Protein fingerprinting and quantification of β-casein variants by ultra-performance liquid chromatography-high-resolution mass spectrometry
Protein fingerprinting and quantification of β-casein variants by ultra-performance liquid chromatography-high-resolution mass spectrometry
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DOI：
10.3168/jds.2019-16273
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发表时间：
2020-02-01
影响因子：
3.5
通讯作者：
Affolter, M.
中科院分区：
文献类型：
--
作者：
Fuerer, C.;Jenni, R.;Affolter, M.
婴儿配方食品不断发展，因为添加了新的蛋白质成分，使其更接近母乳的蛋白质谱;然而，目前缺乏用于表征和定量此类配方食品中主要乳蛋白的精确分析方法。本文描述了一种用于完整蛋白质的超高效液相色谱-高分辨率质谱法，该方法可以有效地检测、识别和表征成分和最终产品中的主要乳蛋白及其蛋白质型（磷酸化状态、糖化程度、遗传变异等），重点是检测和定量婴儿配方奶粉中β-酪蛋白的特定遗传变异。方法灵敏度允许检测A2基婴儿配方奶粉中的β-酪蛋白A1，检测限为2%（每100 g总β-酪蛋白中β-酪蛋白A1的克数）。蛋白质糖化以线性方式影响信号强度，这允许在根据其测量的糖化指数校正后从其质谱信号定量蛋白质。该方法用于婴儿配方奶粉中β-酪蛋白的定量。重复性范围为2 - 3%，中间重现性范围为5 - 9%。计算的β-酪蛋白量范围为基于配方和公布的牛奶蛋白质谱的值的77%至110%。总而言之，该方法可用于一般指纹图谱以及乳基成分和产品中单个主要乳蛋白的特定表征和定量。
Infant formulations arc constantly evolving as novel protein ingredients are added to make them more closely mimic the protein profile of human milk; however, precise analytical methods for characterizing and quantifying the major milk proteins in such formulations are currently lacking. This article describes an ultra-performance liquid chromatography-high-resolution mass spectrometry method for intact proteins that can efficiently detect, identify, and characterize the major milk proteins and their proteoforms (phosphorylation status, degree of glycation, genetic variants among others) in ingredients and final products, with an emphasis on detecting and quantifying specific genetic variants of beta-casein in infant formulas. Method sensitivity allows detection of beta-casein A1 in A2-based infant formulas with a limit of detection of 2% (grams of beta-casein A1 per 100 g of total beta-casein). Protein glycation affects signal intensity in a linear fashion, which permits proteins to be quantified from their mass spectrometry signals after correction according to their measured glycation index. The method was validated for the quantification of beta-casein in infant formulas. Repeatability ranged from 2 to 3% and intermediate reproducibility from 5 to 9%. Calculated beta-casein amounts ranged between 77 and 110% of the values based on formulations and published protein profiles for milk. Altogether, this method can be used for general fingerprinting as well as specific characterization and quantification of individual major milk proteins in dairy-based ingredients and products.