SELECTIVE DETECTION OF PHOSPHOPEPTIDES IN COMPLEX-MIXTURES BY ELECTROSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY
SELECTIVE DETECTION OF PHOSPHOPEPTIDES IN COMPLEX-MIXTURES BY ELECTROSPRAY LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY
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DOI：
10.1016/1044-0305(93)80049-5
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发表时间：
1993-09-01
影响因子：
3.2
通讯作者：
CARR, SA
中科院分区：
文献类型：
--
作者：
HUDDLESTON, MJ;ANNAN, RS;CARR, SA
发展了一种基于质谱学的方法，该方法不涉及使用放射性标记来选择性地检测复杂混合物中的磷酸肽。使用C-18填充熔融硅胶色谱柱(小于或等于320微米直径)，用负离子电喷雾液相色谱/质谱仪分析低皮摩尔水平的磷酸化肽和非磷酸化肽的混合物。在质量分析四极杆之前，层析洗脱液中的多肽和磷酸多肽在自由喷射膨胀区域发生碰撞诱导解离。利用相对较高的碰撞激发电位，含有磷酸丝氨酸、磷酸苏氨酸和磷酸酪氨酸的磷酸多肽在m/z 63和79分别对应于PO2-和PO3-产生诊断离子。含有磷酸肽的色谱峰显示在这些诊断离子最大的位置。使用m/z 63和79的选择离子监测获得了最高的磷酸肽检测灵敏度。可以通过在每次扫描期间将碰撞激发电势从高值步进到较低值来获得显示诊断磷肽片段离子以及伪分子离子的全扫描质谱图，在每个扫描中检测低质量电荷比诊断标记离子，同时扫描较高质量电荷比范围。用含标准三氟乙酸的标准流动相进行反相高效液相色谱分析，获得了良好的检测磷肽的灵敏度。对于含有三种常见的磷酸化氨基酸的多肽和磷酸肽的混合物，提供了说明该方法的选择性和灵敏度的数据。
A mass spectrometry-based method that does not involve the use of radiolabeling was developed for selective detection of phosphopeptides in complex mixtures. Mixtures of phosphorylated and nonphosphorylated peptides at the low picomole level are analyzed by negative ion electrospray liquid chromatography/mass spectrometry using C-18 packed fused-silica columns (less-than-or-equal-to 320-mum i.d.). Peptides and phosphopeptides in the chromatographic eluant undergo collision-induced dissociation in the free-jet expansion region prior to the mass analyzing quadrupole. Using relatively high collisional excitation potentials, phosphopeptides containing phosphoserine, phosphothreonine, and phosphotyrosine fragment to yield diagnostic ions at m/z 63 and 79 corresponding to PO2- and PO3-, respectively. Chromatographic peaks containing phosphopeptides are indicated where these diagnostic ions maximize. The highest sensitivity for phosphopeptide detection is obtained using selected-ion monitoring for m/z 63 and 79. Full-scan mass spectra that exhibit the diagnostic phosphopeptide fragment ions, together with pseudomolecular ions, may be obtained by stepping the collisional excitation potential from a high value during the portion of each scan in which the low-mass-to-charge ratio diagnostic marker ions are being detected to a lower value while the upper mass-to-charge ratio range is being scanned. Good sensitivity for phosphopeptide detection was achieved using standard trifluoroacetic acid containing mobile phases for reversed-phase high-performance liquid chromatography. Data illustrating the selectivity and sensitivity of the approach are presented for mixtures of peptides and phosphopeptides containing the three commonly phosphorylated amino acids.