Panchaud Alexandre, Hansson Jenny, Affolter Michael, Bel Rhlid Rachid, Piu Stéphane, Moreillon Philippe, Kussmann Martin
Département de Microbiologie Fondamentale, Université de Lausanne, Quartier UNIL-Sorge, Bâtiment Biophore, CH-1015 Lausanne, Switzerland.
Mol Cell Proteomics. 2008 Apr;7(4):800-12. doi: 10.1074/mcp.M700216-MCP200. Epub 2007 Dec 13.
Identification and relative quantification of hundreds to thousands of proteins within complex biological samples have become realistic with the emergence of stable isotope labeling in combination with high throughput mass spectrometry. However, all current chemical approaches target a single amino acid functionality (most often lysine or cysteine) despite the fact that addressing two or more amino acid side chains would drastically increase quantifiable information as shown by in silico analysis in this study. Although the combination of existing approaches, e.g. ICAT with isotope-coded protein labeling, is analytically feasible, it implies high costs, and the combined application of two different chemistries (kits) may not be straightforward. Therefore, we describe here the development and validation of a new stable isotope-based quantitative proteomics approach, termed aniline benzoic acid labeling (ANIBAL), using a twin chemistry approach targeting two frequent amino acid functionalities, the carboxylic and amino groups. Two simple and inexpensive reagents, aniline and benzoic acid, in their (12)C and (13)C form with convenient mass peak spacing (6 Da) and without chromatographic discrimination or modification in fragmentation behavior, are used to modify carboxylic and amino groups at the protein level, resulting in an identical peptide bond-linked benzoyl modification for both reactions. The ANIBAL chemistry is simple and straightforward and is the first method that uses a (13)C-reagent for a general stable isotope labeling approach of carboxylic groups. In silico as well as in vitro analyses clearly revealed the increase in available quantifiable information using such a twin approach. ANIBAL was validated by means of model peptides and proteins with regard to the quality of the chemistry as well as the ionization behavior of the derivatized peptides. A milk fraction was used for dynamic range assessment of protein quantification, and a bacterial lysate was used for the evaluation of relative protein quantification in a complex sample in two different biological states.
随着稳定同位素标记与高通量质谱联用技术的出现，在复杂生物样品中鉴定和相对定量成百上千种蛋白质已成为现实。然而，尽管本研究的计算机分析表明，针对两个或更多氨基酸侧链可大幅增加可量化信息，但目前所有化学方法都只针对单一氨基酸官能团（最常见的是赖氨酸或半胱氨酸）。虽然现有方法的组合，如ICAT与同位素编码蛋白质标记的组合在分析上是可行的，但这意味着成本高昂，而且两种不同化学方法（试剂盒）的联合应用可能并不简单。因此，我们在此描述一种基于稳定同位素的新型定量蛋白质组学方法——苯胺苯甲酸标记法（ANIBAL）的开发与验证，该方法采用双化学方法针对两个常见的氨基酸官能团，即羧基和氨基。使用两种简单且廉价的试剂——苯胺和苯甲酸，它们分别以（12）C和（13）C形式存在，具有方便的质量峰间距（6 Da），且在色谱分离或碎片行为上无差异或修饰，用于在蛋白质水平上修饰羧基和氨基，从而使两个反应产生相同的肽键连接的苯甲酰修饰。ANIBAL化学方法简单直接，是第一种使用（13）C试剂对羧基进行通用稳定同位素标记的方法。计算机分析以及体外分析均清楚地表明，使用这种双方法可增加可用的可量化信息。通过模型肽和蛋白质对ANIBAL在化学质量以及衍生化肽的电离行为方面进行了验证。使用乳清组分评估蛋白质定量的动态范围，并使用细菌裂解物评估复杂样品中处于两种不同生物学状态下的蛋白质相对定量。
