N-glycosylation modification of proteins has reported to improve the health of living organisms through antibacterial activity [68], antioxidant activity [69], and antihypertension [70]. For instance, glucose oxidase acts as a natural preservative and a bactericide by reducing oxygen to a hydrogen peroxide formation [71]. Venom dipeptidyl peptidase 4 precursor could enhance immune response

activity by stimulating the T-cells of mammalia [39,40]. Antithrombin-III, Apolipophorin-III protein precursor, and toll-like receptor 13 all play key roles in promoting the innate immunity of honeybee larvae [11,72-77]. MRJP 1 has potential antitumor effects by stimulating macrophages to release TNF-α [61]. In addition, the glycosylated protein affects cell proliferation and regulates circadian rhythm [78]. Chitinase, as a growth factor, stimulates the proliferation and polarization in Drosophila [79]. Protein takeout helps regulation of circadian rhythms and feeding behavior in Drosophila [80]. Overall, the glycosylation of these RJ proteins suggests that they may be involved in the above biological roles benefitting both honeybee and humans.

An oligosaccharide unit attached to the polypeptide at the site of occupancy has reported to improve solubility, folding, and half-life of the glycoprotein [81]. Most glycosylated RJ glycoproteins (~ 50%) carried a single N-glycosylation site, ~ 20% carried two or three sites, and only a few carried four or five sites. In addition, the identified conservative motif of amino acid sequence of N-glycosylated RJ peptides may have structural and functional importance for RJ proteins in future studies [82,83]. Although the glycan linkages associated with the glycosylation sites demand further investigation, this new catalog of knowledge may prove helpful in elucidating the biological implications of glycosylation for the RJ proteins through synthesizing the glycan to the identified sites. This is possible because N-glycosylation is a conserved process of post-translational modification in a diversity of proteins in eukaryotic organisms [18], and the established N-linked glycosylation system in the Campylobacter system could transfer a functional N-linked glycoprotein into Escherichia coli [84]. This provides promising glycoengineering possibilities for producing modified RJ peptides that could produce a protein with desirable pharmacokinetics and biological activity.

Conclusions

A total of 13 novel proteins and 42 novel N-linked glycosylation sites in 25 N-glycosylated RJ proteins have been identified here. Of the glycosylated proteins, most were related to metabolic activities and carry multiple N-linked glycosylation sites. This is important for young larvae and the fertile egg-laying queen, since their high metabolic fuel demands may be achieved through the regulation of the enzymatic activities related to the metabolic process. The glycosylated proteins related to the improvement of human health suggest N-glycosylation plays a key role in helping RJ proteins accomplish their biological functions. The large scale assignment of N-glycosylated sites represents a crucial first step toward systematically revealing the functionality of N-glycosylated RJ proteins. In

addition, the identification of novel proteins mainly associated with metabolic process and promoting human health significantly extend the proteome coverage of RJ.

Methods

Sample preparation

RJ was collected as a pooled samples from 250 queen cell cups from each of five colonies of Apis mellifera ligustica at the apiary of the Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing. RJ proteins were extracted immediately after harvest according to previously described methods [72]. The resulting pellets were divided into three parts for the following analyses.

In-solution digestion

The first part of the above protein pellets (1 mg RJ/100 μl buffer) was dissolved in 40 mM of (NH4)HCO3 (Sigma). The sample was used for in-solution digestion (trypsin, modified sequencing grade, Promega) according to our previous methods [72]. Finally, the peptide-containing solution containing peptides was concentrated using a Speed-Vac system (RVC 2-18, Marin Christ) for MS/MS

analysis.

N-linked glycopeptide enrichment with hydrazide chemistry

The second part of the protein pellet (1 mg RJ/100 μl buffer) was suspended in a coupling buffer [100 mM sodium acetate (Sigma), 150 mM NaCl (Sigma), pH 5.5] and then prepared by enriching the N-linked glycopeptides with hydrazide resin according to the method of Zhang et al. [54]. Briefly, the glycoproteins were oxidized, and these oxidized proteins were captured by hydrazide resin (Bio Rad). The captured glycoproteins were digested overnight by trypsin. Afterwards, the digested glycopeptides were further digested by PNGase F (NEB) to remove the glycans attached to the proteins, and were labeled by H2 18O (Sigma) to confidently assign the N-glycosylation sites. Finally, the collected supernatant was concentrated using a Speed-Vac system for MS/MS analysis.

N-linked glycopeptide enrichment with lectin

The remaining third of the protein pellets (1 mg RJ/100 μl buffer) was suspended in 8 M of urea in 100 mM of Tris-HCl (pH 8.5) and the mixture was transferred into an Ultracel YM-10 10,000 MWCO centrifugal filter unit (Millipore) and digested by trypsin overnight. Following this, the digested peptides were prepared for enrichment by the N-linked glycopeptides with lectin (mixture with Concanavalin A, wheat germ agglutinin, and RCA120 agglutinin) (Sigma) and a second digestion

by PNGase F and H2 18O, labeled according to N-Glyco- FASP [85]. Finally, the labeled peptide sample was concentrated using a Speed-Vac system for MS/MS analysis.

Mass spectrometric analysis

The three peptide samples were analyzed on the QExactive mass spectrometer (Thermo Fisher Scientific) coupled to an Easy-nLC 1000 (Thermo Fisher Scientific) via a nanoelectrospray ion source. Full MS scans were acquired with a resolution of 70,000 at m/z 400 in the orbitrap analyzer. The 20 most intense ions were fragmented by higher energy collisional dissociation (HCD). The HCD fragment ion spectra were acquired in the orbitrap analyzer with a resolution of 17,500 at m/z 400.

Reverse phase chromatography was performed with a binary buffer system consisting of buffer A (0.1% formic acid, 2% acetonitrile in water) and buffer B (0.1% formic acid in acetonitrile). The peptides were separated with a flow rate of 350 nl/min in the EASY-nLC 1000 system by the following gradient program: from 3 to 8% buffer B for 5 min, from 8 to 20% buffer B for 55 min, from 20 to 30% buffer B for 10 min, from 30 to 90% buffer B for 5 min, and 90% buffer B for 15 min.

To obtain a comprehensive map of N-glycosylation sites in RJ proteins, the glycopeptide samples were also analyzed by electrospray ionization, quadruple time-offlight system (Triple TOF 5600, AB SCIEX) coupled with an Eksigent nano liquid chromatography system (Eksigent Technologies). Separation was performed using a selfpacked in-house 150 × 0.075 mm 300A pore C18 column, at a flow rate of 330 nl/min. The peptides were eluted with a spectral acquisition speed of 20 MS/MS per second, using the following gradient program: from 5 to 8% buffer B (0.1% formic acid in acetonitrile) for 0.1 min, from 8 to 30% buffer B for 22 min, from 30 to 48% buffer B for 6 min, from 48 to 80% buffer B for 1 min, and 80% buffer B for 5 min.