Selection of possible marker peptides for the detection of major ruminant milk proteins in food by liquid chromatography-tandem mass spectrometry
Selection of possible marker peptides for the detection of major ruminant milk proteins in food by liquid chromatography-tandem mass spectrometry
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DOI：
10.1007/s00216-010-4422-0
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发表时间：
2011-01-01
影响因子：
4.3
通讯作者：
Baumgartner, Sabine
中科院分区：
文献类型：
--
作者：
Ansari, Parisa;Stoppacher, Norbert;Baumgartner, Sabine
这项工作的目的是确定肽，它可以作为标志物，用于识别食品样品中的牛奶过敏原。重点放在两个酪蛋白（α-和β-酪蛋白）和两个乳清蛋白（α-乳白蛋白和β-乳球蛋白）。计算机胰蛋白酶消化提供了有关预期肽的初步信息。四个牛奶过敏原的胰蛋白酶消化后，通过反相高效液相色谱和四极杆串联质谱（LC-MS/MS）的组合获得的分析数据，导致26肽的鉴定。合成了这些肽中的7种，并用于LC-MS/MS系统的校准。通过BLAST搜索寻求所选肽的种属特异性。在所选的肽中，发现仅来自β-乳球蛋白的LIVTQTMK（m/z 467.6，电荷2+）是牛乳特异性的，并且可以用作标记物。另外两种肽，来自α-酪蛋白的FFVAPF-PEVFGK（m/z 693.3，电荷2+）和来自β-酪蛋白的GPFPIIV（m/z 742.5，电荷1+）也存在于布法罗乳中。其他四种肽也出现在其他物种的乳中，可用作反刍动物乳的标记物。使用这七种肽，开发了基于多分析物MS的方法。在建立该方法时，首先将其应用于不同的乳制品样品，然后应用于巧克力和空白样品，食品样品中的肽含量可低至1 ng/mL。最后，测量加标样品，其中可以以高回收率（超过50%）检测到靶肽。
The aim of this work was the determination of peptides, which can function as markers for identification of milk allergens in food samples. Emphasis was placed on two casein proteins (alpha- and beta-casein) and two whey proteins (alpha-lactalbumin and beta-lactoglobulin). In silico tryptic digestion provided preliminary information about the expected peptides. After tryptic digestion of four milk allergens, the analytical data obtained by combination of reversed-phase high performance liquid chromatography and quadrupole tandem mass spectrometry (LC-MS/MS) led to the identification of 26 peptides. Seven of these peptides were synthesized and used for calibration of the LC-MS/MS system. Species specificity of the selected peptides was sought by BLAST search. Among the selected peptides, only LIVTQTMK from beta-lactoglobulin (m/z 467.6, charge 2+) was found to be cow milk specific and could function as a marker. Two other peptides, FFVAPF-PEVFGK from alpha-casein (m/z 693.3, charge 2+) and GPFPIIV from beta-casein (m/z 742.5, charge 1+), occur in water buffalo milk too. The other four peptides appear in the milk of other species also and can be used as markers for ruminant species milk. Using these seven peptides, a multianalyte MS-based method was developed. For the establishment of the method, it was applied at first to different dairy samples, and then to chocolate and blank samples, and the peptides could be determined down to 1 ng/mL in food samples. At the end, spiked samples were measured, where the target peptides could be detected with a high recovery (over 50%).