Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis-assisted open-tubular liquid chromatography-mass spectrometry

We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LPS, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.     

Fragmentation studies on monensin A and B by accurate-mass electrospray tandem mass spectrometry

Monensin A and B were studied by electrospray ionisation tandem mass spectrometry (ESI-MS/MS) and the fragment ions were confirmed by accurate-mass measurements. Analyses were performed on both a quadrupole time-of-flight (QTOF) and a Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer. The analysis revealed that fragment ions were produced by Grob-Wharton fragmentations and pericyclic rearrangements in addition to various simple neutral losses. A study of the protonated and sodiated sodium salt revealed different fragmentation pathways for these species, thus complementary structural information could be gained. A complete fragmentation pathway of monensin A and B protonated sodium salt [(M-H+Na)+H])+) and sodiated sodium salt [(M-H+Na)+Na](+) is proposed. MS(3) analysis confirmed the separate fragmentation pathways.     

Analysis of sialyl oligosaccharides by high-performance liquid chromatography-electrospray ionisation-mass spectrometry with differentiation of alpha2-3 and alpha2-6 sialyl linkages

A method for analysing sialyl oligosaccharides from bovine colostrum using high-performance liquid chromatography-electrospray ionisation-mass spectrometry (HPLC-ESI-MS) is described. Under positive ionisation mode, mass spectra of alpha2-3 and alpha2-6 linkages were different, and the former produced a prominent B2 (or B3 in disialyl lactose) mass fragment. This fragment was absent from mass spectra with alpha2-6 linkages. Two sialyl oligosaccharides, which have not been reported previously, were tentatively identified. One comprises a N-acetyl neuraminic acid (Neu5Ac), two hexoses (Hex), and one N-acetyl hexosamine (HexNAc) residue ((Neu5Ac)1 (Hex)2 (HexNAc)1), and the other comprises one Neu5Ac and one Hex residue ((Neu5Ac)1(Hex)1).     

Characterization of limonin glucoside metabolites from human prostate cell culture medium using high-performance liquid chromatography/electrospray ionization mass spectrometry and tandem mass spectrometry

The metabolism of limonin 17-beta-D-glucopyranoside (LG) by non-cancerous (RWPE-1) and cancerous (PC-3) human prostate epithelial cells was investigated using high-performance liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with in-source fragmentation and tandem mass spectrometry (MS/MS). During positive ion LC/ESI-MS, LG formed an abundant sodiated species ([M+Na]+) while the protonated molecule was barely observable. [M+Na]+ further fragmented into the less abundant [LARL+H]+ and a predominantly protonated aglycone molecule (limonin) due to in-source fragmentation. The major metabolite, limonin A-ring lactone (LARL), formed an abundant protonated molecule that was fragmented into a protonated molecule of limonin by loss of one molecule of water. In MS/MS by collisionally activated dissociation (CAD), LG produced the sodiated aglycone, [aglycone+Na]+, while LARL fragmented into [M+H]+ of limonin and fragment ions resulted by further loss of water, carbon monoxide and carbon dioxide, indicating the presence of oxygenated-ring structures. The limits of detection of LG were 0.4 and 20 fmol in selected-ion monitoring (SIM) and selected-reaction monitoring (SRM) detection, respectively.     

Electron Capture Dissociation Studies of the Fragmentation Patterns of Doubly Protonated and Mixed Protonated-Sodiated Peptoids

The fragmentation patterns of a group of doubly protonated ([P + 2H]²⁺) and mixed protonated-sodiated ([P + H + Na]²⁺) peptide-mimicking oligomers, known as peptoids, have been studied using electron capturing dissociation (ECD) tandem mass spectrometry techniques. For all the peptoids studied, the primary backbone fragmentation occurred at the N-C_α bonds. The N-terminal fragment ions, the C-ions (protonated) and the C′-ions (sodiated) were observed universally for all the peptoids regardless of the types of charge carrier. The C-terminal ions varied depending on the type of charge carrier. The doubly protonated peptoids with at least one basic residue located at a position other than the N-terminus fragmented by producing the Z^?-series of ions. In addition, most doubly protonated peptoids also produced the Y-series of ions with notable abundances. The mixed protonated-sodiated peptoids fragmented by yielding the Z^?′-series of ions in addition to the C′-series. Chelation between the sodium cation and the amide groups of the peptoid chain might be an important factor that could stabilize both the N-terminal and the C-terminal fragment ions. Regardless of the types of the charge carrier, one notable fragmentation for all the peptoids was the elimination of a benzylic radical from the odd-electron positive ions of the protonated peptoids ([P + 2H]^?+) and the sodiated peptoids ([P + H + Na]^?+). The study showed potential utility of using the ECD technique for sequencing of peptoid libraries generated by combinatorial chemistry.

Fragmentation studies on lasalocid acid by accurate mass electrospray mass spectrometry

Lasalocid acid is an important polyether ionophore veterinary drug. Polyether ionophores have been the subject of MS study for many years, but this is the first rigorous study of the complex fragmentation processes occurring in ESI MS/MS for lasalocid, underpinned by high-resolution accurate-mass measurement. Initial low-resolution analyses were performed on an ion-trap instrument. High-resolution analyses were performed on a Fourier-transform ion cyclotron resonance mass spectrometer. The MS/MS analysis of the pseudo-molecular ion shows that fragment ions are produced either by beta-elimination or by neutral losses of water. Additional ions were observed in the source dissociation analysis, indicating that additional fragmentation reactions occur in the source region. Some of these ions can then undergo additional ion-ion or ion-molecule reactions before being extracted from the source. The study of both the protonated and sodiated sodium salts shows the same fragmentation pathways, with fragment ions containing two sodiums at low intensity. A fragmentation pathway of the lasalocid acid protonated sodium salt [(M-H+Na)+H]+ (m/z 613) and sodiated sodium salt [(M-H+Na)+Na]+ (m/z 635) is presented. The increased understanding afforded by this study will help in the development of unequivocal analytical methods for lasalocid and related polyether ionophore veterinary drugs.     

Electrospray ionization for analysis of platelet-activating factor.

Platelet-activating factor (PAF) was analyzed by electrospray-ionization mass spectrometry (ESI-MS) using a single quadrupole mass spectrometer. The positive-ion spectrum was dominated by an ion corresponding to a sodiated molecule when a low potential difference between the capillary exit (nozzle) and the skimmer was employed, but when the capillary exit voltage was increased, fragmentation of PAF was observed. Initial fragmentation involved the loss of the elements of trimethylamine from the sodiated molecule to yield [M+Na-59]+. An intense ion at m/z 147, generated by the loss of trimethylamine from the sodiated phosphocholine portion of the molecule was also detected, along with a lower intensity ion at m/z 184 which is representative of a protonated phosphocholine moiety. With negative-ion detection the major molecular species was [M+Cl]-. Interpretation of the mass spectral fragments was verified by ESI tandem mass spectrometry on a triple-quadrupole tandem mass spectrometer.     

Multiple-stage mass spectrometric analysis of complex oligosaccharide antibiotics (everninomicins) in a quadrupole ion trap

Electrospray ionization (ESI) quadrupole ion-trap tandem mass spectrometry (MS/MS) was utilized to characterize a class of complex oligosaccharide antibiotics (everninomicins) that include SCH 27899, everninomicin-D, amino everninomicin (SCH 27900), and SCH 49088 (containing a hydroxylamino-ether sugar). The addition of sodium chloride (approximately 1 microg/mL) facilitates the formation of abundant metal complex ions, and this was used because protonation does not readily occur for most of these compounds. The multiple-stage mass analysis (MS(n)) of the sodiated species provides an important series of fragment ions that are specific for sugar sequence and for some sugar-ring opening. These data suggest a general charge-remote fragmentation pattern with the sodium cation residing in a specific, central location of the sugar chain and fragmentation occurring to trim the end of the molecule. For protonated everninomicin (SCH 27900), however, the proton appears to be mobile during the collisional activation process, opening different fragmentation pathways depending on the proton location. The use of water and acetonitrile with 0.1% acetic acid as the solvent in ESI-MS promotes rapid hydrolysis of the central ortho ester, resulting in the formation of abundant sodiated products that are hydrated. These product ions of the hydrated molecules are likely formed by the same charge-remote fragmentation processes as those that occur for the unhydrolyzed precursor.     

Structural analysis of permethylated oligosaccharides using electrospray ionization quadrupole time-of-flight tandem mass spectrometry and deutero-reduction

Deutero-reduced permethylated oligosaccharides were analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, fitted with a nanoflow ESI source. Under these ionization conditions such derivatives preferentially form sodiated molecular species in addition to protonated molecular species. Under collision-induced dissociation, protonated and sodiated molecular species yield simple and predictable fragment mass spectra. A systematic study was conducted on a series of deutero-reduced permethylated glycans to allow rationalization of the fragmentation processes. MS/MS spectra were characterized by fragments resulting from the cleavage of glycosidic bonds. These fragments originating from both the reducing and the non-reducing ends of the glycan yield information on sequence and branching. Furthermore, the substituent 3-linked to a HexNAc unit was readily eliminated. Special attention was devoted to a systematic study of fucosylated glycans. The fucosylated deutero-reduced permethylated glycans were submitted to an acidic hydrolysis, releasing specifically the fucosyl residues. The nascent free hydroxyl groups were subsequently CD3-labelled in order to determine the positions initially bearing the fucosyl residues along the oligosaccharide backbone. This methodology was finally applied to characterize a glycan pool enzymatically released from glycoproteins. The present data show that structural elucidation can be achieved at the 50 fmol level.     

Multiple losses of neutral C14H14 in the tandem mass spectrometry of several perbenzyl ether intermediates in the synthesis of green tea constituents

Electrospray and matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) experiments were used to investigate an unusual fragmentation in collision-induced dissociation (CID) of sodiated and potassiated perbenzyl ether intermediates obtained in the total synthesis of gallate ester constituents of green tea. Prominent fragments correspond to multiple sequential losses of neutral C14H14 that were not observed in the protonated and ammoniated species, that instead present fragment ion series in which members are separated by C7H6. High-resolution MALDI quadrupole time-of-flight (Q-TOF) and electrospray-Fourier transform mass spectrometry (FTMS) were used to confirm elemental compositions of these and related ions.     

Fragmentation study of iridoid glucosides through positive and negative electrospray ionization, collision-induced dissociation and tandem mass spectrometry

Mass spectrometric methodology based on the combined use of positive and negative electrospray ionization, collision-induced dissociation (CID) and tandem mass spectrometry (MS/MS) has been applied to the mass spectral study of a series of six naturally occurring iridoids through in-source fragmentation of the protonated [M+H]+, deprotonated [M--H]- and sodiated [M+Na]+ ions. This led to the unambiguous determination of the molecular masses of the studied compounds and allowed CID spectra of the molecular ions to be obtained. Valuable structural information regarding the nature of both the glycoside and the aglycone moiety was thus obtained. Glycosidic cleavage and ring cleavages of both aglycone and sugar moieties were the major fragmentation pathways observed during CID, where the losses of small molecules, the cinnamoyl and the cinnamate parts were also observed. The formation of the ionized aglycones, sugars and their product ions was thus obtained giving information on their basic skeleton. The protonated, i.e. [M+H]+ and deprotonated [M--H]-, ions were found to fragment mainly by glycosidic cleavages. MS/MS spectra of the [M+Na]+ ions gave complementary information for the structural characterization of the studied compounds. Unlike the dissociation of protonated molecular ions, that of sodiated molecules also provided sodiated sugar fragments where the C0+ fragment corresponding to the glucose ion was obtained as base peak for all the studied compounds.     

Direct structural assignment of neutral and sialylated N-glycans of glycopeptides using collision-induced dissociation MSn spectral matching

Mass spectrometric analyses of various N-glycans binding to proteins and peptides are highly desirable for elucidating their biological roles. An approach based on collision-induced dissociation (CID) MS(n) spectra acquired by electrospray ionization linear ion trap time-of-flight mass spectrometry (ESI-LIT-TOFMS) in the positive- and negative-ion modes has been proposed as a direct method of assigning N-glycans without releasing them from N-glycopeptides. In the positive-ion mode of this approach, the MS(2) spectrum of N-glycopeptide was acquired so that a glycoside-bond cleavage occurs in the chitobiose residue (i.e., GlcNAcbeta1-4GlcNAc, GlcNAc: N-acetylglucosamine) attached to asparagine (N), and two charges on the [M+H+Na](2+) precursor ion are shared with both of the resulting fragments. These fragments are sodiated B(n)-type fragment ions of oligosaccharide (N-glycan) and a protonated peptide ion retaining one GlcNAc residue on the asparagine (N) residue. The structure of N-glycan was assigned by comparing MS(3) spectra derived from both the sodiated B(n)-type fragment ions of N-glycopeptide and the PA (2-aminopyridine) N-glycan standard (i.e., MS(n) spectral matching). In a similar manner, the structural assignment of sialylated N-glycan was performed by employing the negative-ion CID MS(n) spectra of deprotonated B(n)-type fragment ions of N-glycopeptide and the PA N-glycan standard. The efficacy of this approach was tested with chicken egg yolk glycopeptides with a neutral and a sialylated N-glycan, and human serum IgG glycopeptides with neutral N-glycan isomers. These results suggest that the approach based on MS(n) spectral matching is useful for the direct and simple structural assignment of neutral and sialylated N-glycans of glycopeptides.     

Stereochemical effects on the mass spectrometric behavior of native and derivatized trisaccharide isomers: comparisons with results from molecular modeling

Differentiation of oligosaccharide isomers by mass spectrometry (MS) is a challenging task. For native, permethylated and peracetylated trisaccharides, matrix-assisted laser desorption/ionization time-of-flight (MALDI/TOF) MS and liquid secondary ionization (LSI) MS experiments can produce complementary results that are useful for molecular mass and sugar sequence determination and isomer differentiation. Linear MALDI/TOF-MS analysis of native and derivatized oligosaccharides usually produces cationized molecular ions. Characterization by LSI-MS and tandem mass spectrometry (LSI-MS/MS) typically may yield only low-abundance protonated molecular ions but produces dominant B-type ions by elimination of ROH (R = Me, Ac) from the C-1 position at the reducing end and distinctive sequence-related fragments. Results for four milk trisaccharides, two neutral (fucosyllactoses) and two sialylated (sialyllactoses), are presented to demonstrate the utility of microscale permethylation and gas-to-solid phase peracetylation for high sensitivity structural elucidation. For the pairs of carbohydrates investigated in this study by LSI-MS, LSI-MS/MS and linear MALDI/TOF-MS, the fragmentation patterns of the native, permethylated and peracetylated isomer pairs are shown to differ markedly as a consequence of their limited dissimilarities. In addition, the tendency of sialylated carbohydrates to form lactones upon peracetylation has been exploited to take advantage of the variation in the extent of lactonization with orientation of the sialic acid moiety relative to the adjacent sugar rings. Lactone formation is favored for 3'-sialyllactose compared with its 6'-isomer; Hyperchem was employed to indicate the relative stabilities of the molecular and fragment ions and to visualize the molecules in 3D (rather than to obtain absolute conformational energy values). The relative conformational energies of lactonized and non-lactonized ions were calculated using the Hyperchem software; their values support the trends observed by MS.     

Fragmentation of pentacoordinate spirobicyclic aminoacyl-phosphoranes (P-AAs) by electrospray ionization tandem mass spectrometry concerning P-O and P-N bond cleavage

The fragmentation pathways of both protonated and sodiated pentacoordinate spirobicyclic aminoacylphosphoranes (P‐AAs) have been studied by electrospray ionization multi‐stage mass spectrometry (ESI‐MSⁿ) in positive mode. The possible pathways and their mechanisms are elucidated through the combination of ESI‐MS/MS, isotope (¹⁵ N and ²H) labeling and high‐resolution Fourier transform ion cyclotron resonance (FTICR)‐MS/MS. The relative Gibbs free energies (ΔG) of the product ions and possible fragmentation pathways are estimated at the B3LYP/6‐31 G(d) level of theory. The theoretical calculations show that both protonated and sodiated P‐AAs would quickly fragment before Berry pseudorotation. For protonated P‐AAs, they have different tendencies to P–O or P–N bond cleavage. For sodiated P‐AAs, the P–N bond is easier to cleave and produces the tetracoordinated phosphorus ion H. These results to some extent may give a clue to the chemistry of the active sites of phosphoryl transfer enzymes and will enrich the gas‐phase ESI‐MS ion chemistry of pentacoordinate phosphoranes. Copyright © 2011 John Wiley & Sons, Ltd.     

Mass spectrometry of proton adducts of fucosylated N-glycans: fucose transfer between antennae gives rise to misleading fragments

Fragmentation behavior of fucosylated N-glycans in both protonated and sodiated form was studied by low-energy collision-induced dissociation with an ion trap mass spectrometer as well as by laser-induced dissociation with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Diantennary, core-(alpha1-6)-fucosylated N-glycans with Lewis X (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAcbeta1-) and/or fucosylated LacdiNAc antennae (GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAcbeta1-) were obtained from the human parasite Schistosoma mansoni and used as model substances, after labeling with 2-aminobenzamide, or as native reducing glycans. While fragment spectra of sodiated as well as protonated species obtained in both mass spectrometers resulted in B- and Y-type ions, fragmentation of proton adducts additionally gave rise to various fragment ions which had acquired fucose residues from other parts of the molecule. In particular, fucose was transferred efficiently to the Lewis X antennae suggesting the occurrence of difucosylated antennae, which could erroneously be interpreted as Lewis Y epitopes. By studying two additional model substances, this fucose gain was shown to occur by transfer of fucose between the antennae, but not by transfer of a core-(alpha1-6)-fucose. Despite the drastically different lifetimes of the ions, protonated species analyzed on the ion trap (millisecond range) and by MALDI-TOF/TOF-MS (microsecond range) showed similar rearrangement patterns, suggesting that the fucose mobility goes hand in hand with decomposition. Notably, permethylation of the model N-glycans seemed to completely preclude fucose migration. This study indicates that caution should be applied with the interpretation of tandem mass spectrometric (MS/MS) data of protonated glycoconjugates, including glycopeptides, because of the potential occurrence of fucose rearrangements.     

Energy-dependent collision-induced dissociation study of buprenorphine and its synthetic precursors

The collision-induced dissociation (CID) of protonated buprenorphine ([M+H](+) ) and four related compounds was studied by electrospray quadrupole/time-of-flight mass spectrometry (ESI-QTOF MS). The fragmentation pathways were investigated by using energy-dependent CID and pseudo-MS(3) (in-source CID combined with tandem mass spectrometry (MS/MS)) methods. The first steps of the fragmentation are the parallel losses of the substituents from the non-aromatic ring moieties. Depending on the applied collision energies, a large number of further fragment ions arising from the cross-ring cleavages of the core-ring structure were observed. Based on the experimental results, a generalized fragmentation scheme was developed for the five buprenorphine derivatives highlighting the differences for the alternatively substituted compounds. The collision-energy-dependent fragmentation profile of buprenorphine is visualized in a two-dimensional plot to aid its fingerprint identification.     

Structure and further fragmentation of significant [a3 + Na − H]+ ions from sodium‐cationized peptides

A good understanding of gas‐phase fragmentation chemistry of peptides is important for accurate protein identification. Additional product ions obtained by sodiated peptides can provide useful sequence information supplementary to protonated peptides and improve protein identification. In this work, we first demonstrate that the sodiated a₃ ions are abundant in the tandem mass spectra of sodium‐cationized peptides although observations of a₃ ions have rarely been reported in protonated peptides. Quantum chemical calculations combined with tandem mass spectrometry are used to investigate this phenomenon by using a model tetrapeptide GGAG. Our results reveal that the most stable [a₃ + Na ? H]⁺ ion is present as a bidentate linear structure in which the sodium cation coordinates to the two backbone carbonyl oxygen atoms. Due to structural inflexibility, further fragmentation of the [a₃ + Na ? H]⁺ ion needs to overcome several relatively high energetic barriers to form [b₂ + Na ? H]⁺ ion with a diketopiperazine structure. As a result, low abundance of [b₂ + Na ? H]⁺ ion is detected at relatively high collision energy. In addition, our computational data also indicate that the common oxazolone pathway to generate [b₂ + Na ? H]⁺ from the [a₃ + Na ? H]⁺ ion is unlikely. The present work provides a mechanistic insight into how a sodium ion affects the fragmentation behaviors of peptides. Copyright © 2015 John Wiley & Sons, Ltd.     

Electrospray ionization ion-trap time-of-flight tandem mass spectrometry of two furofurans: sesamin and gmelinol

High-resolution electrospray ionization multistage tandem mass spectrometry (MS(1-7)) in positive ion mode was used to determine the accurate masses and the fragmentation pathways of two furofurans, sesamin and gmelinol. The protonated molecules [M+H]+ were absent in the conventional mass spectra of both compounds, but two characteristic ions, [M+H-H(2)O]+ and [M+H-H(2)]+, were always observed. Successive losses of CH(2)O and CO are the common characteristic fragmentations. Based on the exact masses acquired from 21 different tandem mass spectra, two or three fragmentation pathways for each compound are proposed. The consecutive losses of two H(2)O molecules and one H(2) molecule readily take place from the furan rings for both sesamin and gmelinol, resulting in the absence of the protonated molecules in the single-stage experiments. HCHO loss is observed at least three times in the tandem mass spectra, mainly from methylenedioxy groups (OCH(2)O) for sesamin but only from tetrahydrofuran rings for gmelinol. Moreover, CO loss is found at least three times in the tandem mass spectra of both sesamin and gmelinol from the 3,4-methylenedioxyphenyl (ArOCH(2)O) moieties for sesamin and from both the dimethoxyphenyl and the tetrahydrofuran ring moieties for gmelinol. In addition, the disubstituted benzyl cation ArCH(2)+ at m/z 135 for sesamin and at m/z 151 for gmelinol was found in the MS(3) spectra of both sesamin and gmelinol, which is very helpful in the identification of the compositions of 3,4-disubstituted groups on the benzene rings of the furofurans.     

A tandem mass spectrometric study of saccharides at high mass resolution

Nine monosaccharides and four disaccharides were mass analyzed using a quadrupole time-of-flight tandem mass spectrometer combined with an electrospray ionization source. Product ion mass spectra of deprotonated, protonated, and sodiated saccharides were observed and were compared within each group of saccharides. Each of the deprotonated pentoses, hexoses and disaccharides yielded a significantly different product ion mass spectrum with the exception of alpha-lactose and beta-lactose. The disaccharides alpha- and beta-lactose differ only at the glycosidic linkage. Product ion mass spectra of protonated and sodiated alpha- and beta-lactose were indistinguishable also.     

Constant neutral loss scanning for the characterization of glycerol phosphatidylcholine phospholipids

The product-ion spectra of the sodiated molecules of glycerol phosphatidylcholine phospholipids (GPC) were obtained, using fast-atom bombardment (FAB) as the ionization method, in a tandem mass spectrometer. The product-ion spectra of these sodiated molecules of GPCs were found to differ significantly from those of the protonated GPC molecules. This difference is due to the absence of the ion of m/z 184 (protonated-phosphocholine moiety) and to the presence of an ion resulting from the loss of trimethylamine (m=59 Da) from the polar head group, which is the dominant fragmentation. This characteristic neutral loss provides a means of identification of this class of phospholipids and of differentiation from other phospholipid classes in complex mixtures by performing a constant-neutral-loss scan of 59.