Qian Wei-Jun, Goshe Michael B, Camp David G, Yu Li-Rong, Tang Keqi, Smith Richard D
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, P.O. Box 999, MSIN: K8-98, Richland, Washington 99352, USA.
Anal Chem. 2003 Oct 15;75(20):5441-50. doi: 10.1021/ac0342774.
Many cellular processes are regulated by reversible protein phosphorylation, and the ability to broadly identify and quantify phosphoproteins from proteomes would provide a basis for gaining a better understanding of these dynamic cellular processes. However, such a sensitive, efficient, and global method capable of addressing the phosphoproteome has yet to be developed. Here we describe an improved stable-isotope labeling method using a phosphoprotein isotope-coded solid-phase tag (PhIST) for isolating and measuring the relative abundances of phosphorylated peptides from complex peptide mixtures resulting from the enzymatic digestion of extracted proteins. The PhIST approach is an extension of the previously reported phosphoprotein isotope-coded affinity tag (PhIAT) approach developed by our laboratory, where phosphoseryl and phosphothreonyl residues were derivatized by hydroxide ion-mediated beta-elimination followed by the Michael addition of 1,2-ethanedithiol (EDT). Instead of using the biotin affinity tag, peptides containing the EDT moiety were captured and labeled in one step using isotope-coded solid-phase reagents containing either light (12C6, 14N) or heavy (13C6, 15N) stable isotopes. The captured peptides labeled with the isotope-coded tags were released from the solid-phase support by UV photocleavage and analyzed by capillary liquid chromatography-tandem mass spectrometry. The efficiency and sensitivity of the PhIST labeling approach for identification of phosphopeptides from mixtures were determined using casein proteins. Its utility for proteomic applications was demonstrated by the labeling of soluble phosphoproteins from a human breast cancer cell line.
许多细胞过程受可逆性蛋白质磷酸化调控，从蛋白质组中广泛鉴定和定量磷酸化蛋白质的能力将为更好地理解这些动态细胞过程提供基础。然而，一种能够处理磷酸化蛋白质组的灵敏、高效且全面的方法尚未开发出来。在此，我们描述了一种改进的稳定同位素标记方法，该方法使用磷酸化蛋白质同位素编码固相标签（PhIST），用于从提取蛋白质经酶消化产生的复杂肽混合物中分离并测量磷酸化肽的相对丰度。PhIST方法是我们实验室先前报道的磷酸化蛋白质同位素编码亲和标签（PhIAT）方法的扩展，在PhIAT方法中，磷酰丝氨酸和磷酰苏氨酸残基通过氢氧根离子介导的β-消除反应进行衍生化，随后通过1,2-乙二硫醇（EDT）的迈克尔加成反应进行修饰。与使用生物素亲和标签不同，含有EDT部分的肽使用含有轻（12C6, 14N）或重（13C6, 15N）稳定同位素的同位素编码固相试剂在一步中进行捕获和标记。用同位素编码标签标记的捕获肽通过紫外光裂解从固相支持物上释放出来，并通过毛细管液相色谱-串联质谱进行分析。使用酪蛋白确定了PhIST标记方法从混合物中鉴定磷酸化肽的效率和灵敏度。通过对人乳腺癌细胞系中的可溶性磷酸化蛋白质进行标记，证明了其在蛋白质组学应用中的实用性。
