Characterization of cardiolipin as the sodiated ions by positive-ion electrospray ionization with multiple stage quadrupole ion-trap mass spectrometry

The application of multiple-stage ion-trap (IT) mass spectrometric methods for the structural characterization of cardiolipin (CL), a 1,3-bisphosphatidyl-sn-glycerol that consists of four fatty acyl chains and three glycerol backbones (designated as A, B, and central glycerol, respectively), as the sodiated adduct ions in the positive-ion mode was evaluated. Following collisionally activated dissociation (CAD), the [M - 2H + 3Na]+ ions of CL yield two prominent fragment ion pairs that consist of the phosphatidyl moieties attached to the 1'- and 3'-position of the central glycerol, respectively, resulting from the differential losses of the diacylglycerol moieties containing A and B glycerol, respectively. The results are consistent with those previously described for the [M - H]- and [M - 2H + Na]- ions in the negative-ion mode, thus permitting assignment of the two phosphatidyl moieties attached to the 1'- or 3'-position of the central glycerol. The identities of the fatty acyl substituents and their positions on the glycerol backbones (glycerol A and B) are deduced from further degradation of the above ion pairs that give the fragment ions reflecting the fatty acid substituents at the sn-1 (or sn-1') and sn-2 (or sn-2') positions. The ions that arise from losses of the fatty acid substituents at sn-1 and sn-1', respectively, are prominent, but the analogous ions from losses of the fatty acid substituents at sn-2 and sn-2', respectively, are of low abundance in the MS2 product-ion spectra. This feature further confirms the assignment of the positions of the fatty acid substituents. The similar IT multiple-stage mass spectrometric approaches including MS2 and MS3 for structural characterization of CL using its [M + Na]+ and the [M - H + 2Na]+ ions are also readily applicable. However, their uses for structural characterization are less desirable because formation of the [M + Na]+ and the [M - H + 2Na]+ ions for CL is not predictable.     

Characterization of cardiolipin from Escherichia coli by electrospray ionization with multiple stage quadrupole ion-trap mass spectrometric analysis of [M−2H+Na]− ions

We report a multiple-stage ion-trap (IT) mass spectrometric approach with electrospray ionization (ESI) for structural characterization of the [M - 2H + Na]- ion of cardiolipin (CL), a 1,3-bisphosphatidyl-sn-glycerol that consists of four fatty acyl chains and three glycerol backbones designated as A, B, and central glycerol, respectively (see Scheme 1). Following collisionally activated dissociation (CAD), the [M - 2H + Na]- ions of CL yield two prominent fragment ions that arise from the differential losses of the diacylglycerol moieties containing A or B glycerol, respectively. The tentative assignment of the two phosphatidyl moieties attached to the 1'- or 3'-position of the central glycerol is based on the observation that the ions arising from loss of the diacylglycerol moiety containing glycerol B is more abundant than that containing glycerol A. The structures of the above two ions, including the identities of the fatty acyl substituents and the position of fatty acyl substituents on the glycerol backbones (glycerol A and B) are determined by MS3 experiments that give spectra comprising several sets of prominent ions informative for the structural assignment of the fatty acyl substituents on the glycerol A and glycerol B. This method permits the structures of CL in a mixture isolated from Escherichia coli, including species that consist of various isomers, to be unveiled in detail.     

Studies on sulfatides by quadrupole ion-trap mass spectrometry with electrospray ionization: Structural characterization and the fragmentation processes that include an unusual internal galactose residue loss and the classical charge-remote fragmentation

The structural characterization of sulfatides by collisional-activated dissociation (CAD) quadrupole ion-trap tandem mass spectrometric methods with electrospray ionization is described. When subjected to CAD in the negative-ion mode, the [M - H]- ions of sulfatides yield abundant structurally informative ions that permit unequivocal assignments of the long-chain base, and fatty acid constituent including the location of double bond. The identification of the position of the double bond on the fatty acyl substituent is based on the observation of the series of the ions arising from classical charge-remote fragmentation processes similar to those observed by high-energy CAD and by tandem quadrupole mass spectrometry. An unusual internal galactose residue loss due to a rearrangement process was also observed. The [M - H]- ions of sulfatides also dissociates to a ceramide anion, which undergoes consecutive fragmentation processes to yield ions informative for identification of the ceramide moiety and permits distinction the sulfatide with a sphingosine subclass from that with a sphinganine long-chain base subclass. The MS(2)-spectra of the sulfatide subclass with a sphingosine LCB and a alpha-hydroxy fatty acyl substituent (d18:1/hFA-sulfatide) are featured by the prominent ion sets of m/z 568, 550, 540, and 522, originated from a primary cleavage of the fatty acyl CO-CH(OH) bond, and are readily differentiable from those arising from the non-hydroxy sulfatide subclass (d18:1/nFA-sulfatide), in which the ion sets are of low abundance. The fragmentation pathways of sulfatides under low-energy CAD are proposed. The pathways are supported by the MS(2)- and MS(3)-spectra of various compounds, and of their H-D exchanged analogs.     

Electrospray ionization/tandem quadrupole mass spectrometric studies on phosphatidylcholines: the fragmentation processes

We applied low-energy collisionally activated dissociation (CAD) tandem quadrupole mass spectrometry to study the fragmentation pathways of the [M + H](+) and [M + Li](+) ions of phosphatidylcholine (PC), generated by electrospray ionization (ESI). It is revealed that the fragmentation pathways leading to loss of the polar head group and of the fatty acid substituents do not involve the hydrogens attached to the glycerol backbone as previously reported. The pathway for formation of the major ion of m/z 184 by loss of the polar head group from the [M + H](+) precursor of a diacyl PC involves the participation of the alpha-hydrogen of the fatty acyl substituents, whereas the H(+) participates in the loss of fatty acid moieties. The alpha-hydrogens of the fatty acid substituents also participate in the major fragmentation processes, including formation of [M + Li-R(x)CO(2)H](+) and [M + Li-59-R(x)CO(2)H](+) ions for the [M + Li](+) ions of diacyl PCs, when subjected to low-energy CAD. These fragmentation processes are deterred by substitution of the fatty acyl moieties with alkyl, alkenyl, or hydroxyl groups and consequentially, result in a distinct product-ion spectrum for various PC, including diacyl-, plasmanyl- plasmenyl-, and lyso-PC isomers. The alpha-hydrogens of the fatty acyl substituents at sn-2 are more labile than those at sn-1. This is reflected by the preferential loss of the R(1)CO(2)H over the R(2)CO(2)H observed for the [M + Li](+) ions of diacyl PCs. The spectrum features resulting from the preferential losses permit identification and assignment of the fatty acid moieties in the glycerol backbone. The new fragmentation pathways established by tandem and source CAD tandem mass spectra of various PC molecules, including deuterium-labeling analogs, were proposed. These pathways would clarify the mechanisms underlying the ion formations that lead to the structural characterization of PC molecules.     

Characterization of inositol phosphorylceramides from <Emphasis Type="Italic">Leishmania major</Emphasis> by tandem mass spectrometry with electrospray ionization

We describe tandem mass spectrometric approaches, including multiple stage ion-trap and source collisionally activated dissociation (CAD) tandem mass spectrometry with electrospray ionization (ESI) to characterize inositol phosphorylceramide (IPC) species seen as [M - H](-) and [M - 2H + Li](-) ions in the negative-ion mode as well as [M + H](+), [M + Li](+), and [M - H + 2Li](+) ions in the positive-ion mode. Following CAD in an ion-trap or a triple-stage quadrupole instrument, the [M - H](-) ions of IPC yielded fragment ions reflecting only the inositol and the fatty acyl substituent of the molecule. In contrast, the mass spectra from MS(3) of [M - H - Inositol](-) ions contained abundant ions that are readily applicable for assignment of the fatty acid and long-chain base (LCB) moieties. Both the product-ion spectra from MS(2) and MS(3) of the [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) ions also contained rich fragment ions informative for unambiguous assignment of the fatty acyl substituent and the LCB. However, the sensitivity of the ions observed in the forms of [M - 2H + Alk](-), [M + H](+), [M + Alk](+), and [M - H + 2Alk](+) (Alk = Li, Na) is nearly 10 times less than that observed in the [M - H](-) form. In addition to the major fragmentation pathways leading to elimination of the inositol or inositol monophosphate moiety, several structurally informative ions resulting from rearrangement processes were observed. The fragmentation processes are similar to those previously reported for ceramides. While the tandem mass spectrometric approach using MS(n) (n = 2, 3) permits the structures of the Leishmania major IPCs consisting of two isomeric structures to be unveiled in detail, tandem mass spectra from constant neutral loss scans may provide a simple method for detecting IPC in mixtures.     

Structural characterization of triacylglycerols as lithiated adducts by electrospray ionization mass spectrometry using low-energy collisionally activated dissociation on a triple stage quadrupole instrument

We describe features of tandem mass spectra of lithiated adducts of triacylglycerol (TAG) species obtained by electrospray ionization mass spectrometry (ms) with low-energy collisionally activated dissociation (CAD) on a triple stage quadrupole instrument. The spectra distinguish isomeric triacylglycerol species and permit assignment of the mass of each fatty acid substituent and positions on the glycerol backbone to which substituents are esterified. Source CAD-MS2 experiments permit assignment of double bond locations in polyunsaturated fatty acid substituents. The ESI/MS/MS spectra contain [M + Li - (RnCO2H)]+, [M + Li - (RnCO2Li)]+, and RnCO+ ions, among others, that permit assignment of the masses of fatty acid substituents. Relative abundances of these ions reflect positions on the glycerol backbone to which substituents are esterified. The tandem spectra also contain ions reflecting combined elimination of two adjacent fatty acid residues, one of which is eliminated as a free fatty acid and the other as an alpha, beta-unsaturated fatty acid. Such combined losses always involve the sn-2 substituent, and this feature provides a robust means to identify that substituent. Fragment ions reflecting combined losses of both sn-1 and sn-3 substituents without loss of the sn-2 substituent are not observed. Schemes are proposed to rationalize formation of major fragment ions in tandem mass spectra of lithiated TAG that are supported by studies with deuterium-labeled TAG and by source CAD-MS2 experiments. These schemes involve initial elimination of a free fatty acid in concert with a hydrogen atom abstracted from the alpha-methylene group of an adjacent fatty acid, followed by formation of a cyclic intermediate that decomposes to yield other characteristic fragment ions. Determination of double bond location in polyunsaturated fatty acid substituents of TAG is achieved by source CAD experiments in which dilithiated adducts of fatty acid substituents are produced in the ion source and subjected to CAD in the collision cell. Product ions are analyzed in the final quadrupole to yield information on double bond location.     

Toward total structural analysis of cardiolipins: multiple-stage linear ion-trap mass spectrometry on the [M − 2H + 3Li]<Superscript>+</Superscript> ions

ESI multiple-stage linear ion-trap (LIT) mass spectrometric approaches for a near-complete structural characterization of cardiolipins (CLs), including identification of the fatty acyl substituents, assignment of the fatty acid substituents on the glycerol backbone, and location of the double-bond(s) or cyclopropyl group along the fatty acid chain are described. Upon collisionally activated dissociation (CAD) on the [M − 2H + 3Li]⁺ ions of CL in an ion-trap (MS²), two sets of fragment ions (designated as (a + 136) and (b + 136) ions) analogous to those previously reported for the [M − 2H + 3Na]⁺ ions were observed, leading to assignment of the phosphatidyl moieties attached to 1′- or 3′-position of the central glycerol. Further dissociation of the (a + 136) (or (b + 136)) ions (MS³) gives rise to the (a + 136 − R_{1(or 2)}CO₂Li) (or b + 136 − R_{1(or 2)}CO₂Li) ion pairs that identify the fatty acid moieties and their position on the glycerol backbone. This is followed by MS⁴ on the (a + 136 − R_{1(or 2)}CO₂Li) (or b + 136 − R_{1(or 2)}CO₂Li) ion to eliminate a tricylic glycerophosphate ester residue (136 Da) to yield the (a − R_{1(or 2)}CO₂Li) ion, which is then subjected to MS⁵. The MS5 spectrum contains the structural information that locates the double-bond(s) or cyclopropyl group of the fatty acid substituents. Finally, the subsequent MS⁶ on the dilithiated fatty acid ions generated from MS⁵ also yields feature ions that confirm the assignment.     

Electrospray ionization multiple stage quadrupole ion-trap and tandem quadrupole mass spectrometric studies on phosphatidylglycerol from arabidopsis leaves

Phosphatidylglycerol (PG) is the major phospholipid of plant chloroplasts. PG from Arabidopsis thaliana has an unusual fatty acyl chain, 3-trans-hexadecenoyl (Delta(3)16:1) in the sn-2 position of the major 18:3/Delta(3)16:1-PG species, as well as in 18:2/Delta(3)16:1-PG and 16:0/Delta(3)16:1-PG. Upon low-energy collisionally activated dissociation (CAD) in a tandem quadrupole or in an ion-trap mass spectrometer, the [M - H]- ions of the PG molecules containing Delta(3)16:1 give product-ion spectra that are readily distinguishable from those arising from PGs without the Delta(3)16:1 species. The Delta(3)16:1-fatty acyl-containing PGs are characterized by MS(2) product-ion mass spectra that contain predominant [M - H - 236]- ions arising from loss of the Delta(3)16:1-fatty acyl substituent as a ketene. This is attributable to the fact that the alpha-hydrogen of the Delta(3)16:1-fatty acid substituent involved in the ketene loss is an allylic hydrogen, which is very labile. This leads to preferential neutral loss of 236 and drastic decline in the neutral loss of 254 (i.e., loss as a fatty acid), the unique features that signify the presence of Delta(3)16:1-fatty acyl containing PGs. The neutral loss scan of 236, thus, provides a sensitive tandem quadrupole mass spectrometric means to identify Delta(3)16:1-containing PG species in lipid mixtures. This low-energy tandem mass spectrometric approach also permits the structures of the Arabidopsis PGs that consist of two isomeric structures to be unveiled.     

Structural characterization of phosphatidyl-<Emphasis Type="Italic">myo</Emphasis>-inositol mannosides from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> bacillus calmette gúerin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. II. Monoacyl- and diacyl-PIMs

The multiple-stage ion-trap mass spectrometric approaches towards to the structural characterization of the monoacyl-PIM (triacylated PIM) and the diacyl-PIM (tetracylated PIM), namely, the PIM (diacylated PIM) consisting of one or two additional fatty acid substituents attached to the glycoside, respectively, were described. While the assignment and confirmation of the fatty acid substituents on the glycerol backbone can be easily achieved by the methods described in the previous article, the identity of the glycoside moiety and its acylation state can be determined by the observation of a prominent acylglycoside ion arising from cleavage of the diacylglycerol moiety ([M - H - diacylglycerol](-)) in the MS(2)-spectra of monoacyl-PIM and diacyl-PIM. The distinction of the fatty acid substituents on the 2-O-mannoside (i.e., R(3)CO(2)H) from that on the inositol (i.e., R(4)CO(2)H) is based on the findings that the MS(3)-spectrum of [M - H - diacylglycerol](-) contains a prominent ion arising from further loss of the fatty acid at the 2-O-mannoside (i.e., the [M - H - diacylglycerol - R(3)CO(2)H](-) ion), while the ion arising from loss of the fatty acid substituent at the inositol (i.e., the [M - H - diacylglycerol - R(4)CO(2)H](-) ion) is of low abundance. The fatty acyl moiety on the inositol can also be identified by the product-ion spectrum from MS(4) of the [M - H - diacylglycerol - R(3)CO(2)H](-) ion, which gives rise to a prominent ion corresponding to loss of R(4)CO(2)H. An [M - H - acylmannose](-) ion was also observed in the MS(2)-spectra and, thus, the identity of the fatty acid substituent attached to 2-O-mannoside can be confirmed. The combined information obtained from the multiple-stage product-ion spectra from MS(2), MS(3), and MS(4) permit the assignment of the complex structures of monoacyl-PIMs and diacyl-PIMs in a mixture isolated from M. bovis Bacillus Calmette Guérin.     

A study of resonance electron capture ionization on a quadrupole tandem mass spectrometer

Procedures that allow the realization of resonance electron capture (REC) mode on a commercial triple-quadrupole mass spectrometer, after some simple modifications, are described. REC mass spectrometry (MS) and tandem mass spectrometry (MS/MS) experiments were performed and spectra for some compounds were recorded. In particular, the charge-remote fragmentation (CRF) spectra of [M - H](-) ions of docosanoic and docosenoic acids under low-energy collisionally activated dissociation (CAD) conditions were obtained, and showed that there were no significant differences for [M - H](-) ions produced at different resonances (i.e. for [M - H](-) ions with different structures). This observation was explained on the basis of results obtained from deuterium-labeled fatty acids, which showed that different CRF ions (but with the same m/z value in the absence of labels) could be produced by different mechanisms, and all of them were obviously realized under CAD conditions that made spectra practically indistinguishable. The other example, which compared the REC-MS/MS spectrum of [M - H](-) ions and EI-MS/MS spectrum of M(+.) ions of daidzein, demonstrated the potential of the REC-MS/MS technique for more complex structure elucidation.     

Structural characterization of phosphatidyl-<Emphasis Type="Italic">myo</Emphasis>-inositol mannosides from <Emphasis Type="Italic">Mycobacterium bovis</Emphasis> bacillus calmette guérin by multiple-stage quadrupole ion-trap mass spectrometry with electrospray ionization. I. PIMs and lyso-PIMs

We described a multiple-stage ion-trap mass spectrometric approach to characterize the structures of phosphatidylinositol and phosphatidyl-myoinositol mannosides (PIMs) in a complex mixture isolated from Mycobacterium bovis Bacillus Calmette Guérin. The positions of the fatty acyl substituents of PIMs at the glycerol backbone can be easily assigned, based on the findings that the ions arising from losses of the fatty acid substituent at sn-2 as molecules of acid and of ketene, respectively (that is, the [M - H - R(2)CO(2)H](-) and [M - H - R(2)CHCO](-) ions), are respectively more abundant than the ions arising from the analogous losses at sn-1 (that is, the [M - H - R(1)CO(2)H](-) and [M - H - R(1)CHCO](-) ions) in the MS(2) product-ion spectra of the [M - H](-) ions desorbed by electrospray ionization (ESI). Further dissociation of the [M - H - R(2)CO(2)H](-) and [M - H - R(1)CO(2)H](-) ions gives rise to a pair of unique ions corresponding to losses of 74 and 56 Da (that is, [M - H - R(x)CO(2)H - 56](-) and [M - H - R(x)CO(2)H - 74](-) ions, x = 1, 2), respectively, probably arising from various losses of the glycerol. The profile of the ion-pair in the MS(3) spectrum of the [M - H - R(2)CO(2)H](-) ion is readily distinguishable from that in the MS(3) spectrum of the [M - H - R(1)CO(2)H](-) ion and thus the assignment of the fatty acid substituents at the glycerol backbone can be confirmed. The product-ion spectra of the [M - H](-) ions from 2-lyso-PIM and from 1-lyso-PIM are discernible and both spectra contain a unique ion that arises from primary loss of the fatty acid substituent at the glycerol backbone, followed by loss of a bicyclic glycerophosphate ester moiety of 136 Da. The combined structural information from the MS(2) and MS(3) product-ion spectra permit the complex structures of PIMs that consist of various isomers to be unveiled in detail.     

Characterization of phosphatidylinositol, phosphatidylinositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate by electrospray ionization tandem mass spectrometry: A mechanistic study

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.     

Charge-driven fragmentation processes in diacyl glycerophosphatidic acids upon low-energy collisional activation. A mechanistic proposal

A mechanistic study of diacyl glycerophosphatidic acid (GPA) under low energy collisionally activated decomposition (CAD) with electrospray ionization tandem mass spectrometry is reported. The fragmentation pathways leading to the formation of carboxylate anions [RxCO2-], (x = 1, 2) and the formation of the ions representing neutral loss of fatty acid ([M-H-RxCO2H] ) and neutral loss of ketene ([M-H-R'xCH-C=O] ) (Rx=R'xCH2) are charge-driven processes that are governed by the gas-phase basicity and the steric configuration of the molecules. The preferential formation of the ions of [M-H-R2CO2H]- > [M-H-R1CO2H]- and [M-H-R'2CH=C=O]- > [M-H-R'1CH=C=O]- are attributed to the fact that loss of fatty acid and loss of ketene are sterically more favorable at sn-2. While the observation of the abundance of [M-H-RxCO2H]- > [M-H-R'xCH=C=O]- is attributed to the acidity of the gas phase ion of GPA, which undergoes a more facile neutral loss of acid than loss of ketene. The major pathway leading to the formation of RxCO2- ion under low energy CAD arises from further fragmentation of the [M-H-RxCO2H]- ions by neutral loss of 136, resulting in an abundance of R1CO2- > R2CO2-. The differential formation of the carboxylate anions permits accurate assignment of the regiospecificity of the fatty acid substituents of GPA molecules by tandem mass spectrometry.     

Studies on phosphatidylglycerol with triple quadrupole tandem mass spectrometry with electrospray ionization: Fragmentation processes and structural characterization

Negative-ion low-energy collisionally activated dissociation (CAD) tandem mass spectrometry of electrospray-produced ions permits structural characterization of phosphatidylglycerol (PG). The major ions that identify the structures arise from neutral loss of free fatty acid substituents ([M − H − R _x CO₂H]⁻) and neutral loss of the fatty acids as ketenes ([M − H − R′ _x CH = C = O]⁻), followed by consecutive loss of the glycerol head group. The abundances 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′₂CH = C = O]⁻) are greater than those of the product ions from the analogous losses at sn-1. Nucleophilic attack of the anionic phosphate site on the C-1 or the C-2 of the glycerol to which the carboxylates attached expels the sn-1 (R₁CO₂⁻) or the sn-2 (R₂CO₂⁻) carboxylate anion, resulting in a greater abundance of R₂COO⁻ than R₁COO⁻. These features permit assignments of fatty acid substituents and their position in the glycerol backbone. The results are also consistent with our earlier findings that pathways leading to those losses at sn-2 are sterically more favorable than those at sn-1. Fragment ions at m/z 227, 209 and 171 reflect the glycerol polar head group and identify the various PG molecules. Both charge-remote fragmentation (CRF) and charge-drive fragmentation (CDF) processes are the major pathways for the formation of [M − H − R _x COOH]⁻ ions. The CRF process involves participation of the hydrogen atoms on the glycerol backbone, whereas the CDF process involves participation of the exchangeable hydrogen atoms of the glycerol head group. The proposed fragmentation pathways are supported by CAD tandem mass spectrometry of the analogous precursor ions arising from the H-D exchange experiment, and further confirmed by source CAD in combination with tandem mass spectrometry.     

Charge-remote and charge-driven fragmentation processes in diacyl glycerophosphoethanolamine upon low-energy collisional activation: A mechanistic proposal

A mechanistic study of diacyl glycerophosphoethanolamine fragmentation under low energy collision-activated dissociation with electrospray ionization tandem mass spectrometry is reported. The fragmentation pathways leading to the formation of carboxylate anions (RxCO2-) (x = 1, 2) and the formation of the ions representing neutral loss of ketene ([M - H - Rx'CH=C=O]-) are charge-driven processes, which are governed by the gas-phase basicity and the steric configuration of the molecules. The fragmentation pathway for the formation of the [M - H - RxCO2H]- ions, reflecting neutral loss of fatty acid, is a charge-remote process, which involves the participation of the hydrogens at C-1 and C-2 of the glycerol, resulting in [M - H - R2CO2H]- > [M - H - R1CO2H]-. The preferential formations of R2CO2- > R1CO2-, and of [M - H - R2'CH=C=O]- > [M - H - R1'CH=C=O]- are attributed to the findings that charge-driven processes are sterically more favorable at sn-2. The observation of the abundance of [M - H - Rx'CH=C=O]- > [M - H - RxCO2H]- is attributed to the fact that the [M - H]- ions of GPE are basic precursor ions, which undergo preferential loss of ketene than loss of acid. The major pathway for the formation of RxCO2- ions arises from the nucleophilic attack of the anionic charge site of the phosphate on the C-1 or C-2 of the glycerol to render a charge transfer. The sterically more favorable attack on the C-2 than C-2 of the glycerol results in the abundance of R2CO2- > R1CO2-. These features of tandem spectra readily identify and locate the fatty acid substituents of GPE in the glycerol backbone.     

Structural assignment of isomeric 2-aminopyridine-derivatized monosialylated biantennary N-linked oligosaccharides using negative-ion multistage tandem mass spectral matching

To investigate the possibility of structural assignment based on negative-ion tandem multistage (MSn) mass spectral matching, four isomers of 2-aminopyridine (PA)-derivatized monosialylated oligosaccharides (i.e., complex-type N-glycans with an alpha2-3- or alpha2-6-linked sialic acid on alpha1-6 or alpha1-3 antennae) were analyzed using high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry (HPLC/ESI-IT-TOFMS). The negative ion [M-2H]2- is observed predominantly in the MS1 spectra without the loss of a sialic acid. The MS2 spectra derived from it are sufficiently reproducible that MS2 spectral matching based on correlation coefficients can be applied to the assignment of these isomers. The isomers containing a sialic acid on alpha1-6 or alpha1-3 antennae can be distinguished by MS2 spectral matching, but the alpha2-3 and alpha2-6 linkage types of sialic acid cannot be distinguished by their MS2 spectra. However, MS3 spectra derived from fragment ions containing a sialic acid (i.e., C4- and D-type ions) clearly differentiate the alpha2-3 and alpha2-6 linkage types of sialic acid in their MS3 spectral patterns. This difference might be rationalized in terms of a proton transfer from the reducing-end mannose to the negatively charged sialic acid. These two moieties are very close in the structural conformations of the precursor C4-type fragment ions of alpha2-6 linkage type, as predicted by molecular mechanics calculations. Thus, negative-ion MSn (n = 2, 3) spectral matching was demonstrated to be useful for the structural assignment of these four monosialylated PA N-glycan isomers.     

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−R₂CO₂H]⁻ than [M−2H+Alk−87−R₁CO₂H]⁻ and a greater abundance of [M−2H+Alk−87−R₂CO₂Li]⁻ than [M−2H+Alk−87−R₁CO₂Li]⁻; 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 (R₁CO₂⁻) 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−R₁CO₂H]⁺ than [M−H+2Alk−87−R₂CO₂H]⁺. 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.     

Fragmentation mechanisms of oligodeoxynucleotides: Effects of replacing phosphates with methylphosphonates and thymines with other bases in T-rich sequences

This article reports another step in an ongoing effort to understand the fragmentation of T-rich oligodeoxynucleotides. We extended an earlier investigation of T-rich 4-mers to T-rich 6-mers, 8-mers and 10-mers by using four different tandem mass spectrometric methods. The methods include low-energy collisionally activated decomposition (CAD) of electrospray ionization (ESI)-produced ions, source-CAD of ESI-produced ions, post-source decay (PSD), and CAD of matrix assisted laser desorption ionization (MALDI)-generated ions. The most abundant fragment ions produced from [M - 2H]2- precursors upon low-energy CAD in an ion trap are the [a - B]- and their complementary w ions. The predominant cleavage sites for T-rich oligodeoxynucleotides are always the 3' C-O bonds adjoining a non-T nucleobase (i.e., a base with a higher proton affinity (PA) than that of T). The relative abundance of [a - B]- correlates with the PAs of the nucleobases, underscoring the importance of proton transfer to the base. The propensity to form [a - B]- ions falls in the order of G > C approximately A > T. Structural isomers up to 10-mers can be readily sequenced and distinguished with each of the four tandem mass spectrometric methods applied. The fragmentation of oligodeoxynucleotides in which various phosphates were replaced with methylphosphonate is a measure of the participation of the phosphate proton in the formation of [a - B] ions. For 4 and 5-mers, transfer of an acidic proton from the 5'-phosphate to the departing base is the initiating step in the formation of [a - B]- ions.     

Gas-phase chemistry of the negative ions of fully-protected peptides by high-resolution electrospray ionization tandem mass spectrometry

Fully-protected C-terminal free peptides can be conveniently analyzed by high-resolution electrospray tandem mass spectrometry (ESI-MS/MS) in a quadrupole quadrupole time-of-flight tandem hybrid mass spectrometer, operated in the negative (-) ionizaionization mode. The unusual choice of negative ions in mass spectrometry applications to peptide analysis was needed to obtain exhaustive sequence and structural data. The low-energy collision-induced dissociation (CID) experiments provided, in fact, tandem mass spectra displaying highly diagnostic fragments with a good signal-to-noise ratio. The method is applied to segments of porcine calcitonin (Cal), Cal (1016, 1), Cal (1724, 2) and Cal (2528, 3) whose [M H]- deprotonated molecular ions provided low-energy CID mass spectra which allow the evaluation either of the primary structure of the peptide and of the location of the side-chain protective groups. ESI (+) MS can be conveniently used, in the high resolution mode, to achieve precise information on the elemental composition of the examined peptides.     

Characterization of acylphosphatidylglycerols from <Emphasis Type="Italic">salmonella typhimurium</Emphasis> by tandem mass spectrometry with electrospray ionization

Acylphosphatidylglycerol (Acyl-PG), a polar lipid class containing three fatty acyl groups, was isolated from Salmonella bacteria and characterized by tandem quadrupole and quadrupole ion-trap mass spectrometric methods with electrospray ionization. The structural characterization of the acyl-PG with various acyl groups (A-B/C-PG, where A not equal B not equal C) is based on the findings that the carboxylate anions (R(x)CO(2)(-)) arising from sn-2 (R(2)CO(2)(-)) is more abundant than that arising from sn-3' (R(3')CO(2)(-)), which is much more abundant than that arising from sn-1 (R(1)CO(2)(-)). This information provides a simple method for determination of the fatty acyl moieties and their positions in the molecule. The structural identification of the molecule can also be achieved by the findings that the fragment ion reflecting the ketene loss at sn-2 is more prominent than that reflecting the acid loss (i.e., [M - H - R'(2)CH=CO](-) > [M - H - R(2)CO(2)H](-)), while the ion arising from acid loss at sn-1 or sn-3' is, respectively, more abundant than the corresponding ketene loss (i.e., [M - H - R(1)CO(2)H](-) > [M - H - R'(1)CH=CO](-); [M - H - R(3')CO(2)H](-) > [M - H -R'(3')CH=CO](-)). The identity of the acyl moiety at sn-3' can be confirmed by an acyl-glycerophosphate anion observed in the product-ion spectrum obtained with a triple-stage quadrupole (TSQ) instrument, but not in that obtained with an ion-trap mass spectrometer (ITMS). However, the MS(2)-spectrum obtained with an ITMS is featured by the ion series that abundances of [M - H - R'(2)CH=CO - R(3)CO(2)H - 74](-) > [M - H - R'(2)CH=CO - R(1)CO(2)H - 74](-) z.Gt; [M - H - R'(1(or 3'))CH=CO - R(3'(or 1))CO(2)H - 74](-). This information also facilitates structural elucidation of the acyl-PG subclass that contains various acyl substituents. Structural identifications of molecular species having two identical fatty acyl substituents at sn-1, sn-2, or sn-3' or consisting of more than one isomeric structures are also demonstrated. The identities of the minor isomeric species in the molecules can be revealed by the aforementioned structural information arising from the various ion series combined.