Quintana Luis F, Campistol Josep M, Alcolea Maria P, Bañon-Maneus Elisenda, Sol-González Amandaé, Cutillas Pedro R
Servicio de Nefrología y Trasplante renal, Hospital Clinic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universidad de Barcelona, Barcelona, Spain.
Mol Cell Proteomics. 2009 Jul;8(7):1658-73. doi: 10.1074/mcp.M900059-MCP200. Epub 2009 Apr 7.
The advent of quantitative proteomics opens new opportunities in biomedical and clinical research. Although quantitative proteomics methods based on stable isotope labeling are in general preferred for biomolecular research, biomarker discovery is a case example of a biomedical problem that may be better addressed by using label-free MS techniques. As a proof of concept of this paradigm, we report the use of label-free quantitative LC-MS to profile the urinary peptidome of kidney chronic allograft dysfunction (CAD). The aim was to identify predictive biomarkers that could be used to personalize immunosuppressive therapies for kidney transplant patients. We detected (by LC-M/MS) and quantified (by LC-MS) 6000 polypeptide ions in undigested urine specimens across 39 CAD patients and 32 control individuals. Although unsupervised hierarchical clustering differentiated between the groups when including all the identified peptides, specific peptides derived from uromodulin and kininogen were found to be significantly more abundant in control than in CAD patients and correctly identified the two groups. These peptides are therefore potential biomarkers that might be used for the diagnosis of CAD. In addition, ions at m/z 645.59 and m/z 642.61 were able to differentiate between patients with different forms of CAD with specificities and sensitivities of 90% in a training set and, significantly, of approximately 70% in an independent validation set of samples. Interestingly low expression of uromodulin at m/z 638.03 coupled with high expression of m/z 642.61 diagnosed CAD in virtually all cases. Multiple reaction monitoring experiments further validated the results, illustrating the power of our label-free quantitative LC-MS approach for obtaining quantitative profiles of urinary polypeptides in a rapid, comprehensive, and precise fashion and for biomarker discovery.
定量蛋白质组学的出现为生物医学和临床研究带来了新机遇。尽管基于稳定同位素标记的定量蛋白质组学方法在生物分子研究中通常更受青睐，但生物标志物发现是一个生物医学问题的实例，使用无标记质谱技术可能能更好地解决。作为这一范例的概念验证，我们报告了使用无标记定量液相色谱 - 质谱来分析肾脏慢性移植功能障碍（CAD）患者的尿肽组。目的是识别可用于为肾移植患者个性化免疫抑制治疗的预测性生物标志物。我们通过液相色谱 - 串联质谱（LC - M/MS）检测并通过液相色谱 - 质谱（LC - MS）定量了39例CAD患者和32例对照个体的未消化尿液样本中的6000个多肽离子。尽管在纳入所有鉴定出的肽时，无监督层次聚类能够区分不同组，但发现源自尿调节蛋白和激肽原的特定肽在对照组中比CAD患者中明显更丰富，并能正确区分这两组。因此，这些肽是可能用于CAD诊断的潜在生物标志物。此外，质荷比为645.59和642.61的离子能够区分不同形式的CAD患者，在训练集中特异性和敏感性为90%，在独立验证样本集中显著约为70%。有趣的是，在几乎所有病例中，质荷比为638.03的尿调节蛋白低表达与质荷比为642.61的高表达相结合可诊断CAD。多反应监测实验进一步验证了结果，说明了我们的无标记定量液相色谱 - 质谱方法能够以快速、全面和精确的方式获得尿液多肽的定量图谱并用于生物标志物发现的能力。
