Professor Shen Lirong, Zhejiang University: The ultra-efficient liquid chromatography series triple four-stage bar mass spectrometry quantitative method of Wang pulp main protein and its application in honey authenticity detection

Original title: Professor Shen Lirong, Zhejiang University: Ultra-efficient liquid chromatography series triple four-stage mass spectrometry quantitative method of Wang pulp main protein and application in honey authenticity detectionhighlights
developed the ultra-efficient liquid chromatography series triple four-stage chromatography (UPLC-TQMS) detection method
using
(UPLC-TQQ) detection method The MS) method screened out the quantitative detection specific peptide markers of MRJPs
and constructed the MRJP 1
to 3 content threshold database of 70 real honey samples
and showed the application prospect of MRJP 1 to 3, the endogenemic marker of real honey analysis.
adulteration
is a long-standing problem in the international honey industry, which has a very serious impact on the healthy development of the honey industry. Although the existing honey adulteration detection method can effectively and targeted control the known adulteration phenomenon, but the lack of early warning and rapid response capacity for new adulteration, there are detection technology development behind adulteration technology and adulteration supervision costs are too high and other shortcomings, resulting in difficult to completely eliminate the occurrence of honey adulteration phenomenon. In recent years, biomarker detection technology based on proteomics and metabolomics has been widely used in the field of food authenticity detection, which has laid the foundation for the study of the honey endo-protein-major jelly proteins (MAJOR royal jelly proteins, MRJPs) as the authenticity detection marker of honey. However, due to the low content of the main protein in honey, there is still a lack of accurate quantitative detection and reliable methods at home and abroad.
In recent years, Professor Shen Lirong of Zhejiang University's Department of Food Science and Nutrition, in cooperation with Zhang Jinjie Researcher, led the Qinhuangdao Customs Technology Center National Honey Key Laboratory, Zhejiang Grain and Oil Quality Testing Center, Hangzhou Bee Language Bee Industry Co., Ltd., carried out MRJPs in honey. Sex markers, using ultra-efficient liquid chromatography series triple four-stage bar mass spectrometrography (UPLC-TQMS) detection technology to establish three main members of the main protein in honey - MRJP 1 to 3 quantitative analysis method system research. After a large number of UPLC-TQMS testing by carbon isotope ratio mass spectrometization and pollen testing and determination as authentic honey samples, a database of MRJP 1 to 3 content in real honey was established, and a honey authenticity detection threshold was set. Through analog adulteration, commodity honey detection, compared with carbon isotope ratio mass spectrometrometization and pollen detection method, the effectiveness of this method in honey authenticity detection is verified, providing key techniques and basis for the international accurate detection and judgment of honey authenticity, and the establishment of new precision testing standards.
qualitative identification of Results
's main protein M
RJP

using ultra-efficient liquid chromatography in series flight time mass spectrometry (UPLC-Q-TOF) The main protein peptide of the bee king pulp of trypsinase is qualitatively tested, and the data obtained are compared and analyzed successfully by ProteinLynx Global Server (PLGS) software, using the peptide sequence information of MRJPs obtained from the UnProt database. A total of 108 peptides were identified in the first-stage sequence data of 8 king pulp main proteins (MRJP1 to 7 and MRJP 9), of which 24 belonged to MRJP1 and 28 belonged to MRJP2,2 1 belongs to MRJP3, 14 belongs to MRJP4, 11 belongs to MRJP5, 3 belongs to MRJP6, 6 belongs to MRJP7, 1 belongs to MRJP9. The polypeptide coverage of 8 king pulp main proteins (MRJPs) was distributed from 1.7 to 76.6% (Table 1).
Table
1MRJPs and their enzyme-dissected peptides Analysis Results
-
Peptide Coverage Estimated by Protein Lynx Global Server Version 2.5 Software
M
RJP in Honey
s candidate marker peptide screening
based on UPLC-TQMS on all the candidate peptides in honey pulp protein repetitive detection and analysis, from 108 candidate peptides in the initial screening peak area is higher, of which the reproducible has 34 peptides. Then, using the two indicators of easy-to-enzymatic and post-enzymatic stability of peptides as appropriate indicators of peptide screening criteria, 34 peptides of 7 king pulp proteins were analyzed. According to two evaluation indicators, all candidate peptides are divided into five categories: A. easy-to-enzyme and stable; B.easy-to-enzymatic but unstable; C. not enzymatic but stable; D. non-enzymatic and unstable; and E. irregular. After a comparative analysis from the high level, it was finally successfully selected for the screening of an MRJP 1 specific marker peptide belonging to Class B: YNGVPSSLNVISK, belonging to Class A for the screening of an MRJP 2 specific marker peptide: TLQMIAGMK;
Table 2Multi-reaction monitoring (MRM)
-
isotope-labeled peptides
-
quantitative ions, L-
-
-Fmoc-Leu-OH (
13
C
6,
,
15
N) The establishment and methodological verification of MRJP 1 to 3 quantitative methods in
honey
3 specific marker peptide standards and their isotope-labeled peptides of MRJP 1 to 3 are synthesized respectively, and the UPLC-TQMS detection method of MRJP 1 to 3 quantitative detection is established. And through the standard curve, precision, recovery rate, quantitative limit analysis, the method is methodologically verified, the results show that the relevant requirements of CLSI C62-A of the American Association of Clinical Laboratory Standards are met. Using UPLC-TQMS method, MRJP 1 to 3 quantitative analysis was carried out on three common honey adulterated syrups, such as artichoke honey, rapeseed honey, jingle honey and lychee honey. The results showed that mrJP 1 to 3 and other 3 specific marker peptides were contained in artichoke honey, jingle honey, rape honey and lychee honey, which could not be detected in any kind of honey adulteration such as corn syrup, rice syrup or high fructose syrup, and confirmed that MRJP 1 to 3 belongs to the specific endogenetic protein of honey. The results of the detection and analysis of simulated syrup adulterated honey showed that the concentration of mixed high fructose syrup was negatively related to the content of MRJP 1 to 3 in the honey sample (
R
2
>0.99), which proved that the use of MRJP 1 to 3 as the honey endogenic marker protein had the feasibility of being applied to adulterated honey testing (Figure 1).