Gouw Joost W, Tops Bastiaan B J, Mortensen Peter, Heck Albert J R, Krijgsveld Jeroen
Biomolecular Mass Spectrometry and Proteomics Group, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, The Netherlands.
Anal Chem. 2008 Oct 15;80(20):7796-803. doi: 10.1021/ac801249v. Epub 2008 Sep 23.
Comparative proteomics has emerged as a powerful approach to determine differences in protein abundance between biological samples. The introduction of stable-isotopes as internal standards especially paved the road for quantitative proteomics for comprehensive approaches to accurately determine protein dynamics. Metabolic labeling with (15)N isotopes is applied to an increasing number of organisms, including Drosophila, C. elegans, and rats. However, (15)N-enrichment is often suboptimal (<98%), which may hamper identification and quantitation of proteins. Here, we systematically investigated two independent (15)N-labeled data sets to explore the influence of heavy nitrogen enrichment on the number of identifications as well as on the error in protein quantitation. We show that specifically larger (15)N-labeled peptides are under-represented when compared to their (14)N counterparts and propose a correction method, which significantly increases the number of identifications. In addition, we developed a method that corrects for inaccurate peptide ratios introduced by incomplete (15)N enrichment. This results in improved accuracy and precision of protein quantitation. Altogether, this study provides insight into the process of protein identification and quantitation, and the methods described here can be used to improve both qualitative and quantitative data obtained by labeling with heavy nitrogen with enrichment less than 100%.
比较蛋白质组学已成为一种强大的方法，用于确定生物样品之间蛋白质丰度的差异。引入稳定同位素作为内标尤其为定量蛋白质组学铺平了道路，使其能够采用综合方法准确测定蛋白质动态变化。用（15）N同位素进行代谢标记已应用于越来越多的生物体，包括果蝇、秀丽隐杆线虫和大鼠。然而，（15）N富集往往并不理想（<98%），这可能会妨碍蛋白质的鉴定和定量。在这里，我们系统地研究了两个独立的（15）N标记数据集，以探讨重氮富集对鉴定数量以及蛋白质定量误差的影响。我们发现，与（14）N对应物相比，特别是较大的（15）N标记肽代表性不足，并提出了一种校正方法，该方法显著增加了鉴定数量。此外，我们开发了一种方法，用于校正由不完全（15）N富集引入的不准确肽比率。这提高了蛋白质定量的准确性和精密度。总之，本研究深入了解了蛋白质鉴定和定量过程，这里描述的方法可用于改进通过重氮标记且富集小于100%获得的定性和定量数据。
