change |
---|---|---|---|---|---|
1 | Folate biosynthesis | map00790 | H9IVS0 | aldehyde reductase [31] | 3.11 |
B2ZZX0 | alkaline phosphatase [28] | 4.18 | |||
H9JTG9 | aldehyde reductase [32] | 0.50 | |||
H9IVT6 | aldehyde reductase | 0.06 | |||
2 | Lysosome | map04142 | Q69FX2 | niemann-Pick C2 protein [33,34] | 2.34 |
A4PHN6 | hexosaminidase [35] | 0.50 | |||
H9IWR3 | lysosomal alpha-mannosidase [36] | 0.47 | |||
3 | Galactose metabolism | map00052 | H9IVS0 | aldehyde reductase [37] | 3.11 |
H9JTG9 | aldehyde reductase [32] | 0.50 | |||
H9IVT6 | aldehyde reductase | 0.06 | |||
4 | Fructose and mannose metabolism | map00051 | H9IVS0 | aldehyde reductase [37] | 3.11 |
H9JTG9 | aldehyde reductase [32] | 0.50 | |||
H9IVT6 | aldehyde reductase | 0.06 | |||
5 | Pentose and glucuronate interconversions | map00040 | H9IVS0 | aldehyde reductase [37] | 3.11 |
H9JTG9 | aldehyde reductase [32] | 0.50 | |||
H9IVT6 | aldehyde reductase | 0.06 | |||
6 | Glycerolipid metabolism | map00561 | H9IVS0 | aldehyde reductase [37] | 3.11 |
H9JTG9 | aldehyde reductase [32] | 0.50 | |||
H9IVT6 | aldehyde reductase | 0.06 | |||
7 | Purine metabolism | map00230 | H9JFH1 | xanthine dehydrogenase/oxidase [30] | 3.06 |
H9JDV4 | nucleoside-diphosphate kinase [38] | 0.26 | |||
H9JFX4 | xanthine dehydrogenase/oxidase [30] | 0.38 | |||
8 | Peroxisome | map04146 | H9JFH1 | xanthine dehydrogenase/oxidase [30] | 3.06 |
Q08J22 | superoxide dismutase, Cu-Zn family [39,40] | 0.33 | |||
H9JFX4 | xanthine dehydrogenase/oxidase [30] | 0.38 | |||
9 | Starch and sucrose metabolism | map00500 | H9IX93 | alpha-amylase [41] | 2.78 |
H9J822 | alpha-trehalase [42] | 0.42 | |||
10 | Glutathione metabolism | map00480 | H9JES0 | aminopeptidase N [43] | 2.37 |
H9JEW2 | aminopeptidase N [43] | 4.47 |
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Xu, P.; Zhang, M.; Qian, P.; Li, J.; Wang, X.; Wu, Y. iTRAQ-Based Quantitative Proteomic Analysis of Digestive Juice across the First 48 Hours of the Fifth Instar in Silkworm Larvae. Int. J. Mol. Sci. 2019, 20, 6113. https://doi.org/10.3390/ijms20246113
Xu P, Zhang M, Qian P, Li J, Wang X, Wu Y. iTRAQ-Based Quantitative Proteomic Analysis of Digestive Juice across the First 48 Hours of the Fifth Instar in Silkworm Larvae. International Journal of Molecular Sciences. 2019; 20(24):6113. https://doi.org/10.3390/ijms20246113
Chicago/Turabian StyleXu, Pingzhen, Meirong Zhang, Ping Qian, Jiawei Li, Xueyang Wang, and Yangchun Wu. 2019. "iTRAQ-Based Quantitative Proteomic Analysis of Digestive Juice across the First 48 Hours of the Fifth Instar in Silkworm Larvae" International Journal of Molecular Sciences 20, no. 24: 6113. https://doi.org/10.3390/ijms20246113