Protein Analysis Facility, Center for Integrative Genomics, Faculty of Biology and Medicine, University of Lausanne, Switzerland.
Rapid Commun Mass Spectrom. 2009 Nov;23(22):3570-8. doi: 10.1002/rcm.4287.
Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large-scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse-labeled with a stable isotope by metabolic incorporation of 'heavy' amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple-quadrupole ion trap mass spectrometer. PIS-based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several-fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS-based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique.
稳定同位素标记物通常被引入蛋白质组中进行定量研究。通过对不同生物学状态的细胞进行全标记，然后进行样品混合、分级和大量数据采集，可获得总蛋白质水平的准确大规模定量。然而，生物过程通常仅在短时间内影响一小部分蛋白质，导致难以在总蛋白质组背景下检测到变化。另一种方法可以是针对特定生物刺激合成的蛋白质进行靶向分析。可以通过“重”氨基酸的代谢掺入来对这些蛋白质进行脉冲标记。在这项研究中，我们使用三重四极杆离子阱质谱仪上基于前体离子扫描（PIS）的采集方法，研究了标记蛋白的特异性检测和鉴定。PIS 方法用于检测源自标记肽的独特的亚铵离子。我们在标准混合物中测试了不同的标签和方法，以优化性能。我们表明，与在同一仪器上进行的非靶向分析相比，该方法可将标记蛋白相对于未标记蛋白的检测特异性提高数倍。该技术应用于鉴定在含有牛血清蛋白的生长培养基中分泌的人细胞蛋白，可优先检测到标记的细胞蛋白而不是未标记的牛蛋白。然而，与在两台不同仪器上进行的非靶向采集相比，基于 PIS 的策略在灵敏度方面显示出一些局限性。我们讨论了该技术的可能前景。
