DVMT OF METHODS TO ELUCIDATE POST TRANSLATIONAL MOD OF PROTEINS: PHOSPHORYLATION

我们正在开发快速、高度敏感的测绘技术 蛋白质中的磷酸化位置和跟随进化 磷酸化/去磷酸化事件的复杂阵列 蛋白质。我们的磷酸化位点图谱的总体策略是 具体步骤如下：1)获得化合物的MALDI-TOF质谱图 完整的磷酸化蛋白质。2)如果可行，除磷化和 获得去磷酸化蛋白的MALDI-TOF质谱图。3) 减去在步骤I和步骤2中获得的质量，以计算平均值 连接到蛋白质上的磷酸基团的数量(或至少 由磷酸酶释放的磷酸基团的数目)。4)如果 有足够的蛋白质可用，使用ESI-四极杆重复步骤1-3 ESI-MS提供了更高分辨率的测量，但要求更高 蛋白质和更仔细的前端样品准备和清理。我们 通过将样品微型化，提高了我们的灵敏度和样品处理能力 我们的脱盐塔和优化的填料用于 将蛋白质固定在一起，仔细调整 ESI-四极仪器参数。我们目前可以获得很高的 某些蛋白质负载-I pmoL后的质量质谱图 蛋白质进入脱盐盒。5)对蛋白质进行酶处理 或化学降解，并获得MALDI-TOF和MALDI-ITMS地图 由此产生的蛋白质分解产物。6)使用以下几个选项重复步骤5 不同的降解剂，以确保良好的蛋白质覆盖率。 7)在MALDI-TOF谱中搜索未修改的和 通过将测得的质量与 根据已知蛋白质序列和碎裂规则进行计算 酶的活性。8)在MALDI-ITMS谱中搜索所有A=98 Da 成对的。我们发现所有的磷肽离子都会发生易失性丢失 离子陷阱中的磷酸元素，因此A =98 Da对为磷肽提供了一个方便的标记 复杂的混合物。这种损失发生在磷酸丝氨酸中， 磷酸苏氨酸和磷酸酪氨酸。9)确认 MALDI-ITMS/MS测定磷酸化多肽的磷酸化位点(S) 离子--再次使用98 Da的损失作为签名。另一种选择 步骤8-9涉及用以下物质处理多肽混合物 磷酸酶：处理组与未处理组的比较 多肽图。10)通常，感兴趣的多肽包含 可能被磷酸化的多个候选位点。至 精确定位磷酸化的实际位置，w，正在测试 -(I)LC-电喷雾电离-MS/MS的实用价值 三重四极分析器使用恒定中性点损失扫描和 79次扫描的父母(Carr等人)。(Ii)关于我们芬兰人的LC-ESI-MS/MS 细心利用信息的LCQ离子陷阱质谱计 从MALDI-TOF-MS实验中获得。(Iii)阶梯排序 感兴趣的多肽

We are developing rapid, highly sensitive techniques for mapping sites of phosphorylation in proteins and for following the evolution of complex arrays of phosphorylation/de-phosphorylation events in proteins. Our overall strategy for phosphorylation site mapping is outlined in the steps below: 1) Obtain MALDI-TOF mass spectrum of the intact phosphorylated protein. 2) If feasible, dephosphorylate and obtain MALDI-TOF mass spectrum of the de-phosphorylated protein. 3) Subtract the masses obtained in steps I and 2 to compute the average number of phosphate groups attached to the protein (or at least the number of phosphate-groups released by the phosphotase). 4) If sufficient protein is available, repeat steps 1-3 using ESI-Quadrupole MS. ESI-MS provides a higher resolution measurement but requires more protein and more careful front-end sample preparation and cleanup. We have improved our sensitivity and sample handling by miniaturizing of our desalting columns and optimizing the packing materials used to immobilize the proteins together with careful tuning of the ESI-quadrupole instrument parameters. We can currently obtain high quality mass spectra of certain proteins after loading -I pmol of the protein onto the desalting cartridge. 5)Subject protein to enzymatic or chemical degradation and obtain a MALDI-TOF and MALDI-ITMS map of the resultant proteolysis products. 6)Repeat step 5 with several different degradation reagents to ensure good coverage of the protein. 7) Search the MALDI-TOF spectrum for both unmodified and phosphorylated peptides by comparing the measured masses with those calculated from the known protein sequence and the fragmentation rules of the enzyme. 8) Search the MALDI-ITMS spectrum for all A = 98 Da pairs. We have found that all phosphopeptide ions undergo facile loss of the elements of phosphoric acid in the ion trap and therefore the A = 98 Da pairs provide a convenient signature for phosphopeptides in complex mixture. This loss occurs from phosphoserines, phosphothreonines, and phosphotyrosines. 9) Confirm the phosphorylation site(s) by MALDI-ITMS/MS of the phosphorylated peptide ion - again using the loss of 98 Da as the signature. An alternative to steps 8-9 involves treatment of the peptide mixtures with phosphatase followed by comparison of the treated versus untreated peptide maps. 10) Frequently, the peptide of interest contains multiple candidate sites that potentially may be phosphorylated. To pinpoint the actual site of phosphorylation, w, are testing the practical utility of-(i) LC-Electrospray ionization MS/MS on the triple quadrupole analyzer using constant neutral loss scans and parent of 79- scans (Carr et al). (ii) LC-ESI-MS/MS on our Finnigan LCQ ion trap mass spectrometer making careftil use of the information obtained from the MALDI-TOF-MS experiments. (iii) Ladder sequencing of the peptide of interest