Gall aphid saliva LC-MS/MS
Aphid saliva plays an essential role in the interaction between aphids and their host plants. Several aphid salivary proteins have been identified and analyzed. However, none of the characterized salivary proteins are from galling aphids. Here we analyzed the salivary proteins from the Chinese gall aphid, Schlechtendalia chinensis using LS-MS/MS analysis. A total of 31 proteins were identified directly from secreted saliva collected via artificial diet, and 141 proteins were identified from protein extracts derived from dissected salivary glands. Among these identified proteins, 17 were found in both secreted saliva and dissected salivary glands. In comparison with salivary proteins identified from three other free living aphids, the most striking feature of the salivary protein from S. chinensis is the existence of high proportion of proteins with binding activity, including DNA binding, protein binding, ATP binding and ion binding proteins et al. We speculate that these binding proteins may be involved in induction of gall formation. Our results provide a framework for future research to elucidate the molecular basis for gall induction by S. chinensis.
Sample Processing Protocol
Insects: A S. chinensis colony was maintained on the hosts, R. chinensis and Plagiomnium maximovicgii under natural conditions at the Resources insect Research Institute, Chinese Academy of Forestry, Kunming, China. Wingless fundatrigeniae were obtained from fresh galls on R. chinensis trees. Secreted saliva from wingless fundatrigeniae was collected following a modified protocol of Carolan et al. (2009). Salivary glands were dissected from wingless fundatrigenia transferred from the fresh gall following a modified protocol of Mutti et al (2006). Saliva collection and quantification: Wingless fundatrigeniae were obtained from fresh galls and put into a Petri dish. The fundatrigeniae were hungered for 24 hours. After that, about 1000 fundatrigeniae were transferred onto a ParafilmTM sachet with ultra pure water as the diet for 24 hours in 20℃ with relative humidity 90%. Secreted saliva contained in the diet was collected using a syringe. Twenty four sachets were pool together and combined with equal volume of PBS, and then centrifuged for 30 minutes with 7500 rpm through a 5000 molecular weight cut-off (MWCO) polyethersulfone (PES) membrane at 4℃ (Sigma 3K30 Centrifuges, Germany). The concentrate was then transferred to another centrifuge tube, and centrifuged for 50 minutes with 10000 rpm through a 3000 MWCO PES membrane. The extract was resuspended in 100 ul lysis buffer (LB; 9.5 M urea, 2% CHAPS, 0.8% Pharmalyte pH 3-10, 1% DTT) for gel electrophoresis.
Data Processing Protocol
The 1-dimensional gel electrophoresis-separated proteins were subjected to LC-MS/MS on a Q Exactive mass spectrometer (Thermo Fisher Scientific, UK). Peptides were eluted off the trap and separated using a Biobasic C18 at a flow rate of 200 nl/min and gradient of 5-95% ACN over 45 min. All data were acquired with the mass spectrometer operating in automatic data-dependent switching mode. A zoom scan was performed on the five most intense ions to determine charge state prior to MS/MS analysis. Protein identification from the MS/MS data was performed using the TurboSEQUEST algorithm in BioWork v. 3.2 to correlate the data against ACYPI proteins V2.1b, the official protein models available at http://www.aphidbase.com/aphidbase/downloads. The genomic sequence and databases used for peptide/ protein searches were produced and made available by the International Aphid Genomics Consortium (IAGC; www.aphidbase.com) and the UniProt (www.aphidbase.org) (Beijing proteome research center, China). The following search parameters were used: precursor ions mass tolerance of ±0.1 Da, fragment ions tolerance of ±0.2 Da, and a maximum of two missed cleavages sites allowed. Peptides obtained from the tryptic-digested bands were matched to peptides tags originating from the data base of Pea aphid protein (ACYPI proteins V2.1b) (http://www.aphidbase.com), UniProt (http://www.uniprot.org) and Flybase (http://www.flybase.org).
Yang Z, Ma L, Francis F, Yang Y, Chen H, Wu H, Chen X. Proteins Identified from Saliva and Salivary Glands of the Chinese Gall Aphid Schlechtendalia chinensis. Proteomics. 2018 18(9):e1700378 PubMed: 29577599