Data analysis

Tandem mass spectra were retrieved using Xcalibur (version 2.2, Thermo Fisher Scientific) and AnalystTF (version 1.6, AB SCIEX) software. The MS/MS spectra files were searched against the sequence database (72,672 entries) using in-house PEAKS software (version 6.0, Bioinformatics Solutions Inc.). The database was generated from protein sequences of Apis (downloaded April 2012),augmented with sequences from Sacharomyces cerevisiae (downloaded April 2012), and a common repository of adventitious proteins (cRAP, from The Global Proteome Machine Organization, downloaded April 2012). The precursor and fragment mass tolerances were set to 50 ppm and 0.05 Da, respectively; tryptic cleavage specificity was set for up to two missed cleavages; carbamidomethyl (C, +57.02) as a fixed modification; and oxidation (M, +15.99) and deamidation (N, +0.98) as the only variable modifications for the RJ sample and oxidation (M, +15.99); deamidation (N, +0.98) and deamidation 18O (N, +2.998) for the glycopeptide enriched RJ sample. False discovery rate (FDR) was controlled using a target/decoy database approach for both protein identification and modified peptide identification, applying the cut-off FDR of 0.2%. Protein identification was accepted only if it contained at least two unique peptides. All of the identified glycopeptides and assigned sites were manually checked by applying the cut-off criteria: PEAKS score (-log10P) > 30 and FDR < 0.2%, and the majority of y or b ions could be detected with continuous and strong intensity peaks. To localize protein to the subcellular position, newly identified protein sequences were analyzed by PSORT II Prediction [30] (http://psort.hgc.jp/form2.html). To verify the presence of an N-terminal secretion signal peptide, the SignalP 4.1 Server [31] (http://www.cbs.dtu.dk/services/SignalP/) was also used. The D-cut off for signal-TM networks was set to 0.35. The putative functions of identified proteins and glycoproteins were annotated by searching against the Uniprot database (http://www.uniprot.org/) and grouped on the basis of their molecular behavior and biological process in gene ontology terms. All unique sequences of N-glycopeptides were submitted online to WebLogo [86] in order to extract the N-glycosylated site motif of RJ proteins.

Availability of supporting data

The data sets supporting the results of this article (Additional file 1: Table S1 and Additional file 2: Figure S1) are included within the article and its additional files.

Additional files

Additional file 1: Table S1. Identification of Proteins and Peptides in Royal Jelly Proteins. 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). “-10logP” is the score calculated by PEAKS software (version 6.0, Bioinformatics Solutions Inc.). Z is the number of the carrying charge of the peptide. “# Spec” is the number of the

spectrum of the peptide. “Start” and “end” correspond to the position of the N-terminal and C-terminal amino acids of the peptide in the protein sequence, respectively. RT is the retention time of the peptide in the mass spectrometry. “ppm” is the deviation value between the experimental mass and the theoretical mass of the peptide. C(+57.02) is the carbamidomethyl modification, M(+15.99) is the oxidation modification, and NQ(+0.98) is the deamidation modification.

Additional file 2: Figure S1. Spectra of N-glycosylated peptide in royal jelly proteins. The tandem mass spectrum of the N-glycosylated site is identified in peptide using 18O-water labeling.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

LZ and BH performed experiments related to RJ protein isolation and N-glycoprotein/peptide enrichment, data analysis and the writing manuscript. RLL, XSL, YF and MF performed bioinformatic data analysis. AYN and LHG performed the experiments of liquid chromatography tandem mass spectrometry. JKL participated in the design, coordination and interpretation of the results and the writing manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank Dr. Meghan Milbrath from Michigan State University, USA, for her help with the language of the manuscript. This work is supported by the earmarked fund for Modern Agro-industry Technology Research System (CARS-45).

Author details

1Institute of Apicultural Research, Chinese Academy of Agricultural Science, Beijing 100093, China. 2College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China. 3Bioengineering Department, Zhengzhou University, Zhengzhou 450001, China. 4Thermo Fisher Scientific (China) Co., Ltd, Shanghai 200021, China. 5Shanghai AB Sciex Analytical Instrument Trading Co., Ltd, Shanghai 200023, China.

Received: 18 October 2013 Accepted: 12 February 2014

Published: 16 February 2014