Optimization of matrix-assisted laser desorption/ionization time-of-flight collision-induced dissociation using poly(ethylene glycol)

Details of the optimization of the collision-induced dissociation (CID) process, using a collision cell on a matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometer, are described using poly(ethylene glycol) 1000 (PEG 1000) as a model analyte. The effects of collision gas identity (helium, air, and argon), as well as collision gas pressure, on the resulting MS/MS data were investigated. With PEG 1000, helium was found to give the best results with respect to signal-to-noise (S/N) ratio. The optimum pressure for each gas was found to be in the range where the precursor ion signal was attenuated to approximately 30-50% for helium and 40-60% for argon. The effect of cation choice (Li, Na, and K) on the CID of PEG was also studied. CID spectra were produced for each, but PEG cationized with lithium was found to produce the spectra with the highest S/N ratio. The MALDI-TOF CID spectra that were generated for PEG were compared with the high-energy and low-energy MS/MS spectra obtained from a sector mass spectrometer and from a triple quadrupole mass spectrometer, respectively. The results observed for PEG confirm that CID on a MALDI-TOF mass spectrometer is a high-energy MS/MS technique.     

Lithium and transition metal ions enable low energy collision-induced dissociation of polyglycols in electrospray ionization mass spectrometry

Electrospray ionization tandem mass spectrometry has the potential to be widely used as a tool for polymer structural characterization. However, the backbones or molecular chains of many industrial polymers including functional polyglycols are often difficult to dissociate in tandem mass spectrometers using low energy collision-induced dissociation (CID). We present a method that uses Li+ and transition metal ions such as Ag+ as the cationization reagents for electrospray ionization in an ion trap mass spectrometer. It is shown that lithium and transition metal polyglycol adduct ions can be readily fragmented with low energy CID. Comparative results from different cationization reagents in their abilities of producing both MS spectra and CID spectra are shown. This method opens the possibility of using conventional and readily available low energy CID tandem MS to study polyglycol structures.     

External ion accumulation of low molecular weight poly(ethylene glycol) by electrospray ionization fourier transform mass spectrometry

The combination of electrospray ionization (ESI) with Fourier transform mass spectrometry (FTMS) is a powerful tool in characterizing synthetic polymers. ESI permits the generation of intact, multiply charged high mass ions, whereas FTMS provides high mass resolution and correspondingly improved mass accuracy. However, under “nonideal” empirically determined operating conditions, a mass discrimination effect occurs as a function of accumulation time that can result in significant differences for calculated average mass values (M_n, M_w) and polydispersities. A multidimensional tuning process to eliminate the deleterious effects of mass bias is demonstrated for several sodiated poly(ethylene glycol) samples containing oligomers with masses covering a 600–3350 Da mass regime. In addition, experiments are performed in order to elucidate the possible mechanism(s) that cause the mass discrimination effect. It is proposed that extended collisions (reactive and nonreactive) occur in the hexapole to alter the energy (and velocity) distributions of the ions before injection into the trap. By choosing higher skimmer potentials, ions over a narrower energy “window” are preselected in the hexapole and exhibit lower overall mass bias effects.     

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.     

Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry

Three samples of albumin derived from human plasma (pharmaceutical grade, HSA) obtained from different commercial sources were investigated for their micro-heterogeneities by means of electrospray ionization (ESI) ion trap mass spectrometry (ITMS). The study covered MS analyses of the intact proteins as well as on the tryptic peptide level. The intact protein samples were analyzed without any separation step except for simple desalting. With these samples we observed in the positive ion ESI mass spectra that the multiply charged ion signals of HSA consisted of a number of fully or partly resolved peaks with relative intensities depending on the analyzed sample. The non-modified form of HSA was detected in the three HSA preparations at m/z values of 66448 +/- 3.6, 66450 +/- 0.6 and 66451 +/- 3.2 ([MH]+), respectively. The value calculated from the amino acid sequence was 66439. The second compound present with high intensity (in two cases the base peak in the deconvoluted mass spectrum) is interpreted as a modified HSA, and the molecular mass increase in relation to the unmodified HAS was between 116 and 118 Da (m/z of 66 564, 66 567 and 66 569), suggesting the presence of a covalently bound cysteine residue. A further peak in the deconvoluted ESI spectra was found in all three samples with rather low signal/noise ratio at m/z 66 619, 66 621 and 66 613, respectively, which may correspond to a non-enzymatic glycation described in the literature. The verification of the proposed covalent HSA modifications was subsequently done on the peptide level using high-performance liquid chromatography (HPLC)/ESI-MS and HPLC/ESI-MS/MS including low-energy collision-induced dissociation (CID). Prior to the tryptic digestion, the HSA samples were alkylated without a prior reduction step. Following this procedure we detected peptides of the sequence T21-41 that included the Cys-34 residue in both forms: cysteinylated (m/z 639.15 [M+4H]4+) as well as vinylpyridine-alkylated (m/z 635.69 [M+4H]4+, which means in its previously native free SH form). In the next step on-line LC/ESI low-energy CID MS/MS experiments were performed to verify these two proposed structures. By means of MS/MS analysis of the mentioned ions the described modification (cysteinylation) at the Cys-34 residue could be proven. This abundant modification of HSA in pharmaceutical-grade preparations could be unambiguously identified as cysteinylation at the Cys-34 residue. On the other hand, the proposed non-enzymatic glycation was not detectable on the peptide level in the on-line HPLC/ESI-MS mode, maybe due to the low concentration in the three samples under investigation.     

Characterization of ginseng saponins using electrospray mass spectrometry and collision-induced dissociation experiments of metal-attachment ions

Electrospray mass spectrometry (ESMS) and collision-induced dissociation (CID) methodologies have been developed for the structural characterization of ginseng saponins (ginsenosides). Ginsenosides are terpene glycosides containing a triterpene core to which one to four sugars may be attached. They are neutral molecules which readily form molecular metal-attachment ions in positive ion ESMS experiments. In the presence of ammonium hydroxide intense deprotonated ions are generated. Both positive and negative ion ESMS experiments were found to be useful for molecular mass and structure determination of ten ginsenoside standards. Negative ion experiments made possible the determination of the molecular mass of each ginsenoside standard, the mass of the triterpene core and the masses and sequences of the sugar residues. Positive ion ESMS experiments with the alkali metal cations Li+ or Na+ and the transition metal cations Co2+, Ni2+ and Zn2+ were also useful in determining molecular masses. These alkali and transition metal cations form strongly bonded attachment ions with the ginsenosides. As a result, the CID mass spectra of the metal attachment ions show a variety of (structure characteristic) fragmentations. These experiments can be used to determine the identity of the triterpene core, the types and attachment points of sugars to the core and the nature of the O-glycosidic linkages in the appended disaccharides. Combining the results from the negative and positive ion experiments provides a promising approach to the structure analysis of this class of natural products.     

The characterization of dendronized poly(ethylene glycol)s and poly(ethylene glycol) multi-arm stars using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The synthesis of branched poly(ethylene glycol) (PEG) derivatives, namely star PEG and dendronized PEG, can be challenging and their purity can be difficult to ascertain using traditional techniques, such as NMR and GPC. Herein, the detailed characterization of these branched PEGs using MALDI-ToF MS was investigated in order to confirm their structural purity. In this light, mass spectrometry offers a number of advantages for polymer characterization, including the ability to get detailed structural data, such as end group masses, from microgram-scale samples. In addition, the ability to rapidly acquire data from crude reaction aliquots makes MALDI-ToF MS ideal for monitoring end group transformations.     

Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.     

Characterization of electrospray ionization mass spectrometry for N-diisopropyloxyphosphoryl dipeptide methyl esters

A systematic study of the fragmentation pattern of N-diisopropyloxyphosphoryl (DIPP) dipeptide methyl esters in an electrospray ionization (ESI) tandem mass spectrometry (MS/MS) was presented. A combination of accurate mass measurement and tandem mass spectrometry had been used to characterize the major fragment ions observed in the ESI mass spectrum. It was found that the alkali metal ions acted as a fixed charge site and expelled the DIPP group after transferring a proton to the amide nitrogen. For all the N-phosphoryl dipeptide methyl esters, under the activation of a metal ion, the rearrangement product ion at m/z 163 was observed and confirmed to be the sodium adduct of phosphoric acid mono-isopropyl esters (PAIE), via a specific five-membered penta-co-ordinated phosphorus intermediate. However, no rearrangement ion was observed when a beta-amino acid was at the N-terminal. This could be used to develop a novel method for differentiating isomeric compounds when either alpha- or beta-amino acid are at the N-terminus of peptides. From the [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters (DIPP Xaa1 Xaa2 OMe), the peaks corresponding to the [M+Na Xaa1 C3H6]+ were observed and explained. The [M+Na]+ ESI-MS/MS spectra of N-phosphoryl dipeptide methyl esters with Phe located in the C-terminal, such as DIPPValPheOMe, DIPPLeuPheOMe, DIPPIlePheOMe, DIPPAlaPheOMe and DIPPPhePheOMe, had characteristic fragmentation. Two unusual gas-phase intramolecular rearrangement mechanisms were first proposed for this fragmentation. These rearrangements were not observed in dipeptide methyl ester analogs which did not contain the DIPP at the N-terminal, suggesting that this moiety was critical for the rearrangement.     

Structural characterization of flavonol di-O-glycosides from Farsetia aegyptia by electrospray ionization and collision-induced dissociation mass spectrometry

This study reports the application of mass spectrometric methods to characterize unknown flavonoids of the herb Farsetia aegyptia Turra (Crucifereae). High-performance liquid chromatography was performed in combination with UV-photodiode array detection (LC/UV-DAD) and electrospray ionization mass spectrometry (LC/ESI-MS) in both positive and negative ion modes. Collision-induced dissociation (CID) mass spectral data were obtained off-line by nanospray (nano-ESI) analysis, which provided a wealth of information and led to the structural proposal of the flavonol di-O-glycosides present in the herb extract. In addition to the mass spectral data, we also report NMR data for the major compound which allowed the completion of its structural elucidation. The Farsetia aegyptia Turra herb extract was found to contain three flavonol di-O-glycosides containing a monosaccharidic residue linked to the 3-O position and a disaccharidic residue linked to the 7-O position; the major compound was characterized as the new flavonoid, isorhamnetin 3-O-alpha-L-arabinoside 7-O-[beta-D-glucosyl-1 --> 2]-alpha(L)rhamnoside. Different types of CID spectra, i.e., low-energy [M+H]+, [M+Na]+ and [M--H]- spectra as well as high-energy [M+Na]+ spectra, were evaluated with respect to their utility to locate the O-linked saccharidic residues in flavonol di-O-glycosides and to determine the sequence in the disaccharidic part. In agreement with previously published data, the 3-O-glycosyl residue was more readily lost from the protonated molecule than the 7-O-glycosyl residue. The opposite behavior was noted for the fragmentation of the deprotonated and sodiated molecules. Radical ions were observed in the high-energy [M+Na]+ CID spectra which provided supporting information on the glycosylation positions.     

The fundamentals of applying electrospray ionization mass spectrometry to low mass poly(methyl methacrylate) polymers

Electrospray ionization (ESI) is capable of ionizing many soluble polymers. The ESI spectra are complex because of overlap of the multiply charged ions of the oligomer distribution, causing current computer transform programs to fail. However, it is possible to determine the origin of the multiply charged ions, making it feasible to write a program designed to transform ESI polymer spectra. To assess the value of such a program for polymer analysis, isolated monodisperse methyl methacrylate (MMA) oligomers (25 and 50 repeat units) were used to determine molar signal response and propensity for fragmentation. The sum of the peak areas for the multiply charged MMA 50-mer was found to be only about 66% of the summed peak areas for the 25-mer for the same molar concentration. However, conversion of the multiply charged peak areas to the singly charged representations, with peak area compression taken into account, gave equal signal responses for the 25-and 50-mers. Signal response variations due to the tacticity of the MMA oligomers were not observed. Fragmentation of the MMA oligomers also was shown not to occur under normal ESI conditions. Therefore, transformation of the polymer spectra to the singly charged molecular ion distribution should allow accurate calculation of average molecular weights, polydispersity, end group mass, and repeat unit mass.     

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.     

Tandem mass spectrometry of poly(ethylene glycol) lithium-attachment ions

A study of the fast-atom bombardment tandem mass spectrometry behavior of a number of ethylene glycol polymers (PEGs) has been carried out. Both linear (hydroxyl, amino, and/or alkyl end groups) and cyclic (crown ether) polymers were studied. One of the materials is a block copolymer of ethylene and propylene oxides. Collisional activation was carried out in the collision octapole of a BEoQ hybrid mass spectrometer at a translational energy of 50 eV, with collision gas air. For the linear polymers, the most intense product ions are lithiated, linear polyglycol oligomers. These ions are formed via internal hydrogen transfer reactions that are facilitated (charge-induced) by lithium. This series of product ions allows for the observation of consecutive losses of monomer units from the chain end; this is useful to determine the sequence of monomers in a copolymer. The most abundant product ions from cyclic PEGs are lithiated radical cations. An especially interesting finding in this work is the preferential loss of two internal ethylene oxide (EO) units (dioxane, 88 u) from some [M + Li]⁺ precursors. Factors that influence this loss include (a) the sequence length of EO repeat units in the oligomer and (b) the identity of the end group(s) on the oligomer. It is proposed that this elimination of dioxane involves a six-membered ring intermediate; this decomposition reaction is believed to be a lithium-mediated (charge-induced) rearrangement. (J Am Soc Mass Spectrom 1994, 5, 1072-1080)     

Structural characterization of fatty acids cationized with copper by electrospray ionization mass spectrometry under low-energy collision-induced dissociation

Fatty acids have for many years been characterized by mass spectrometry using electron ionization after chemical derivatization. When fatty acids are ionized using desorption/ionization methods such as electrospray ionization or fast atom bombardment, structural information is usually obtained through high-energy collision-induced dissociation (CID) using sector instruments. It has been shown that copper displays very interesting properties in the gas phase during CID. In this study, the reactivity of saturated and unsaturated fatty acid-copper [M-H+Cu(II)]+ complex and the role of the copper ion in promoting fragmentations were investigated under low-energy collisional activation conditions. The decomposition of these species in an ion trap instrument led to diagnostic ion series that reflect C--C bond cleavage, which involves Cu(II) reduction followed by the release of an alkyl radical. It was demonstrated that in this way the localization of one or two homoconjugated double bonds is possible using low-energy CID. Moreover, the distinction of cis and trans isomers is possible through characteristic product ions related to a specific loss of CO2. When these experiments are repeated using a triple-quadrupole instrument with argon as collision gas, a different behavior is observed as in this case, in addition to the product ion distributions observed in the ion trap, other distributions are observed that reflect the influence of the different kinetic shifts and the occurrence of consecutive decompositions. Different examples are presented with various saturated and unsaturated fatty acid chains. Mechanisms are proposed in order to rationalize the experimental observations.     

In vitro and in vivo metabolism of pyronaridine characterized by low-energy collision-induced dissociation mass spectrometry with electrospray ionization

The in vitro and in vivo metabolism of pyronaridine, an antimalarial agent, was investigated in rats and humans. In vitro incubation of pyronaridine with rat and human liver microsomes resulted in the formation of 11 metabolites, with pyronaridine quinoneimine (M3) as the major metabolite. The structures of pyronaridine metabolites were characterized on the basis of the product ion mass spectra obtained under low-energy collision-induced dissociation (CID) ion trap mass spectrometry. Both pyronaridine (m/z 518) and M3 (m/z 516) produced the same product ion (m/z 447). These results could be explained by the characteristic neutral loss of a 69 Da fragment from M3 via gamma-H rearrangement and 1,7 sigmatropic shift, whereas the neutral loss of a 71 Da fragment from the pyronaridine occurred by charge site-initiated heterolytic cleavage. These fragmentations were further supported by the tandem mass spectrum of D(3)-pyronaridine. Other metabolites generated in the microsomal incubations were carbonylated, hydroxylated and O-demethylated derivatives. Pyronaridine and its metabolites were detected in both feces and urine after intraperitoneal administration to rats. The in vivo metabolic profile in rats was different from the in vitro profile. M3, a chemically reactive quinonimine, was not detected whereas O-demethylated derivatives (M14, M15, M16, and M19) were identified in fecal and urinary extracts. The role of quinoneimine metabolites in pyronaridine-caused toxicity should be further evaluated, although these metabolites or their conjugates were not detected in urine and feces.     

Use of high-performance liquid chromatography/electrospray ionization collision-induced dissociation mass spectrometry for structural identification of monohydroxylated progesterones

For the structural identification of monohydroxylated progesterones synthesized by microorganisms, a method was developed using a combination of high-performance liquid chromatography and electrospray ionization collision-induced dissociation mass spectrometry (HPLC/ESI-CIDMS). The retention times and MS/MS spectra of 11 different standards at 30 eV were collected and compared. The identification of D-ring-hydroxylated progesterones (15beta-, 16alpha-, 17alpha- and 21-OH-P) using ESI-CIDMS was not possible. However, they were separated chromatographically using a 65:35 mixture of water and acetonitrile containing 0.5% acetic acid. The other hydroxylated progesterones (2alpha-, 6beta-, 7beta-, 9alpha-, 11alpha-, 11beta-, and 19-OH-P) could be identified by comparison of eight fragments. The complete separation of 11 standards was achieved chromatographically. The developed assay was evaluated by the identification of monohydroxylated progesterones produced by CYP106A2 from Bacillus megaterium ATCC 13368.     

Characterization of pyrene end-labeled poly(ethylene glycol) by high resolution MALDI time-of-flight mass spectrometry

We report the characterization of a sample of poly(ethylene glycol) (PEG, M _n = 3841, M _w/M _n = 1.01), and its derivative end-labeled with pyrenebutyrate groups, using high resolution MALDI time-of-flight mass spectrometry. A matrix of 2-(4-hydroxyphenyl-azo)benzoic acid containing a trace of either sodium chloride or potassium chloride was employed for laser desorption. Peaks due to the sodium or potassium cationized polymers were obtained, equally spaced at 44 mass units apart. For the pyrenebutyrate diester, the analysis shows that 80 ± 2% of the chains were doubly labeled, with the ramaining chains containing only a single pyrene group. Molecular weight determinations for both sets of samples were entirely consistent with size-exclusion chromatography measurements, but were obtained with greater accuracy and less ambiguity about the influence of the end groups on the hydrodynamic radius of the polymer.     

Qualitative assessment of monomer ratios in putative ionic terpolymer samples by electrospray ionization mass spectrometry with collision-induced dissociation

Collision-induced dissociation in the source of an electrospray (ES) mass spectrometer was employed to characterize putative samples of the ionic terpolymer poly(styrene sulfonate-co-acrylic acid-co-2-acrylamido-2-methyl-1-propanesulfonic acid). Qualitative and semi-quantitative information about the monomer content was quickly obtainable from ES spectra, and indicated that some samples contained little or none of one or two expected comonomers. For two representative samples, confirmatory nuclear magnetic resonance (NMR) data were acquired. The NMR experiments required sample clean-up (to remove additives) and long acquisition times (up to 720 min) for 13C NMR. Cleanup also improved the ES results, providing better agreement with the NMR data. However, qualitative and semi-quantitative information was obtainable by ES (but not by NMR) without the cleanup step. Full quantitation of monomer ratios would require suitable standards, but even without such standards the ES measurements provide a rapid (<1 min) means for differentiating these samples (e.g., for process or quality control).     

Charging megadalton poly(ethylene oxide)s by electrospray ionization. A charge detection mass spectrometry study

Ions from compounds of megadalton (MDa) molecular weight were produced in an electrospray ionization source from solutions of poly(ethylene oxide) (PEO) samples with average molecular weights ranging from 1,000,000 to 7,000,000 Da. Charge detection mass spectrometry (CDMS) has been used to determine the mass of the MDa PEOs. Simultaneous measurement of the charge and velocity of individual ions allows the mass determination of the ion, after calibration of the instrument with independent samples. In addition to the mass spectra, CDMS generates charge-versus-mass plots, which allow investigation of the charging of electrosprayed ions over a broad range of masses. The experimental charging capacity of MDa PEOs is compared with a simple model based on the affinity of alkali cations for oxygen sites and on the electrostatic potential energy of the charged polymer. The charging capacity of PEOs was also investigated as a function of the concentration of and the type of alkali ions.