Cuccurullo Manuela, Schlosser Gitta, Cacace Giuseppina, Malorni Livia, Pocsfalvi Gabriella
Proteomic and Biomolecular Mass Spectrometry Centre, Institute of Food Science and Technology, CNR, Avellino, Italy.
J Mass Spectrom. 2007 Aug;42(8):1069-78. doi: 10.1002/jms.1238.
Reversible protein phosphorylation mediated by protein kinases and phosphatases is the most studied post-translational modification. Efficient characterization of phosphoproteomes is hampered by (1) low stoechiometry, (2) the dynamic nature of the phosphorylation process and (3) the difficulties of mass spectrometry to identify phosphoproteins from complex mixtures and to determine their sites of phosphorylation. Combination of the phosphopeptide enrichment method with MALDI-TOFMS, or alternatively, with HPLC-ESI-MS/MS and MS(3) analysis was shown to be a step forward for the successful application of MS in the study of protein phosphorylation. In our study we used phosphopeptide enrichment performed in a simple single-tube experiment using zirconium dioxide (ZrO(2)). A simple protein mixture containing precipitated bovine milk caseins was enzymatically digested and the mixture of tryptic fragments was analysed before and after enrichment using nanoflow HPLC-ESI-MS/MS and surface-enhanced laser desorption/ionization (SELDI)-MS/MS on QqTOF instruments to compare the efficiency of the two methods in the determination of phosphorylation sites. Both approaches confirm the high selectivity obtained by the use of batch-wise, ZrO(2)-based protocol using di-ammonium phosphate as the eluting buffer. More phosphorylation sites (five for beta-casein and three for alpha(S1)-casein) were characterized by SELDI-MS/MS than by nanoflow HPLC-ESI-MS/MS. Therefore, ZrO(2)-based phosphopeptide enrichment combined with SELDI-MS/MS is an attractive alternative to previously reported approaches for the study of protein phosphorylation in mixtures of low complexity with the advance of fast in situ peptide purification. The method was limited to successful analysis of high-abundance proteins. Only one phosphorylation site was determined for the minor casein component alpha(S2)-casein by ESI-MS/MS and none for kappa-casein. Therefore an improvement in enrichment efficiency, especially for successful phosphoproteomic applications, is needed.
由蛋白激酶和磷酸酶介导的可逆蛋白磷酸化是研究最多的翻译后修饰。磷酸化蛋白质组的有效表征受到以下因素的阻碍：（1）低化学计量比；（2）磷酸化过程的动态性质；（3）质谱从复杂混合物中鉴定磷酸化蛋白并确定其磷酸化位点的困难。磷酸肽富集方法与基质辅助激光解吸/电离飞行时间质谱（MALDI-TOFMS）相结合，或者与高效液相色谱-电喷雾串联质谱（HPLC-ESI-MS/MS）及MS(3)分析相结合，已被证明是质谱成功应用于蛋白质磷酸化研究的一个进步。在我们的研究中，我们使用在简单的单管实验中用二氧化锆（ZrO₂）进行的磷酸肽富集。对含有沉淀的牛乳酪蛋白的简单蛋白质混合物进行酶解，并在富集前后使用纳流HPLC-ESI-MS/MS和表面增强激光解吸/电离（SELDI）-MS/MS在QqTOF仪器上分析胰蛋白酶片段混合物，以比较两种方法在确定磷酸化位点方面的效率。两种方法都证实了使用基于ZrO₂的分批方案并以磷酸二铵作为洗脱缓冲液所获得的高选择性。与纳流HPLC-ESI-MS/MS相比，SELDI-MS/MS鉴定出了更多的磷酸化位点（β-酪蛋白有5个，α(S1)-酪蛋白有3个）。因此，基于ZrO₂的磷酸肽富集与SELDI-MS/MS相结合，是一种有吸引力的替代方法，可用于低复杂度混合物中蛋白质磷酸化的研究，且具有快速原位肽纯化的优势。该方法仅限于成功分析高丰度蛋白质。通过电喷雾串联质谱（ESI-MS/MS）仅确定了次要酪蛋白成分α(S2)-酪蛋白的一个磷酸化位点，而κ-酪蛋白未确定任何磷酸化位点。因此，需要提高富集效率，特别是对于成功的磷酸化蛋白质组学应用。
