Objective To analyze differential proteins between excretory-secretory products (ESPs) of Trichinella spiralis and Trichinella pseudospiralis muscle larvae, and find out the causes for different immunosuppression between the two species and the potential functional proteins involved in the formation of cysts. Methods ESPs were collected from T. spiralis and T. pseudospiralis at the muscle larval stage. The bicinchoninic acid (BCA) detection method was used to determine the ESP protein concentrations. The quality of ESP proteins were assessed by sodium lauryl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), then the proteins were digested by filter-aided sample preparation (FASP), and labelled with isobaric tags for relative and absolute quantitation (iTRAQ) reagents. The samples with mixed labeling underwent reversed-phase high performance liquid chromatography (RP-HPLC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Then the uniprot databases for T. spiralis uniport and T. pseudospiralis were searched, to screen for homologous proteins based on the trusted proteins (scores > 80) and compare their differential expression. Standardized description of differential proteins was done with QuickGO. Nine proteins with significant difference were selected for qRT-PCR verification using the ΔΔCt method. Statistical analysis was performed using the SPSS 19.0 software. Results Based on the iTRAQ analysis, we identified 492 credible proteins for T. spiralis, of which 193 were suitable for quantification, and 535 credible proteins for T. pseudospiralis, of which 164 could be quantified. T. spiralis/T. pseudospiralis homology comparison revealed that 162 proteins with up-regulation and 31 with down-regulation. The GO analysis results showed that the molecular functions were mainly involved in ion-binding function (45 proteins), peptidase activity(18), oxidoreductase activity(12), and nuclease activity (8). As regards biological processes, the small molecule metabolism processes involved the largest number of proteins (15), followed by the carbohydrate metabolism processes (12), the biosynthesis processes (12), and DNA metabolism processes (8). Of various cell components, the cytoplasm and protein-containing complexes had highest enrichment of differential proteins. KEGG analysis showed that most of the proteins related to thiamine metabolism, sphingolipid biosynthesis, and phenylalanine, tyrosine and tryptophan biosynthesis were up-regulated. In addition, at the muscle larva stage, E5SXW8 (plancitoxin-1), E5SPA7 (putative trypsin), E5S387 (cystatin), E5SJH4 (squash family serine protease inhibitor), E5S554 (5′-nucleotidase), E5SQX1 (enolase) and E5SBA6 (L-asparaginase) had higher protein and transcription levels in T. spiralis than in T. pseudospiralis (P < 0.05), while E5RZQ6 (heat shock protein beta-1) and E5S7K8 (histone H2B) had a reversed pattern (P < 0.05). Conclusion The bioinformatics analysis on differential excretory-secretory products between T. spiralis and T. pseudospiralis muscle larva stage suggests that serine protease, cystatin, plancitoxin-1 and 14-3-3 protein may involve in the formation of cysts and immunomodulation.
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