Studies on phosphatidylserine by tandem quadrupole and multiple stage quadrupole ion-trap mass spectrometry with electrospray ionization: Structural characterization and the fragmentation processes

Low-energy CAD product-ion spectra of various molecular species of phosphatidylserine (PS) in the forms of [M - H]^- and [M - 2H + Alk]^- in the negative-ion mode, as well as in the forms of [M + H]⁺, [M + Alk]⁺, [M - H + 2Alk]⁺, and [M - 2H + 3Alk]⁺ (where Alk = Li, Na) in the positive-ion mode contain rich fragment ions that are applicable for structural determination. Following CAD, the [M - H]^- ion of PS undergoes dissociation to eliminate the serine moiety (loss of C₃H₅NO₂) to give a [M - H - 87]^- ion, which equals to the [M - H]^- ion of a phoshatidic acid (PA) and give rise to a MS³-spectrum that is identical to the MS²-spectrum of PA. The major fragmentation process for the [M - 2H + Alk]^- ion of PS arises from primary loss of 87 to give rise to a [M - 2H + Alk - 87]^- ion, followed by loss of fatty acid substituents as acids (R_xCO₂H, x = 1,2) or as alkali salts (e.g., R_xCO₂Li, x = 1,2). These fragmentations result in a greater abundance of [M - 2H + Alk - 87 - R₂CO ₂H]^- than [M - 2H + Alk - 87 - R₁CO ₂H]^- and a greater abundance of [M - 2H + Alk - 87 - R₂CO₂Li]^- than [M - 2H + Alk - 87 - R ₁CO₂Li]^-; while further dissociation of the [M - 2H + Alk - 87 - R_{2(or 1)}CO₂Li]^- ions gives a preferential formation of the carboxylate anion at sn-1 (R₁CO ₂^-) over that at sn-2 (R₂CO₂ ^-). Other major fragmentation process arises from differential loss of the fatty acid substituents as ketenes (loss of R_x′CH=CO, x = 1,2). This results in a more prominent [M - 2H + Alk - R₂′CH= CO]^- ion than [M - 2H + Alk - R₁′CH=CO]^- ion. Ions informative for structural characterization of PS are of low abundance in the MS²-spectra of both the [M + H]⁺ and the [M + Alk]⁺ ions, but are abundant in the MS³-spectra. The MS²-spectrum of the [M + Alk]⁺ ion contains a unique ion corresponding to internal loss of a phosphate group probably via the fragmentation processes involving rearrangement steps. The [M - H + 2Alk] ⁺ ion of PS yields a major [M - H + 2Alk - 87]⁺ ion, which is equivalent to an alkali adduct ion of a monoalkali salt of PA and gives rise to a greater abundance of [M - H + 2Alk - 87 - R₁CO ₂H]⁺ than [M - H + 2Alk - 87 - R₂CO ₂H]⁺. Similarly, the [M - 2H + 3Alk]⁺ ion of PS also yields a prominent [M - 2H + 3Alk - 87]⁺ ion, which undergoes consecutive dissociation processes that involve differential losses of the two fatty acyl substituents. Because all of the above tandem mass spectra contain several sets of ion pairs involving differential losses of the fatty acid substituents as ketenes or as free fatty acids, the identities of the fatty acyl substituents and their positions on the glycerol backbone can be easily assigned by the drastic differences in the abundances of the ions in each pair.