Smith Julia R, Olivier Michael, Greene Andrew S
Biotechnology and Bioengineering Center, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.
Physiol Genomics. 2007 Oct 22;31(2):357-63. doi: 10.1152/physiolgenomics.00096.2007. Epub 2007 Aug 7.
We have developed a method to determine the degree of phosphorylation of a peptide in a complex mixture without enrichment or operation of the mass spectrometer in negative ion mode. Yeast lysate containing known amounts of synthetic peptides (VPQLEIVPNSAEERLHSMK and VPQLEIVPN[pS]AEERLHSMK) was labeled with (16)O and (18)O during hydrolysis. After treatment of one sample with a cocktail of phosphatases, the two samples were pooled. The intensity of the dephosphorylated peptide peaks was used to infer the degree of phosphorylation present before treatment. The linear dynamic range of this method is >10-fold before either of the peptide envelopes becomes indistinguishable from the surrounding noise. Since both the site of posttranslational modification and the proportion of the protein population that is modified are vital in protein function, the employment of this technique will provide a valuable tool for the analysis of the functional implications of protein phosphorylation.
我们开发了一种方法，可在不进行富集或在负离子模式下操作质谱仪的情况下，确定复杂混合物中肽段的磷酸化程度。含有已知量合成肽（VPQLEIVPNSAEERLHSMK和VPQLEIVPN[pS]AEERLHSMK）的酵母裂解物在水解过程中用(^{16}O)和(^{18}O)进行标记。用磷酸酶混合物处理一个样品后，将两个样品合并。去磷酸化肽峰的强度用于推断处理前存在的磷酸化程度。在任何一个肽峰包络与周围噪声变得无法区分之前，该方法的线性动态范围大于10倍。由于翻译后修饰位点和被修饰蛋白质群体的比例在蛋白质功能中都至关重要，因此该技术的应用将为分析蛋白质磷酸化的功能影响提供一个有价值的工具。
