Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry
Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry
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DOI：
10.1002/rcm.2154
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发表时间：
2005-01-01
影响因子：
2
通讯作者：
Allmaier, G
中科院分区：
文献类型：
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作者：
Kleinova, M;Belgacem, O;Allmaier, G
用电喷雾电离(ESI)离子陷阱质谱仪(ITMS)研究了来自不同商业来源的三种人血浆(药用级，HSA)白蛋白的微观不均质性。这项研究涵盖了对完整蛋白质以及胰蛋白酶多肽水平的MS分析。除了简单的脱盐外，完整的蛋白质样品不需要任何分离步骤。对于这些样品，我们在正离子ESI质谱图中观察到，HSA的多电荷离子信号由许多完全或部分分辨的峰组成，其相对强度取决于所分析的样品。在m/z值分别为66448+/-3.6m/z值、66450+/-0.6m/z值和66451+/-3.2m/z值([MH](+))的3种人血清白蛋白制剂中均检测到未修饰的HSA。根据氨基酸序列计算的值为66439。第二个高强度的化合物(在两种情况下是去卷曲质谱图中的基峰)被解释为修饰的HSA，相对于未修饰的HSA的相对分子质量增加在116到118 Da之间(m/z分别为66 564，66 567和66 569)，这表明存在共价结合的半胱氨酸残基。在m/z 66 619、66 621和66 613处的三个样品中都发现了另一个去卷曲的ESI峰，信噪比相当低，这可能对应于文献中描述的非酶糖基化。随后使用高效液相色谱/电喷雾串联质谱仪和包括低能碰撞诱导解离(CID)在内的高效液相色谱/电喷雾串联质谱仪在多肽水平上验证了所提议的共价人血清白蛋白修饰。在胰酶消化之前，HSA样品在没有预先还原步骤的情况下被烷化。在此过程之后，我们检测到序列T21-41的肽包括两种形式的半胱氨酸-34残基：半胱氨酸化(m/z 639.15[M+4H](4+))以及乙烯基吡啶烷基化(m/z 635.69[M+4H](4+)，这意味着其以前天然的游离SH形式)。在下一步中，我们进行了在线LC/ESI低能CIDMS/MS实验来验证这两种结构。通过对上述离子的MS/MS分析，可以证明所描述的半胱氨酸化修饰(Cys-34残基)。在制药级制剂中，这种对HSA的大量修饰可以毫不含糊地确定为半胱氨酸化-34残基。另一方面，在在线HPLC/ESI-MS模式下，在多肽水平上没有检测到所提出的非酶糖基化，这可能是由于所研究的三个样品中的低浓度所致。版权所有(C)2005 John Wiley&Sons，Ltd.
Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH](+)), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H](4+)) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H](4+), which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation. Copyright (C) 2005 John Wiley & Sons, Ltd.