Results
Identified novel royal jelly proteins
To expand the number of known proteins in the RJ proteome, RJ proteins were extracted and digested with insolution methods and analyzed with double high LC-MS/MS (orbitrap-based MS). A total of 42 nonredundant proteins were confidentially identified, of which 13 proteins were novel (Table 1 and Additional file 1: Table S1).
The 42 identified proteins in RJ were classified on the basis of their biological process and molecular function and annotated by gene ontology. In the YELLOW/MRJP family, a new protein, yellow-e3 precursor, was identified. Of the 12 proteins related to metabolic processes, five novel proteins were identified: lysosomal pro-X carboxypeptidase, lysosomal aspartic protease, membrane metallo-endopeptidase 1, matrix metalloproteinase 14, and pancreatic triacylglycerol lipase. Among the 14 proteins associated with health improvement, six were reported here for the first time: venom dipeptidyl peptidase 4 precursor, venom serine protease 34, hymenoptaecin precursor, venom protease, hypothetical protein LOC408570, lysozyme isoform 1. One of the four proteins involved in development processes was novel, protein CREG 1 (Table 1and Additional file 1: Table S1). Interestingly, the majority of the newly-identified proteins were related both to metabolic processes (accounting for 38.5% of all novel proteins) and health promotion activities (46.2% of all novel proteins).
Table 1 Identification of proteins in royal jelly
Note: All of the identified proteins are of Apis mellifera origin. Accession is the unique number given to mark the entry of a protein in the database of Apis (downloaded April 2012, version 4.5 of the honeybee genome) using in-house PEAKS software (version 6.0, Bioinformatics Solutions Inc.). “-10logP” is the score calculated by PEAKS software. Sequence coverage is the ratio of the number of amino acids in every peptide that matches with the mass spectrum divided by the total number of amino acids in the protein sequence. Matches are the number of experiment fragmentation spectra paired to a theoretical segment of protein.
The number of unique peptides refers to the peptide sequences that are unique to an individual parent protein sequence. SignalP refers to the result researched with SignalP 4.1. PSORT refers to the result researched with PSORT II. “*” indicates the protein identified as novel in royal jelly. “√” indicates the protein identified with signal peptide by SignalP 4.1. “#” indicates the protein identified as extracellular by PSORT II. “-” indicates the protein did not be researched with SignalP 4.1or PSORT II.
Mapping N-glycosylated sites
To attain a comprehensive map of N-linked glycosylation sites in RJ, RJ proteins were extracted and enriched by two different enrichment methods (hydrazide and lectin), after which the N-glycosylation peptides were analyzed by two different double high LC-MS/MS (orbitrapbased MS and triple TOF-based MS). The introduction of 18O-water in the process of PNGase F digestion added to confidence to the identification of N-glycopeptides. An example spectrum of N-glycopeptide is shown in Figure 1 (for all other spectra see Additional file 2: Figure S1). Overall, 25 N-glycoproteins carrying 53 unique N-linked glycosylation sites represented 60% of the total identified proteins in RJ. Among the 53 identified N-linked glycosylation sites, 42 were confidentially mapped in RJ proteins for the first time (Table 2).
Table 2 Identification of glycosylated proteins, peptides and their glycosylation sites in royal jelly proteins
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