Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA.
Mol Syst Biol. 2011 Apr 12;7:482. doi: 10.1038/msb.2011.15.
The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites-the global pattern of phosphorylation or 'phospho-form'-giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the 'phospho-form distribution': the relative amount of each of the 2(n) phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods-western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)-on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.
多部位蛋白质磷酸化的功能影响可能取决于磷酸化位点的数量和位置——即“磷酸化形式”的全局模式，这使生物系统具有强大的动态信息处理能力。因此，定量系统生物学的一个核心问题是测量“磷酸化形式分布”：即具有 n 个磷酸化位点的蛋白质的 2(n)种磷酸化形式中的每种形式的相对含量。我们比较了四种潜在的方法——针对具有两个磷酸化位点的研究充分的丝裂原活化蛋白激酶 Erk2 的差异磷酸化样品，使用磷酸特异性抗体的 Western blot、基于肽的液相色谱（LC）和质谱（pepMS）、基于蛋白质的 LC/MS（proMS）和核磁共振波谱（NMR）。MS 方法彼此之间以及与 NMR 的定量一致性在 10%以内，但 Western blot 虽然高度敏感，但与 MS 显示出明显的差异。NMR 还揭示了另外两种磷酸化，其中 pepMS 和 proMS 的组合产生了 16 成员磷酸化形式分布的估计值。这种组合 MS 策略为定量分布提供了准确性和覆盖范围的最佳组合，但位置异构体仍然是一个具有挑战性的问题。
