RJ provides efficient energetic fuels for the fast development of larvae and the egg-laying queen through the metabolism of sugars, lipids, and proteins [2]. The identification of a high number of proteins related to the metabolism of sugar, lipids, and proteins suggests that the honeybee has an evolutionary strategy of using RJ to fulfill the enormous energy requirement of the fast-developing

larvae and the egg-laying queen through these metabolic pathways. Noticeably, five of the 13 novel proteins identified were associated with this category, indicating their biological importance as a source of metabolic fuel for ensuring the normal growth of honeybee larvae. Triacylglycerol lipase breaks down dietary fat, mainly triacylglycerol, to monoacylglycerol, and free fatty acids to supply the energy requirements of living organisms [36]. In addition, enzymes of lysosomal pro-x carboxypeptidase, lysosomal aspartic protease, membrane metalloendopeptidase, and matrix metalloproteinase 14, also participate in the metabolism of protein to produce energy [37,38].

RJ has been well to documented enhance immunity forhoneybees and to promote health for humans [2]. Among the 14 RJ proteins related to health promotion activities, six were identified as novel. Dipeptidyl peptidase IV is known to functionally suppress peritoneal dissemination and the progression of ovarian carcinoma, inhibit the malignant phenotype of prostate cancer cells, and promote the human immune system [39,40]. Venom serine protease 34 is part of a defense mechanism against intruding microorganisms and parasites in insects [41-43]. Hymenoptaecin can inhibit the viability of gram-positive and gram-negative bacteria, and provides wide-spectrum antibacterial protection for honeybees and humans [44,45]. Venom protease has fibrinogenolytic activity and is a strong antithrombotic agent in snakes [46]. Lysozyme isoform 1 is an important member related to the innate immunity of insects, efficiently protecting larvae from diseases and pests [47]. The hypothetical protein LOC408570 (93% homology with peroxidasin protein of Harpegnathos saltator) [48] has functions in phagocytosis and in defense against radioiodinations and oxidation [49].

The newly identified protein cellular repressor of E1Astimulated genes (protein CREG) might contribute to the promotion of differentiation of honeybee larvae by the enhancement of cell differentiation [50] as MRJP 1 does [51].

Mapping N-glycosylated sites

By using two complementary enrichment protocols (hydrazide chemistry and lectin resin) and two orbitrapbased and triple TOF-based double high LC-MS/MS systems, we have achieved an in-depth identification of 25 N-glycoproteins that mapped on to 53 sites on RJ proteins. Among these, 42 novel N-linked glycosylation sites were reported in RJ proteins. To the best of our knowledge, this is the most comprehensive assignment of the N-glycosylated sites of RJ.

Capturing the maximum number of glycopeptides is of great importance for the analysis of mapping glycosylated sites [52,53], and is achievable using the complementary enrichment of glycopeptides with techniques such as hydrazide chemistry and lectin based protocols. Hydrazide chemistry can efficiently capture glycoproteins once oxidized by sodium periodate, and is thus extremely useful for the identification of glycopeptides [54]. “Filter aided sample preparation” (FASP) is an N-glycopeptide enrichment protocol that uses a combination of different lectins to efficiently capture glycopeptides [55]. By adopting two different methods based on lectin and hydrazide enrichment, comprehensive glycosylation sites were assigned in RJ, namely 46 by lectin resin and 16 by hydrazide chemistry. Meanwhile, orbitrap-based MS seems to be more robust than Triple TOF-based MS in the identification of glycosylated sites in RJ, and the combined utilization of two different double high LC-MS/MS yielded identification of more number of N-glycosylated sites in RJ. Together, of the 53 N-glycosylation sites assigned in RJ proteins, 42 were mapped as novel. Nine potential N-glycosylation sites predicted by the Uniprot database (updated April 2013) were also verified. In addition, the only two known N-glycosylation sites [16] were repeatedly identified.

It is now known that blocking glycosylation could result in improper or incomplete folding of many polypeptides. These improperly folded polypeptides would not passing ER quality control [56] and would be retained in the ER and eventually degraded [57]. Given that RJ proteins contain 80-90% of MRJPs [9], glycosylation may help MRJPs reach their native conformation to accomplish their biological roles for both honeybees and humans [9]. Glycosylation also allegedly increases the solubility of proteins [58,59]. Therefore, the glycosylated YELLOW/MRJPs suggest their roles in promoting the solubility of YELLOW/ MRJPs in RJ to enhance their nutritive efficiency of assimilation [60,61]. Since glycosylated proteins have roles in immunity [62], the weak immunity of the young honeybee larvae (the first 48 h) may be promoted by feeding glycosylated MRJPs to ensure normal development [63]. This is in line with report that glycosylated MRJP 2 can effectively inhibit Paenibacillus larvae infection [16]. Glycosylation site occupancy modulates enzymatic activities by the attachment of glycans to peptide backbones [64]. Interestingly, the majority of glycosylated proteins identifed here are enzymes associated with the metabolic pathways of carbohydrates and proteins. For instance, three enzymes, lysosomal alpha-mannosidase, alpha-glucosidase, and glucosylceramidase, are involved in carbohydrate metabolism [65-67]. Four other enzymes, plasma glutamate carboxypeptidase, lysosomal pro-x carboxypeptidase, lysosomal aspartic protease, and membrane metalloendopeptidase, are implicated in the metabolism of proteins [37,38]. The high number of glycosylated proteins related to metabolic processes indicates the production of enough energy through the metabolism of carbohydrates and proteins for queen spawning and larval growth, which may be achieved by modulating the enzymatic efficiency [64].