Trypsin catalyzed 16O-to-18O exchange for comparative proteomics:: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-Ion trap mass spectrometers
Trypsin catalyzed 16O-to-18O exchange for comparative proteomics:: Tandem mass spectrometry comparison using MALDI-TOF, ESI-QTOF, and ESI-Ion trap mass spectrometers
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DOI：
10.1016/s1044-0305(03)00207-1
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发表时间：
2003-07-01
影响因子：
3.2
通讯作者：
Yao, XD
中科院分区：
文献类型：
--
作者：
Heller, M;Mattou, H;Yao, XD
定量或比较蛋白质组分析最初是用二维凝胶电泳进行的，其固有的缺点是偏向于某些蛋白质和劳动密集型。近年来，使用各种稳定同位素标记技术，开发了与无凝胶蛋白质/肽分离结合的替代的基于质谱的方法。所有这些技术的共同之处是生物合成或化学地掺入具有氢、碳、氧或氮（轻形式）的天然同位素分布或分别富集有重同位素如氘、C-13、O-18或N-15的标记部分。通过将等量的具有例如轻形式标记的对照样品与重标记的病例样品混合，通过质谱方法检测差异标记的肽，并且其强度用作直接相对蛋白质定量的手段。虽然每种不同的标记方法都有其优点和缺点，但内切蛋白酶O-16至O-18催化的C-末端羧酸氧交换是非常有前途的，因为酶促反应和基本上每种蛋白酶衍生肽的标记确保了特异性。我们在这里表明，这种方法适用于复杂的生物样品，如人血浆的亚组分。此外，尽管两种标记形式之间的相对小的4 Da的质量差（对应于两个氧原子被两个180同位素交换），但是可以在自动数据依赖性LC-MS/MS采集模式下在质量分辨率为约2000的离子阱质谱仪上定量差异标记的蛋白质。与在线ESI-MS/MS平行的MALDI靶上的柱后样品沉积使得能够通过ESI-MS/MS和MALDI-MS/MS分析相同的化合物。这有可能增加定量结果的置信度，并通过互补肽电离技术增加潜在感兴趣蛋白质的序列覆盖率。此外，O-16/O-18标记肽对的串联质谱中的成对y离子信号提供了一种确认自动蛋白质鉴定结果甚至辅助未知蛋白质从头测序的方法。(C)2003年美国质谱学会。
Quantitative or comparative proteome analysis was initially performed with 2-dimensional gel electrophoresis with the inherent disadvantages of being biased towards certain proteins and being labor intensive. Alternative mass spectrometry-based approaches in conjunction with gel-free protein/peptide separation have been developed in recent years using various stable isotope labeling techniques. Common to all these techniques is the incorporation, biosynthetically or chemically, of a labeling moiety having either a natural isotope distribution of hydrogen, carbon, oxygen, or nitrogen (light form) or being enriched with heavy isotopes like deuterium, C-13, O-18, or N-15, respectively. By mixing equal amounts of a control sample possessing for instance the light form of the label with a heavy-labeled case sample, differentially labeled peptides are detected by mass spectrometric methods and their intensities serve as a means for direct relative protein quantification. While each of the different labeling methods has its advantages and disadvantages, the endoprotease O-16-to-O-18 catalyzed oxygen exchange at the C-terminal carboxylic acid is extremely promising because of the specificity assured by the enzymatic reaction and the labeling of essentially every protease-derived peptide. We show here that this methodology is applicable to complex biological samples such as a subfraction of human plasma. Furthermore, despite the relatively small mass difference of 4 Da between the two labeled forms, corresponding to the exchange of two oxygen atoms by two 180 isotopes, it is possible to quantify differentially labeled proteins on an ion trap mass spectrometer with a mass resolution of about 2000 in automated data dependent LC-MS/MS acquisition mode. Post column sample deposition on a MALDI target parallel to on-line ESI-MS/MS enables the analysis of the same compounds by means of ESI-and MALDI-MS/MS. This has the potential to increase the confidence in the quantification results as well as to increase the sequence coverage of potentially interesting proteins by complementary peptide ionization techniques. Additionally the paired y-ion signals in tandem mass spectra of O-16/O-18-labeled peptide pairs provide a means to confirm automatic protein identification results or even to assist de novo sequencing of yet unknown proteins. (C) 2003 American Society for Mass Spectrometry.