Hsu F F, Turk J
Department of Medicine, Washington University School of Medicine, St Louis, Missouri 63110, USA.
J Am Soc Mass Spectrom. 2000 Nov;11(11):986-99. doi: 10.1016/S1044-0305(00)00172-0.
Structural characterization of phosphatidylinositol (PI), phosphatidylinositol-4-phosphate (PI-4P), and phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2) by collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization is described. In negative ion mode, the major fragmentation pathways under low energy CAD for PI arise from neutral loss of free fatty acid substituents ([M - H - RxCO2H]-) and neutral loss of the corresponding ketenes ([M - H - R'xCH=C=O]-), followed by consecutive loss of the inositol head group. The intensities of the ions arising from neutral loss of the sn-2 substituent as a free fatty acid ([M - H - R2CO2H]-) or as a ketene ([M - H - R'2CH=C=O] ) are greater than those of ions reflecting corresponding losses of the sn-1 substutient. This is consistent with our recent finding that ions reflecting those losses arise from charge-driven processes that occur preferentially at the sn-2 position. These features permit assignment of the position of the fatty acid substituents on the glycerol backbone. Nucleophilic attack of the anionic phosphate onto the C-1 or the C-2 of the glycerol to which the fatty acids attached expels sn-1 (R1CO2-) or sn-2 (R2CO2-) carboxylate anion, respectively. This pathway is sterically more favorable at sn-2 than at sn-1. However, further dissociations of [M - H - RxCO2H - inositol] , [M - H - RxCO2H]-, and [M - H - RxCH=C=O]- precursor ions also yield RxCO2- ions, whose abundance are affected by the collision energy applied. Therefore, relative intensities of the RxCO2- ions in the spectrum do not reflect their positions on the glycerol backbone and determination of their regiospecificities based on their ion intensities is not reliable. The spectra also contain specific ions at m/z 315, 279, 259, 241, and 223, reflecting the inositol head group. The last three ions are also observed in the tandem spectra of the [M - H]- ions of phosphatidylinositol monophosphate (PI-P) and phosphatidylinositol bisphosphate (PI-P2), in addition to the ions at m/z 321 and 303, reflecting the doubly phosphorylated inositol ions. The PI-P2 also contains unique ions at m/z 401 and 383 that reflect the triply phosphorylated inositol ions. The [M - H]- ions of PI-P and PI-P2 undergo fragmentation pathways similar to that of PI upon CAD. However, the doubly charged ([M - 2H]2-) molecular ions undergo fragmentation pathways that are typical of the [M - H]- ions of glycerophosphoethanolamine, which are basic. These results suggest that the further deprotonated gaseous [M - 2H]2 ions of PI-P and PI-P2 are basic precursors.
描述了通过电喷雾电离的碰撞激活解离（CAD）串联质谱法对磷脂酰肌醇（PI）、磷脂酰肌醇-4-磷酸（PI-4P）和磷脂酰肌醇-4,5-二磷酸（PI-4,5-P2）进行结构表征。在负离子模式下，PI在低能量CAD下的主要碎裂途径源于游离脂肪酸取代基的中性丢失（[M - H - RxCO2H]-）和相应烯酮的中性丢失（[M - H - R'xCH=C=O]-），随后依次丢失肌醇头部基团。作为游离脂肪酸（[M - H - R2CO2H]-）或烯酮（[M - H - R'2CH=C=O]）的sn-2取代基中性丢失产生的离子强度大于反映sn-1取代基相应丢失的离子强度。这与我们最近的发现一致，即反映这些丢失的离子源于优先在sn-2位置发生的电荷驱动过程。这些特征允许确定脂肪酸取代基在甘油主链上的位置。阴离子磷酸对连接脂肪酸的甘油的C-1或C-2进行亲核攻击，分别排出sn-1（R1CO2-）或sn-2（R2CO2-）羧酸根阴离子。该途径在sn-2处比在sn-1处空间上更有利。然而，[M - H - RxCO2H - 肌醇]、[M - H - RxCO2H]-和[M - H - RxCH=C=O]-前体离子的进一步解离也产生RxCO2-离子，其丰度受所施加碰撞能量的影响。因此，光谱中RxCO2-离子的相对强度不反映它们在甘油主链上的位置，基于它们的离子强度确定其区域特异性是不可靠的。光谱中还包含m/z 315、279、259、241和223处的特定离子，反映了肌醇头部基团。除了反映双磷酸化肌醇离子的m/z 321和303处的离子外，最后三个离子也在磷脂酰肌醇单磷酸（PI-P）和磷脂酰肌醇双磷酸（PI-P2）的[M - H]-离子的串联光谱中观察到。PI-P2还包含反映三磷酸化肌醇离子的m/z 401和383处的独特离子。PI-P和PI-P2的[M - H]-离子在CAD时经历与PI类似的碎裂途径。然而，双电荷（[M - 2H]2-）分子离子经历的碎裂途径是碱性的甘油磷酸乙醇胺的[M - H]-离子的典型途径。这些结果表明PI-P和PI-P2的进一步去质子化气态[M - 2H]2离子是碱性前体。
