关键词: 液相色谱-串联质谱, 特异性肽段, 稳定同位素标记肽段, 酶解条件, 过敏蛋白质, 稻米

Abstract:

Rice is an important cereal that is consumed as both an energy and protein source by a large proportion of the population worldwide. However, clinical studies have found that rice grains are responsible for cases of severe asthma, eczema, and atopic dermatitis in some adult patients. Several allergenic proteins have been identified and biochemically and immunochemically characterized from rice grains. These include α-amylase/trypsin inhibitors, glyoxalase Ⅰ, and α-globulin. In this study, we proposed an approach for the simultaneous quantification of three allergenic proteins in rice and its products, based on a stable isotope-labeled signature peptide standard and liquid chromatography-tandem mass spectrometry. Samples of rice and products were extracted by a salt solution, hydrolyzed by Lys-C and Trypsin, and purified by C18-SD. The linear ion trap-high resolution mass spectrometry (LTQ-Orbitrap) and Protein Discovery software were used to acquire and identify allergenic proteins in rice samples. In present study, three proteins including seed allergenic protein RAG2, glyoxalase Ⅰ, and 19 kDa globulin were identified. To establish a stable quantitative detection method, the signature peptides selected from the identified enzymatic hydrolysis peptides must have greater abundance and higher specificity as characteristic peptides. Three corresponding signature peptides in rice were screened based on the principles of previous study, and were validated through comparisons of the basic local alignment search tool (BLAST) with the NCBI and UniProt databases. The three signature peptides were successively eluted by liquid chromatography and separated on a Poroshell column. They were then detected by positive electrospray ionization (ESI⁺) in multiple reaction monitoring mode and quantified by an isotope dilution method. To achieve an improvement in the detection sensitivity and specificity, mass spectrometry parameters, such as the collision energy of three ion pairs of each peptide, were optimized. Three recombinant allergenic proteins and the winged stable isotope-labeled signature peptide standard were synthesized. These were then used to compare the effects of different enzymatic conditions, including hydrolysis solvents containing sodium dodecyl sulfate (SDS) with different contents, as well as the enzymes and their amounts, on the digestion efficiency. The data showed that the digestion efficiency of the three proteins could be improved to 65.7%-97.3% when 1 g/L of the SDS-containing hydrolysis solvent, and the combined digestion strategy of Lys-C and Trypsin, were adopted in the enzymatic process. These results indicate the following inferences: a small amount of SDS (1 g/L) in the enzymatic hydrolysis system is beneficial to complete protein denaturation, a Lys-C and Trypsin combined digestion strategy can complement the shortcomings of the two enzymes and improve the digestion efficiency, and the recoveries of the three proteins was not significantly increased by increasing the amount of enzyme when the ratio of protein to enzyme reached more than 20∶1. The method displayed good linearity in the range of 1-200 nmol/L with the correlation coefficients greater than 0.9972. The limits of detection and limits of quantification of the three proteins were 3 mg/kg and 10 mg/kg, respectively. The average recoveries of the three proteins spiked at three levels in different matrices ranging between 80.6%-103.7%, with the intra-day and inter-day precision less than 11.5%. Due to its high stability, excellent sensitivity, and simple operation, this method presents a wide range of application prospects in the analysis of the three allergenic proteins in different rice and rice food products.

Key words: liquid chromatography-tandem mass spectrometry (LC-MS/MS), signature peptide, stable isotope-labeled peptide, digest condition, allergenic protein, rice