Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: A mechanistic study
Ff. Hsu et J. Turk, Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: A mechanistic study, J AM SOC M, 11(11), 2000, pp. 986-999
Citations number
13
Categorie Soggetti
Spectroscopy /Instrumentation/Analytical Sciences
Journal title
JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY
Structural characterization of phosphatidylinositol (PI), phosphatidylinosi
tol-4-phosphate CPI-LP), and phosphatidylinositol-4,5-bisphosphate (PI-4,5-
P-2) 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-x'CH=C=O](-)), followed by consecutiv
e loss of the inositol head group. The intensities of the ions arising from
neutral loss of the sn-2 substutient as a free fatty acid ([M - H - R2CO2H
](-)) or as a ketene ([M - H - R-2'CH=C=O](-)) are greater than those of io
ns reflecting corresponding losses of the sn-l substutient. This is consist
ent with our recent finding that ions reflecting those losses arise from ch
arge-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-l or the C-2 of the glycerol to which the fatty acids attached expel
s sn-l (R1CO2-) or sn-2 (R2CO2-) carboxylate anion, respectively. This path
way is sterically more favorable at sn-2 than at sn-l. However, further dis
sociations of [M - H - RxCO2H - inositol](-), [M - H - RxCO2H](-) and [M -
H - R-x'CH=C=O](-) precursor ions also yield RxCO2- ions, whose abundance a
re affected by the collision energy applied. Therefore, relative intensitie
s of the RxCO2- ions in the spectrum do not reflect their positions on the
glycerol backbone and determination of their regiospecificities based on th
eir 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. Th
e last three ions are also observed in the tandem spectra of the [M - H](-)
ions of phosphatidylinositol monophosphate (PI-P) and phosphatidylinositol
bisphosphate (PI-P-2), in addition to the ions at mit 321 and 303, reflect
ing the doubly phosphorylated inositol ions. The PI-P-2 also contains uniqu
e ions at m/z 401 and 383 that reflect the triply phosphorylated inositol i
ons. The [M - H](-) ions of PI-P and PI-P-2 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 sugge
st that the further deprotonated gaseous [M - 2H](2-) ions of PI-P and PI-P
-2 are basic precursors. (C) 2000 American Society for Mass Spectrometry.