Quantitation and Identification of Intact Major Milk Proteins for High-Throughput LC-ESI-Q-TOF MS Analyses

Cow’s milk is an important source of proteins in human nutrition. On average, cow’s milk contains 3.5% protein. The most abundant proteins in bovine milk are caseins and some of the whey proteins, namely beta-lactoglobulin, alpha-lactalbumin, and serum albumin. A number of allelic variants and post-translationally modified forms of these proteins have been identified. Their occurrence varies with breed, individuality, stage of lactation, and health and nutritional status of the animal. It is therefore essential to have reliable methods of detection and quantitation of these proteins. Traditionally, major milk proteins are quantified using liquid chromatography (LC) and ultra violet detection method. However, as these protein variants co-elute to some degree, another dimension of separation is beneficial to accurately measure their amounts. Mass spectrometry (MS) offers such a tool. In this study, we tested several RP-HPLC and MS parameters to optimise the analysis of intact bovine proteins from milk. From our tests, we developed an optimum method that includes a 20-28-40% phase B gradient with 0.02% TFA in both mobile phases, at 0.2 mL/min flow rate, using 75°C for the C8 column temperature, scanning every 3 sec over a 600–3000 m/z window. The optimisations were performed using external standards commercially purchased for which ionisation efficiency, linearity of calibration, LOD, LOQ, sensitivity, selectivity, precision, reproducibility, and mass accuracy were demonstrated. From the MS analysis, we can use extracted ion chromatograms (EICs) of specific ion series of known proteins and integrate peaks at defined retention time (RT) window for quantitation purposes. This optimum quantitative method was successfully applied to two bulk milk samples from different breeds, Holstein-Friesian and Jersey, to assess differences in protein variant levels.

牛乳是人类膳食中重要的蛋白质来源，平均而言，牛乳的蛋白质含量约为3.5%。牛乳中含量最丰富的蛋白质为酪蛋白（caseins）以及部分乳清蛋白，即β-乳球蛋白（beta-lactoglobulin）、α-乳白蛋白（alpha-lactalbumin）与血清白蛋白（serum albumin）。目前已鉴定出上述蛋白质的多种等位基因变异体与翻译后修饰形式，其出现频率会随动物品种、个体差异、泌乳阶段以及健康与营养状态而变化。因此，建立可靠的该类蛋白质检测与定量方法至关重要。传统上，乳中主要蛋白质的定量分析多采用液相色谱（liquid chromatography, LC）结合紫外检测法。然而，由于这些蛋白质变异体在一定程度上会发生共洗脱，因此采用多维分离技术有助于精准测定其含量。质谱法（mass spectrometry, MS）即可满足此类需求。本研究对多种反相高效液相色谱（reverse phase-high performance liquid chromatography, RP-HPLC）与质谱参数进行了测试，以优化牛乳中完整牛源蛋白质的分析方法。通过本次测试，我们开发出一套最优分析方法：流动相体系中均添加0.02%的三氟乙酸（trifluoroacetic acid, TFA），采用B相梯度为20%-28%-40%的洗脱程序，流速设定为0.2 mL/min，C8色谱柱柱温维持在75℃，质荷比扫描范围设为600~3000 m/z，扫描间隔为3秒。本次方法优化采用商业化外购的外标物质完成，已验证其电离效率、校准曲线线性、检出限（limit of detection, LOD）、定量限（limit of quantitation, LOQ）、灵敏度、选择性、精密度、重现性与质量准确度等各项性能指标。通过质谱分析，我们可利用已知蛋白质的特征离子序列的提取离子色谱图（extracted ion chromatograms, EICs），并在指定的保留时间（retention time, RT）窗口内对峰面积进行积分以实现定量分析。该最优定量方法已成功应用于两个分别来自不同奶牛品种的批量乳样，即荷斯坦-弗里生（Holstein-Friesian）与泽西牛（Jersey），以评估其蛋白质变异体水平的差异。