Identification of Racemization Sites Using Deuterium Labeling and Tandem Mass Spectrometry
Identification of Racemization Sites Using Deuterium Labeling and Tandem Mass Spectrometry
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DOI：
10.1021/ac101348w
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发表时间：
2010-08-01
影响因子：
7.4
通讯作者：
De Felippis, Michael R.
中科院分区：
文献类型：
--
作者：
Huang, Lihua;Lu, Xiaojun;De Felippis, Michael R.
氨基酸的外消旋化是在生物药物中观察到的常见化学降解途径，并且在合成肽中特别普遍。由于异构形式之间的同量异位质，通过质谱法鉴定外消旋氨基酸残基特别具有挑战性。在本文中，我们提出了一种新的方法相结合的稳定氘标记与碰撞诱导解离-串联质谱（CID-MS/MS）来阐明免疫球蛋白样品中的消旋氨基酸残基。免疫球蛋白G亚类IgG 1、IgG 2和IgG 4样品首先在质子化或氘代缓冲液（pH 8或9）中于40或50 ℃储存数天或数周。将这些强制降解的样品还原、S-氨基甲酰甲基化，并在质子化溶液中用胰蛋白酶消化，然后通过液相色谱/质谱（LC-MS）分析胰蛋白酶解产物或通过液相色谱/串联质谱（LC-MS/MS）测序，以检测外消旋肽并阐明外消旋氨基酸残基的位置。该方法成功地鉴定了三种IgG亚类重链恒定区中的几个外消旋氨基酸残基。虽然IgG亚类具有非常相似的主要蛋白质序列，但我们的结果有趣地表明特定氨基酸残基的外消旋化速率不同。
Racemization of amino acids is a common chemical degradation pathway observed in biopharmaceuticals and is particularly prevalent in synthetic peptides. The identification of racemized amino acid residue(s) by mass spectrometry is particularly challenging due to isobaric mass between the isomeric forms. In this paper, we present a novel methodology combining stable deuterium labeling with collisionally induced dissociation-tandem mass spectrometry (CID-MS/MS) to elucidate racemized amino acid residues in immunoglobulin samples. Immunoglobulin G subclasses IgG1, IgG2, and IgG4 samples were first stressed in protonated or deuterated buffer (pH 8 or 9) at 40 or 50 degrees C storage for days or weeks. These forced degraded samples were reduced, S-carbamidomethylated, and digested with trypsin in protonated solution, and the tryptic digests were then analyzed via liquid chromatography/mass spectrometry (LC-MS) or sequenced via liquid chromatography/tandem mass spectrometry (LC-MS/MS) to detect racemized peptides and elucidate the location of racemized amino acid residues. The methodology successfully identified several racemized amino acid residues in the constant region of the heavy chains of the three IgG subclasses. Although the IgG subclasses have very similar primary protein sequences, our results interestingly indicated different racemization rates for specific amino acid residues.