Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode
Detection of tyrosine phosphorylated peptides by precursor ion scanning quadrupole TOF mass spectrometry in positive ion mode
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DOI：
10.1021/ac001318c
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发表时间：
2001-04-01
影响因子：
7.4
通讯作者：
Mann, M
中科院分区：
文献类型：
--
作者：
Steen, H;Kuster, B;Mann, M
磷酸化是蛋白质修饰的常见形式。为了了解其生物学作用，必须确定磷酸化位点。通常，细胞中仅存在有限量的磷酸化蛋白质，因此需要高度灵敏的磷酸化位点测定程序。在这里，介绍了一种新方法，可以将凝胶分离的蛋白质中的酪氨酸磷酸化定位在飞摩尔范围内，该方法利用 m/z 216.043 的磷酸酪氨酸铵离子进行正离子模式前体离子扫描，并结合最近在四极杆 TOF 质谱仪上推出的 Q2 脉冲功能。四极杆 TOF 仪器的高分辨率能够选择性检测磷酸酪氨酸铵离子，而不受相同标称质量的其他肽片段的干扰。在正离子模式下执行前体离子扫描有利于测序，因为不需要极性切换或改变喷雾溶剂的 pH 值。与三重四极杆质谱仪相比，这种方法获得了类似的检测限，但由于碎片离子选择的高精度，选择性明显更好。可以检测到 1 fmol/μL 的合成磷酸肽，并且凝胶中 100 fmol 的酪氨酸磷酸化蛋白足以检测未分离的消化混合物中的磷酸化肽以及明确的磷酸化位点测定。新方法可以应用于未知的蛋白质样品，因为修饰的识别和定位是在同一样品上进行的。
Phosphorylation is a common form of protein modification. To understand its biological role, the site of phosphorylation has to be determined. Generally, only limited amounts of phosphorylated proteins are present in a cell, thus demanding highly sensitive procedures for phosphorylation site determination. Here, a novel method is introduced which enables the localization of tyrosine phosphorylation in gel-separated proteins in the femtomol range, The method utilizes the immonium ion of phosphotyrosine at m/z 216.043 for positive ion mode precursor ion scanning combined with the recently introduced Q2-pulsing function on quadrupole TOF mass spectrometers. The high resolving power of the quadrupole TOF instrument enables the selective detection of phosphotyrosine immonium ions without interference from other peptide fragments of the same nominal mass. Performing precursor ion scans in the positive ion mode facilitates sequencing, because there is a no need for polarity switching or changing pH of the spraying solvent. Similar limits of detection were obtained in this approach when compared to triple-quadrupole mass spectrometers but with significantly better selectivity, owing to the high accuracy of the fragment ion selection. Synthetic phosphopeptides could be detected at 1 fmol/muL, and 100 fmol of a tyrosine phosphorylated protein in gel was sufficient for the detection of the phosphorylated peptide in the unseparated digestion mixture and for unambiguous phosphorylation site determination. The new method can be applied to unknown protein samples, because the identification and localization of the modification is performed on the same sample.