Torres Matthew P, Thapar Roopa, Marzluff William F, Borchers Christoph H
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, NC 27599, USA.
J Proteome Res. 2005 Sep-Oct;4(5):1628-35. doi: 10.1021/pr050129d.
Phosphopeptides can be difficult to detect and sequence by mass spectrometry (MS) due to low ionization efficiency and suppression effects in the MS mode, and insufficient fragmentation in the tandem MS (MS/MS) mode, respectively. To address this problem, we have developed a technique called Phosphatase-directed Phosphorylation-site Determination (PPD), which combines on-target phosphatase reactions, MALDI MS/MS of IMAC beads on target, and hypothesis-driven MS (HD-MS). In this method, on-target dephosphorylation experiments are conducted on IMAC-bound phosphopeptides, because dephosphorylated peptides have, in general, higher MS sensitivities than the corresponding phosphopeptides. The detected dephosphorylated peptides are sequenced by MS/MS, which identifies the potentially phosphorylated peptide and the total number of Ser, Thr, or Tyr residues that could hypothetically be phosphorylated within that peptide. On the basis of this information, a mass list containing every possible phosphorylation state of each observed peptide (where 1 HPO(3) = 80 Da) is used to direct MALDI-MS/MS on the phosphorylated peptides bound to IMAC beads at each theoretical mass from the list. If the peptide is present, the resulting MS/MS spectrum reveals the exact site(s) of phosphorylation in the peptide. We have demonstrated the applicability of PPD to the detection of in vivo phosphorylation sites on the Drosophila Stem Loop Binding Protein (dSLBP), and the complementarity of this new technique to conventional MS phosphorylation site mapping methods, since the phosphorylation sites in dSLBP could not be detected by other methods.
由于质谱（MS）模式下的低电离效率和抑制效应，以及串联质谱（MS/MS）模式下的碎片化不足，磷酸化肽段难以通过质谱进行检测和测序。为了解决这个问题，我们开发了一种名为磷酸酶导向磷酸化位点测定（PPD）的技术，该技术结合了靶上磷酸酶反应、靶上IMAC磁珠的基质辅助激光解吸电离串联质谱（MALDI MS/MS）以及假设驱动质谱（HD-MS）。在该方法中，对IMAC结合的磷酸化肽段进行靶上脱磷酸化实验，因为一般来说，脱磷酸化肽段比相应的磷酸化肽段具有更高的质谱灵敏度。通过MS/MS对检测到的脱磷酸化肽段进行测序，从而确定潜在的磷酸化肽段以及该肽段中理论上可能被磷酸化的丝氨酸（Ser）、苏氨酸（Thr）或酪氨酸（Tyr）残基的总数。基于这些信息，使用包含每个观察到的肽段的每种可能磷酸化状态的质量列表（其中1个磷酸（HPO₃） = 80道尔顿），针对列表中每个理论质量下与IMAC磁珠结合的磷酸化肽段进行MALDI-MS/MS指导。如果该肽段存在，所得的MS/MS谱图将揭示该肽段中磷酸化的确切位点。我们已经证明了PPD在检测果蝇茎环结合蛋白（dSLBP）体内磷酸化位点方面的适用性，以及这项新技术与传统质谱磷酸化位点映射方法的互补性，因为dSLBP中的磷酸化位点无法通过其他方法检测到。
