Department of Biological Sciences, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Anal Chem. 2011 Feb 1;83(3):701-7. doi: 10.1021/ac100775s. Epub 2011 Jan 6.
In this study, the impact of amino acid modifications on the accuracy of the iTRAQ (isobaric tags for relative and absolute quantitation) method was evaluated. MCF-7 breast cancer cells, cultured in the presence of 17β-estradiol and tamoxifen, were used as a model system. The cells were labeled and analyzed by reversed-phase liquid chromatography and pulsed Q dissociation ion trap tandem mass spectrometry detection. Database searching was performed by using various combinations of amino acid modification allowances, i.e, Lys/Tyr/Cys and amino terminal iTRAQ labeling, Lys methylation, acetylation and carbamylation, and Cys/Met oxidation. Other than the intended Lys/amino terminal iTRAQ labeling, such modifications occur as a result of either enzymatic or sample preparation related reactions and are typically ignored in quantitation analysis to minimize the rate of false-positive peptide identifications. The study revealed that the modifications with the greatest impact on protein identification and quantitation pertain to Lys and Tyr amino acid residues, that by enabling such modifications the number and type of identified proteins will change (by up to 10%), and that the rate of false-positive protein identifications can be maintained below an upper threshold of 5% if appropriate data filtering conditions are used. In addition, the interference of possible posttranslational modifications (i.e., phosphorylation) with iTRAQ quantitation was examined.
在这项研究中，评估了氨基酸修饰对 iTRAQ（相对和绝对定量的同重同位素标记）方法准确性的影响。使用 MCF-7 乳腺癌细胞作为模型系统，该细胞在 17β-雌二醇和他莫昔芬的存在下进行培养。通过反相液相色谱和脉冲 Q 解离离子阱串联质谱检测对细胞进行标记和分析。通过使用各种氨基酸修饰允许的组合进行数据库搜索，即 Lys/Tyr/Cys 和氨基末端 iTRAQ 标记、Lys 甲基化、乙酰化和氨甲酰化以及 Cys/Met 氧化。除了预期的 Lys/氨基末端 iTRAQ 标记之外，这些修饰是由于酶或样品制备相关反应而发生的，通常在定量分析中被忽略，以最大程度地减少假阳性肽鉴定的速率。研究表明，对蛋白质鉴定和定量影响最大的修饰涉及 Lys 和 Tyr 氨基酸残基，通过允许这些修饰，可以改变鉴定的蛋白质的数量和类型（最多可达 10%），并且如果使用适当的数据过滤条件，可以将假阳性蛋白质鉴定的速率维持在 5%以下的上限。此外，还检查了可能的翻译后修饰（例如磷酸化）对 iTRAQ 定量的干扰。
