Lehmann Sylvain, Vialaret Jérôme, Combe Guillaume Gras, Bauchet Luc, Hanon Olivier, Girard Marine, Gabelle Audrey, Hirtz Christophe
CHRU de Montpellier, Université de Montpellier and INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France.
Service de Neurochirurgie, CHRU de Montpellier, hôpital Gui de Chauliac, Montpellier, INSERM U 1051 and Université de Montpellier, Montpellier, France.
Rapid Commun Mass Spectrom. 2015 Oct 30;29(20):1917-25. doi: 10.1002/rcm.7289.
Intravenous administration of stable isotope labeled amino acid ((13)C6-leucine) to humans recently made it possible to study the metabolism of specific biomarkers in cerebrospinal fluid (CSF) using targeted mass spectrometry (MS). This labeling approach could be of great interest for monitoring many leucine-containing peptides in parallel, using high-resolution MS. This will make it possible to quantify the rates of synthesis and clearance of a large range of proteins in humans with a view to obtaining new insights into protein metabolism processes and the pathophysiology of diseases such as Alzheimer's disease.
Proteins from human lumbar and ventricular CSF samples collected at different times after intravenous (13)C6-leucine infusion were digested enzymatically with LysC/trypsin after being denatured, reduced and alkylated. Desalted tryptic peptides were fractionated using Strong Cation eXchange chromatography (SCX) and analyzed using nanoflow liquid chromatography (nano-LC) coupled to a QTOF Impact II (Bruker Daltonics) mass spectrometer. Data-dependent acquisition (DDA) mode was used to identify and quantify light and heavy (13)C6-leucine peptides. The ratios of (13)C6-leucine incorporation were calculated using the Skyline software program in order to determine the rates of appearance and clearance of proteins in the CSF.
After SCX fractionation and quadrupole time-of-flight (QTOF) analysis, 4528 peptides containing leucine were identified in five fractions prepared from 40 μL of CSF. Upon analyzing one of these fractions, 66 peptides (2.7%) corresponding to 61 individual proteins had significant and reproducible rate of (13)C6-leucine incorporation at various time points. The plots of the light-to-heavy peptide ratios showed the existence of proteins with different patterns of appearance and clearance in the CSF.
The Stable Isotope Labeling Amino acid in Vivo (SILAV) method presented here, which yields unprecedented information about protein metabolism in humans, constitutes a promising new approach which certainly holds great potential in the field of clinical proteomics.
最近，向人体静脉注射稳定同位素标记的氨基酸（(13)C6-亮氨酸），使得利用靶向质谱法（MS）研究脑脊液（CSF）中特定生物标志物的代谢成为可能。这种标记方法对于使用高分辨率MS并行监测许多含亮氨酸的肽可能非常有意义。这将有可能量化人类多种蛋白质的合成和清除率，以期获得对蛋白质代谢过程以及诸如阿尔茨海默病等疾病病理生理学的新见解。
在静脉注射(13)C6-亮氨酸后不同时间采集的人腰椎和脑室CSF样本中的蛋白质，经变性、还原和烷基化处理后，用LysC/胰蛋白酶进行酶解。脱盐后的胰蛋白酶肽段采用强阳离子交换色谱（SCX）进行分离，并使用纳流液相色谱（nano-LC）与QTOF Impact II（布鲁克道尔顿公司）质谱仪联用进行分析。采用数据依赖采集（DDA）模式来鉴定和定量轻链和重链(13)C6-亮氨酸肽段。使用Skyline软件程序计算(13)C6-亮氨酸掺入率，以确定CSF中蛋白质的出现和清除率。
经过SCX分离和四极杆飞行时间（QTOF）分析，在从40μL CSF制备的五个馏分中鉴定出4528个含亮氨酸的肽段。在分析其中一个馏分时，对应于61种单个蛋白质的66个肽段（2.7%）在不同时间点具有显著且可重复的(13)C6-亮氨酸掺入率。轻链与重链肽段比率的图谱显示CSF中存在具有不同出现和清除模式的蛋白质。
本文介绍的体内稳定同位素标记氨基酸（SILAV）方法提供了有关人类蛋白质代谢的前所未有的信息，是一种有前景的新方法，在临床蛋白质组学领域无疑具有巨大潜力。
