Note: All of the identified proteins are from Apis mellifera. 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). “-10logP” is the score calculated by PEAKS software (version 6.0, Bioinformatics Solutions Inc.). Charge is the number of the carrying charge of the peptide. No. of spectra is the number of the spectrum of the peptide generated by mass spectrometry. Amino acid residue No. corresponds to the position of the N-terminal and C-terminal amino acid of the peptide in the protein sequence. Glycosylation site indicates the position of the N-glycosylated amino acids of the peptide in the protein sequence. Orbitrap refers the peptides analyzed by the Q-Exactive mass spectrometry (Thermo Fisher Scientific). Triple TOF refers the peptides analyzed by Triple TOF 5600 (AB SCIEX). Lectin denotes N-glycopeptides enriched by the lectin method. Hydrazide represents N-glycopeptides enriched by hydrazide chemistry. “√” indicates that peptide is identified by the corresponding enrichment method and mass spectrometer. “a” is the known site in the known protein. “b” is the potential site (bioinformatics has predicted these potential sites in UniProt Database (updated April 2013), and it is experimentally confirmed in this study) in the known protein. “c” is the potential site in the novel protein. “d” denotes the novel site in the known protein. “e” is the novel site in the novel protein.

In the YELLOW/MRJP family, seven proteins were identified as N-glycoproteins, glycosylated on 12 unique peptides, each carrying a single N-glycosylated site (Table 2). Of the proteins involved in metabolic processes, seven were N-glycosylated on 16 unique N-glycopeptides: all but on each contained a single N-glycosylation site and one unique N-glycopeptide carried two sites (Table 2). Of the proteins related to health improvement, seven were found N-glycosylated on 18 unique peptides, and each peptide had a single N-glycosylated site (Table 2). Of the two proteins implicated in the regulation of morphological development, IDGF 4 was N-glycosylated on one unique peptide with a single site, and N-glycosylated protein takeout had one unique peptide carrying two sites (Table 2). Finally, two identified N-glycoproteins with unknown functions each had one unique peptide harboring a single N-glycosylated site (Table 2).

Among those 53 unique N-glycosylated sites, 21 were identified by lectin enrichment alone, eight were uniquely identified by the hydrazide enrichment, and 18 were identified by both enrichment methods using orbitrap-based MS (Figure 2A). Similarly, eight N-glycopeptides were specifically identified by the lectin enrichment protocol, two were specifically identified by the hydrazide chemistry, and six were identified by both enrichment methods using triple TOF-based MS (Figure 2B). In general, 29

N-glycopeptides were uniquely identified by orbitrapbased MS, four were uniquely identified by triple TOFbased MS, and 10 were identified by both MS systems using the lectin enrichment method (Figure 2C). Likewise, 18 N-glycopeptides were identified by orbitrap-based MS alone, and eight were identified by both types of LC-MS/MS instruments with adoption of hydrazide enrichment (Figure 2D).