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Abstract
Manduca sextais a lepidopteran model widely used to study insect physiological processes, including innate immunity. In this study, we explored the proteomes of cell-free hemolymph from larvae injected with a sterile buffer (C for control) or a mixture of bacteria (I for induced). Of the 654 proteins identified, 70 showed 1.67 to >200-fold abundance increases after the immune challenge; 51 decreased to 0-60% of the control levels. While there was no strong parallel between plasma protein levels and their transcript levels in hemocytes or fat body, the mRNA level changes (i.e.I/C ratios of normalized read numbers) in the tissues concurred with their protein level changes (i.e.I/C ratios of normalized spectral counts) with correlation coefficients of 0.44 and 0.57, respectively. Better correlations support that fat body contributes a more significant portion of the plasma proteins involved in various aspects of innate immunity. Consistently, ratios of mRNA and protein levels were better correlated for immunity-related proteins than unrelated ones. There is a set of proteins whose apparent molecular masses differ considerably from the calculatedMr's, suggestive of posttranslational modifications. In addition, some lowMrproteins were detected in the range of 80 to >300 kDa on a reducing SDS-polyacrylamide gel, indicating the existence of highMrcovalent complexes. We identified 30 serine proteases and their homologs, 11 of which are known members of an extracellular immune signaling network. Along with our quantitative transcriptome data, the protein identification, inducibility, and association provide leads toward a focused exploration of humoral immunity inM. sexta.
Highlights
From the ‡Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078; §Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078
Proteomics Workflow and Protein Identification—To identify M. sexta plasma proteins, especially those involved in innate immunity, we collected hemolymph samples from the larvae injected with buffer or bacteria and analyzed them by the gel-LC-MS/MS approach (Fig. 1) [25, 28]
Independent proteins in the database, and 150 were known or predicted to be defense-related based on homology (Table S2). This represents a dramatic increase in coverage of the M. sexta hemolymph proteome, reflecting quality of the protein database, sensitivity increase due to prefractionation, as well as our selection of day 1, fifth instar larvae for injection
Summary
From the ‡Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK 74078; §Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078. We describe the identification of larger hemolymph proteins, their differential expression after a bacteria challenge, and correlations of induced changes between plasma proteins and the corresponding mRNAs in fat body and hemocytes. Proteins in the gel slices were identified by searching MS/MS data against a sequence database of M. sexta proteins.
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