Quantitative analysis of modified proteins by LC-MS/MS of peptides labeled with phenyl isocyanate
Quantitative analysis of modified proteins by LC-MS/MS of peptides labeled with phenyl isocyanate
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DOI：
10.1021/pr0255856
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发表时间：
2003-05-01
影响因子：
4.4
通讯作者：
Liebler, DC
中科院分区：
文献类型：
--
作者：
Mason, DE;Liebler, DC
稳定同位素标记方法使得能够在LC-MS/MS分析中对样品之间的蛋白质进行相对定量。然而，大多数这样的方法不适用于修饰的蛋白质的差异定量，因为同位素标记试剂仅与某些肽反应，或者因为试剂掺入的质量增量太小而不能在低分辨率离子阱MS仪器上进行可靠的定量。在这里，我们描述了使用d（0）-和d（5）-苯基异氰酸酯（PIC）作为N-末端反应标签，基本上所有的肽在蛋白水解酶。PIC与肽N-末端胺在中性pH下在数分钟内定量反应，PIC标记的肽进行信息性MS/MS裂解。几种模型肽的d（0）-和d（5）-PIC标记衍生物的比率在10 000倍的肽浓度比范围内呈线性，因此表明定量的动态范围较宽。PIC标记的应用使得能够在LC-MS/MS分析中对人血红蛋白的几种氧化苯乙烯加合物进行相对定量。PIC标记提供了一种多功能的手段，定量改变成对样品中的修饰或变异蛋白质形式。
Stable isotope tagging methods have enabled relative quantitation of proteins between samples in LC-MS/MS analyses. However, most such methods are not applicable to the differential quantitation of modified proteins because the isotope tagging reagents only react with certain peptides or because the reagents incorporate a mass increment that is too small to allow reliable quantitation on low resolution ion trap MS instruments. Here, we describe the use of d(0)- and d(5)-phenyl isocyanate (PIC) as N-terminal reactive tags for essentially all peptides in proteolytic digests. PIC reacts quantitatively with peptide N-terminal amines within minutes at neutral pH and the PIC-labeled peptides undergo informative MS/MS fragmentation. Ratios of d(0)- and d(5)-PIC-labeled derivatives of several model peptides were linear across a 10 000-fold range of peptide concentration ratios, thus indicating a wide dynamic range for quantitation. Application of PIC labeling enabled relative quantitation of several styrene oxide adducts of human hemoglobin in LC-MS/MS analyses. PIC labeling offers a versatile means of quantifying changes in modified or variant protein forms in paired samples.