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

我们正在开发快速、高灵敏度的测绘技术 蛋白质中的磷酸化位点， 磷酸化/去磷酸化事件的复杂阵列， proteins. 我们磷酸化位点定位的总体策略是 1）获得化合物的MALDI-TOF质谱， 完整的磷酸化蛋白。 2)如果可行，去磷酸化， 获得去磷酸化蛋白质的MALDI-TOF质谱。 第三章 减去步骤I和步骤2中获得的质量，计算平均值 连接到蛋白质的磷酸基团的数量（或至少 由磷酸酶释放的磷酸基团的数量）。 4)如果 有足够蛋白可用，使用ESI-四极杆重复步骤1 - 3 女士 ESI-MS提供了更高的分辨率测量，但需要更多 蛋白质和更仔细的前端样品制备和清理。 我们 我们通过以下方法提高了灵敏度和样品处理能力： 我们的色谱柱和优化用于 将蛋白质混合在一起， ESI-四极杆仪器参数。 目前，我们可以获得高 上样后某些蛋白质的质量质谱-1 pmol 将蛋白质转移到荧光粉盒上。 5)将蛋白质进行酶促处理 或化学降解，并获得MALDI-TOF和MALDI-ITMS图谱， 得到的蛋白水解产物。 6)重复步骤5， 不同的降解试剂，以确保蛋白质的良好覆盖。 7)搜索MALDI-TOF光谱， 通过比较测量的质量与那些磷酸化的肽 根据已知的蛋白质序列和片段化规则计算 的酶。 8)搜索所有A = 98 Da的MALDI-ITMS光谱 对. 我们发现所有的磷酸肽离子都容易丢失 离子阱中的磷酸元素的量，因此A = 98 Da对提供了磷酸肽的方便特征， 复杂混合物 这种损失发生在磷酸丝氨酸， 磷酸苏氨酸和磷酸酪氨酸。 9)确认 通过磷酸化肽的MALDI-ITMS/MS测定的磷酸化位点 离子-再次使用98 Da的损失作为特征。 一个替代 步骤8 - 9涉及用以下物质处理肽混合物 磷酸酶，然后比较处理与未处理 肽图谱 10)通常，感兴趣的肽含有 多个可能被磷酸化的候选位点。 到 精确定位磷酸化的实际位点， （i）LC-电喷雾离子化MS/MS对 使用恒定中性损失扫描的三重四极分析器， 79次扫描的父代（卡尔等人）。 (ii)LC-ESI-MS/MS分析 LCQ型离子阱质谱计信息的合理利用 从MALDI-TOF-MS实验获得。 (iii)阶梯排序 感兴趣的肽的

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