Characterization of ceramides by low energy collisional-activated dissociation tandem mass spectrometry with negative-ion electrospray ionization

Negative-ion electrospray ionization tandem quadrupole mass spectrometry provides a useful method for the structural characterization of ceramides. Fragment ions referring to the identities of the fatty acid substituent and of the long chain base of the molecules are readily available and the structure of ceramides can be easily determined. A unique fragmentation pathway which leads to formation of the fatty acid carboxylate anions (RCO2) was observed. This fragmentation is initiated by cleavage of the C2-C3 bond of the LCB to yield a N-acylaminoethanol anion ([RCONHCH2CH2O]-), followed by rearrangement to a carboxyethylamine ([RCO2CH2CH2NH]-) intermediate, which further dissociates to a RCO2- ion. This pathway is confirmed by the CAD tandem mass spectrum of the synthetic N-acylaminoethanol standard and of the deuterated analogs of ceramides obtained by H-D exchange. The observation of RCO2- ion species permits an unambiguous identification of the fatty acyl moiety of ceramides. Tandem mass spectrometry methods for characterization of structural isomers of ceramides using product-ion scanning and for identification of specific ceramide subclasses in biological mixtures using neutral loss scanning are also demonstrated.     

Novel phosphoryl derivatization method for peptide sequencing by electrospray ionization mass spectrometry

A one-step phosphoryl derivatization method has been used in a peptide sequencing procedure for electrospray ionization tandem mass spectrometry (ESI-MS/MS). The sodiated derivatized peptides exhibit very simple dissociation patterns, in which two kinds of fragment ions, [b(n) + OH + Na]+ and [a(n) + Na]+, are formed. Since the amino acid residues are lost sequentially from the C-terminus, peptide sequences can be identified easily. The fragmentation efficiency of peptides increased as a result of the phosphorylation, and also provided peaks of useful intensity at lower m/z. A peptide with lysine at the C-terminus was derivatized and analyzed by ESI-MS/MS. Similar mass spectra, from which the sequence could be read out, were obtained. This is a novel derivatization method yielding neutral derivatives that should be suitable for peptide sequencing by LC/ESI-MS/MS.     

Characterization of protein-hapten conjugates by mass spectrometry

An hapten 1 designed for the production of catalytic antibodies was synthesized after coupling its precursor 2 to bovine serum albumin (BSA). The conjugates BSA-2 and BSA-1 were characterized by MALDI mass spectrometry. This paper shows that besides the average number of molecules bound to the protein deduced from the molecular ion peak shift, the MALDI technique can also give access to their distribution, bv simulation of the peak.     

Characterization of titanocenium and titanocene by tandem mass spectrometry

Tandem mass spectrometric techniques were used for the characterization of gas-phase titanocenium ions. Decomposition of metastable and collisionally activated C₁₀H₁₀Ti^+˙ ions involved cyclopentadienyl–metal bond rupture, acetylene loss and dehydrogenation as the prominent processes. The intermediate formation of titanium (di)hydride complexes was proposed to explain the selective H₂ molecule loss. The neutralization–reionization mass spectrum showed a very abundant recovery signal, indicating a high stability for the neutral gas-phase C₁₀H₁₀Ti species.     

Characterization of chitosan by pyrolysis—mass spectrometry

Chitosan of different degrees of deacetylation have been prepared from chitin. Pyrolysis-mass spectra of these chitosan samples in the ion source of a mass spectrometer were examined to check for a correlation with the degree of deacetylation, as represented by the amine content. The results indicate that as the degree of deacetylation increases, the peak ratios 80:60, 67:60 and 80:42 increase to a limit, representing the limit of deacetylation of chitin. The 80 and 67 fragments originate from the d-glucosamine moiety of the polymer and the 60 and 42 fragments from the N-ethyl-d-glucosamine moiety. For chitin, the predicted values of these ratios are expected to be low when compared to chitosan, and this is borne out by the experimental data.     

Characterization of internal salts by laser mass spectrometry

An intermolecular alkyl transfer reaction (ATR) leading to ion-pair formation has been observed for internal salts by using laser mass spectrometry (l.m.s.). Positive- and negative-ion spectra both show evidence for alkyl transfer. Both the LAMMA-500 (transmission) and LAMMA-1000 (reflection) laser mass spectrometers were used. The positive-ion laser mass spectra obtained by these two instruments show some significant differences; no significant differences were observed in the negative-ion spectra. Results obtained for quaternary ammoniohexanoates as a function of laser power indicate that the extent of ATR is greater at high laser power. Addition of a small amount of p-toluenesulfonic acid to the ammoniohexanoates reduces fragmentation and enhances the intensity of the quasimolecular ion (M + H)⁺ relative to ATR. Results from deuterated sultaines were used to confirm intermolecular alkyl transfer and to elucidate some fragmentation processes. Field-desorption (f.d.) mass spectra of internal salts show similarities and differences from l.m.s.; not all internal salts showed the alkyl transfer reaction in f.d. Cluster ion formation was observed in f.d.m.s. but not in l.m.s.     

Characterization of metal-labelled peptides by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry

Metal labelling of peptides and proteins using high-affinity metal-chelating compounds has found widespread applications in the medical and bioanalytical fields. In the present study we investigated the analysis of peptides derivatized either with cysteine- or amino group-directed metal-bound DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The metal complexes of DOTA were shown to be stable under MALDI-MS conditions. The introduction of the metal label led in a number of cases to significantly increased signal-to-noise (S/N) values and thus improved sensitivity of the labelled peptides compared to their unlabelled counterparts, especially for multiply labelled peptides. The presence of the labels did alter the tandem mass spectrometric (MS/MS) behaviour, namely the formation of sequence specific a-, b- and y-ion series, in dependence of the position of the label within the peptide sequence. For cysteine-derivatized peptides several label-specific reporter ions and characteristic immonium ions could be identified. Amino-directed labelling led only to the formation of characteristic immonium ions in ε-amino groups of lysine, whereas N-terminal labelling in some cases led to the formation of a(1)- and b(1)-ions. The results clearly show that MALDI-MS is suitable for the analysis of metal-labelled peptides, which was also confirmed in liquid chromatography (LC)/MALDI-based identification of proteins in a model protein mixture labelled with Cys-reactive DOTA. Here, in comparison to a run with alkylated cysteines, more than 50% more cysteine-containing peptides were identified.     

Separation and identification of ceramides in the human stratum corneum by high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and electrospray multiple-stage mass spectrometry profiling

Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum coracum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramdies than that achievable by thin-layer chromatography. Summary A sensitive, selective, and rapid method is described for analysis of ceramides in the human stratum corneum by direct coupling of HPLC with an electrospray ion-trap mass spectrometry. Nonaqueous reversed-phase chromatography stabilizes the electrospray ionization, resulting in sensitivity that enables direct measurement of skin lipid extracts with no special sample preparation. Assignment of individual signals to the corresponding ceramide species is based on interpretation of the fragment spectra from MS-MS experiments. This enables much finer differentiation between ceramides than that achievable by thin-layer chromatography.     

Characterization of linear and branched polyacrylates by tandem mass spectrometry

The unimolecular degradation of alkali-metal cationized polyacrylates with the repeat unit CH₂CH(COOR) and a variety of ester pendants has been examined by tandem mass spectrometry. The fragmentation patterns resulting from collisionally activated dissociation depend sensitively on the size of the ester alkyl substituent (R). With small alkyl groups, as in poly(methyl acrylate), lithiated or sodiated oligomers (M) decompose via free-radical chemistry, initiated by random homolytic C-C bond cleavages along the polymer chain. The radical ions formed this way dissociate further by backbiting rearrangements and β scissions to yield a distribution of terminal fragments with one of the original end groups and internal fragments with 2–3 repeat units. If the ester alkyl group bears three or more carbon atoms, cleavages within the ester moieties become the predominant decomposition channel. This distinct reactivity is observed if R = t-butyl, n-butyl, or the mesogenic group (CH₂)₁₁-O-C₆H₄-C₆H₄-CN. The [M+alkali metal]⁺ ions of the latter polyacrylates dissociate largely by charge-remote 1,5-H rearrangements that convert COOR to COOH groups by expulsion of 1-alkenes. The acid groups may displace an alcohol unit from a neighboring ester pendant to form a cyclic anhydride, unless hindered by steric effects. Using atom transfer radical polymerization, hyperbranched polyacrylates were prepared carrying ester groups both within and between the branches. Unique alkenes and alcohols are cleaved from ester groups at the branching points, enabling determination of the branching architecture. Figure MALDI-CAD tandem mass spectrum of the lithiated 4-mer from a hyperbranched polyacrylate. The fragments marked by green stars diagnose the branched architecture shown on top of the spectrum. The fragments marked by violet stars diagnose a different isomer.     

Characterization of some penicillins and cephalosporins by pyrolysis mass spectrometry

A number of clinically significant penicillins and cephalosporins have been characterized by pyrolysis mass spectrometry. With only a 2-μg sample of formulated drugs, the antibiotics can be clearly differentiated. The structure of the major fragments is assessed.     

Characterization of cationic glycoporphyrins by electrospray tandem mass spectrometry

Novel cationic porphyrin derivatives having a galactose or a bis(isopropylidene)galactose unit linked directly to a pyridine or to an aminophenyl group were characterized by electrospray tandem mass spectrometry (ESI-MS/MS). The electrospray mass spectra (ESI-MS) show the M(+) ions, since these porphyrins are already monocharged in solution. The fragmentation of these ions under ESI-MS/MS conditions was studied and it was found that elimination of the sugar residue as a radical (-163 or -243 Da) is a common fragmentation pathway. Loss of the sugar unit as a neutral fragment (-162 or -242 Da) and cross-ring fragmentations typical of glyco-derivatives are also observed for the pyridinium glycoporphyrins, but they are absent in the case of ammonium glycoporphyrins. The cationic beta-pyridiniumvinyl porphyrins show an atypical fragmentation due to the cleavage of the C(5)-C(6) bond of the sugar unit. Overall, the different patterns of fragmentation observed in the ESI-MS/MS spectra of the sugar pyridinium porphyrins and of the sugar ammonium phenyl porphyrins can give important information about the type of spacer between the porphyrin and the sugar unit.     

Characterization of glycopeptides from HIV-ISF2 gp120 by liquid chromatography mass spectrometry

Previously, we have characterized the HIV-I(SF2) gp120 glycopeptides using matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) and nanospray electrospray ionization (ESI). Although we characterized 25 of 26 consensus glycosylation sites, we could not obtain any information about the extent of sialylation of the complex glycans. Sialylation is known to alter the biological activity of some glycoproteins, e.g., infectivity of some human and nonhuman primate lentiviruses is reduced when the envelope glycoproteins are extensively sialylated, and thus, characterization of the extent of sialylation of complex glycoproteins is of biological interest. Since neither MALDI/MS nor nanospray ESI provided much information about sialylation, probably because of suppression effects inherent in these techniques, we utilized online nanocapillary high performance liquid chromatography (nHPLC) with ESI/MS to characterize the sites and extent of sialylation on gp120. Eight of the known 26 consensus glycosylation sites of HIV-ISF2 gp120 were determined to be sialylated. Two of these sites were previously uncharacterized complex glycans. Thirteen high mannose sites were also determined. The heterogeneity of four of these sites had not been previously characterized. In addition, a peptide containing two consensus glycosylation sites, which had previously been determined to contain complex glycans, was also determined to be high mannose as well.     

Characterization of a model Phillips catalyst by mass spectrometry

A model Phillips catalyst for ethylene polymerization, prepared by spin coating a Cr(III)(Cr(acac)3) precursor on a silicon wafer, was submitted to an oxidative activation. Laser ablation Fourier transform mass spectrometry provided direct information on molecular species at the silicon wafer surface during activation. At 350 degrees C the chromium precursor was degraded, while chromium oxide species were formed. The chromium concentration decreased with temperature. The activated model catalyst was active for ethylene polymerization. Using complementary techniques (Fourier transform infrared spectroscopy, laser desorption/ionization mass spectrometry), the polymer was identified as crystalline polyethylene. After 1 h of polymerization at 160 degrees C, dome-like structures were observed by atomic force microscopy. Their morphologies were constituted of regions of parallel aligned lamellae of polymer.     

Characterization of natural wax esters by MALDI-TOF mass spectrometry

The applicability of matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) to the analysis of wax esters (WEs) was investigated. A series of metal salts of 2,5-dihydroxybenzoic acid (DHB) was synthesized and tested as possible matrices. Alkali metal (Li, Na, K, Rb, Cs) and transition metal (Cu, Ag) salts were studied. The matrix properties were evaluated, including solubility in organic solvents, threshold laser power that should be applied for successful desorption/ionization of WEs, the nature of the matrix ions and the mass range occupied by them, and the complexity of the isotope clusters for individual metals. Lithium salt of dihydroxybenzoic acid (LiDHB) performed the best and matrices with purified lithium isotopes ((6)LiDHB or (7)LiDHB) were recommended for WEs. Three sample preparation procedures were compared: (1) mixing the sample and matrix in a glass vial and deposition of the mixture on a MALDI plate (Mix), (2) deposition of sample followed by deposition of matrix (Sa/Ma), and (3) deposition of matrix followed by deposition of sample (Ma/Sa). Morphology of the samples was studied by scanning electron microscopy. The best sample preparation technique was Ma/Sa with the optimum sample to matrix molar ratio 1 : 100. Detection limit was in the low picomolar range. The relative response of WEs decreased with their molecular weight, and minor differences between signals of saturated and monounsaturated WEs were observed. MALDI spectra of WEs showed molecular adducts with lithium [M + Li](+). Fragments observed in postsource decay (PSD) spectra were related to the acidic part of WEs [RCOOH + Li](+) and they were used for structure assignment. MALDI with LiDHB was used for several samples of natural origin, including insect and plant WEs. A good agreement with GC/MS data was achieved. Moreover, MALDI allowed higher WEs to be analyzed, up to 64 carbon atoms in Ginkgo biloba leaves extract.     

Characterization of sodiated glycerol phosphatidylcholine phospholipids by mass spectrometry

Fast atom bombardment mass spectrometry in the positive mode was used for the characterization of sodiated glycerol phosphatidylcholines. The relative abundance (RA) of the protonated species is similar to the RA of the sodiated molecular species. The sodiated fragment ion, [M + Na - 59](+), corresponding to the loss of trimethylamine, and other sodiated fragment ions, were also observed. The decomposition of the sodiated molecule is very similar for all the studied glycerol phosphatidylcholines, in which the most abundant ion corresponds to a neutral loss of 59 Da. Upon collision-induced dissociation (CID) of the [M + Na](+) ion informative ions are formed by the losses of the fatty acids in the sn-1 and sn-2 positions. Other major fragment ions of the sodiated molecule result from loss of non-sodiated and sodiated choline phosphate, [M + Na - 183](+), [M + Na - 184](+.) and [M + Na - 205](+), respectively. The main CID fragmentation pathway of the [M + Na - 59](+) ion yields the [M + Na - 183](+) ion, also observed in the CID spectra of the [M + Na](+) molecular ion. Other major fragment ions are [M + Na - 205](+) and the fragment ion at m/z 147. Collisional activation of [M + Na - 205](+) results in charge site remote fragmentation of both fatty acid alkyl chains. The terminal ions of these series of charge remote fragmentations result from loss of part of the R(1) or R(2) alkyl chain. Other major informative ions correspond to acylium ions.     

Characterization of multimetallic grid-type complexes by electrospray mass spectrometry

The self-assembly of the terdentate ligands 1a-h, based on terpyridine-like binding sites, with octahedrally coordinated metal ions, such as Fe(II), Co(II), Cu(II), Zn(II), Cd(II), Hg(II) and Pb(II), leads to the formation of the supramolecular grid-type complexes 2a-c(M(II)), 3d-g(M(II)) and 4h(M(II)). The structures and compositions of these coordination complexes in solution were deduced from electrospray mass spectrometry (ESMS) measurements. The results agree with the data available from x-ray radiocrystallography in the solid state and/or NMR spectroscopy in solution. ESMS may be applied in cases where other methods are difficult to use or inconclusive. This study stresses the power of ESMS in supramolecular chemistry.     

Structural analysis of commercial ceramides by gas chromatography-mass spectrometry

A simple method using gas chromatography-mass spectrometry was applied to analyse structures of ceramides. Identification of trimethylsilylated ceramides were obtained in short analysis times (derivatization of ceramides in 30 min at room temperature and 20 min gas chromatography mass spectrometry run) even for complex mixtures. For example in ceramide Type III, 18 peaks were observed which represent 27 various structures. The coeluted compounds were ceramides containing the same functional groups and the same carbon number but with a different distribution on the two alkyl chains of the molecule. They were accurately differentiated by mass spectrometry. Therefore, 83 structures of trimethylsilylated ceramides were identified in 11 different commercial mixtures. For 52 structures of these, mass spectral data were not described in the literature, neither full mass spectra nor characteristic fragments.     

Characterization and identification of isomeric flavonoid O-diglycosides from genus Citrus in negative electrospray ionization by ion trap mass spectrometry and time-of-flight mass spectrometry

Flavonoid O-diglycosides are important bioactive compounds from genus Citrus. They often occur as isomers, which makes the structural elucidation difficult. In the present study, the fragmentation behavior of six flavonoid O-diglycosides from genus Citrus was investigated using ion trap mass spectrometry in negative electrospray ionization (ESI) with loop injection. For the flavonoid O-rutinosides, [M − H − 308]⁻ ion was typically observed in the MS² spectrum, suggesting the loss of a rutinose. The fragmentation patterns of flavonoid O-neohesperidosides were more complicated in comparison with their rutinoside analogues. A major difference was found in the [M − H − 120]⁻ ion in the MS² spectrum, which was a common feature of all the flavonoid O-neohesperidosides. The previous literature for naringin located the loss of 120 Da to the glycan part, whereas the present study for naringin had shown that the [M − H − 120]⁻ ion was produced by a retro-Diels-Alder reaction in ring C, and this fragmentation pattern was confirmed by the accurate mass measurement using an orthogonal time-of-flight mass spectrometer. Combined with high performance liquid chromatography (HPLC) and diode array detection (DAD), the established approach to the structural identification of flavonoid O-diglycosides by ion trap mass spectrometry was applied to the analysis of extracts of two Chinese medicines derived from genus Citrus, namely Fructus aurantii and F. aurantii immaturus. According to the HPLC retention behavior, the diagnostic UV spectra and the molecular structural information provided by multistage mass spectrometry (MSⁿ) spectra, 13 flavonoid O-glycosides in F. aurantii and 12 flavonoid O-glycosides in F. a. immaturus were identified rapidly.     

Analysis of ceramides in cosmetics by reversed-phase liquid chromatography/electrospray ionization mass spectrometry with collision-induced dissociation

A rapid, sensitive and selective method involving reversed-phase liquid chromatography (LC) with electrospray ionization (ESI) mass spectrometry (MS) was employed for determination of commercial ceramides in cosmetics for quality control of the product formulation. Using this LC/ESI-MS technique, simultaneous separation and characterization of ceramides and an impurity substance were possible. Informative fragmentation patterns were obtained by employing LC/ESI-MS in both positive and negative ionization modes to identify the structures of both sphingoid base and N-acyl chains of ceramides, and also of an impurity. The combination of positive and negative mass spectra can be used for unambiguous confirmation of ceramides and for characterization of unknown species. In-source collision-induced fragmentation resulted in characteristic product anions for the ceramides containing a phytosphingosine moiety at m/z 267, 255 and 225, and for those with a sphingosine moiety at m/z 263 and 237, regardless of the length of the fatty acyl chains. The detection limit was about 0.5 pmol in selected-ion monitoring mode. Quantification using internal standards showed good linearity and a relative standard deviation of 4%. These ceramides were more sensitively detected in positive than in negative ion mode.